Journal articles: 'Electrophoretic exclusion'

1

Kenyon, Stacy M., Michael W. Keebaugh, and Mark A. Hayes. "Development of the resolution theory for electrophoretic exclusion." ELECTROPHORESIS 35, no. 18 (July 21, 2014): 2551–59. http://dx.doi.org/10.1002/elps.201300572.

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Meighan, Michelle M., Michael W. Keebaugh, Alicia M. Quihuis, Stacy M. Kenyon, and Mark A. Hayes. "Electrophoretic exclusion for the selective transport of small molecules." ELECTROPHORESIS 30, no. 21 (November 2009): 3786–92. http://dx.doi.org/10.1002/elps.200900340.

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Esplandiu, Maria J., David Reguera, and Jordi Fraxedas. "Electrophoretic origin of long-range repulsion of colloids near water/Nafion interfaces." Soft Matter 16, no. 15 (2020): 3717–26. http://dx.doi.org/10.1039/d0sm00170h.

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Zhu, Fanyi, Brent L. Nannenga, and Mark A. Hayes. "Electrophoretic exclusion microscale sample preparation for cryo-EM structural determination of proteins." Biomicrofluidics 13, no. 5 (September 2019): 054112. http://dx.doi.org/10.1063/1.5124311.

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Meighan, Michelle M., Jared Vasquez, Luke Dziubcynski, Sarah Hews, and Mark A. Hayes. "Investigation of Electrophoretic Exclusion Method for the Concentration and Differentiation of Proteins." Analytical Chemistry 83, no. 1 (January 2011): 368–73. http://dx.doi.org/10.1021/ac1025495.

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Kenyon, Stacy M., Noah G. Weiss, and Mark A. Hayes. "Using electrophoretic exclusion to manipulate small molecules and particles on a microdevice." ELECTROPHORESIS 33, no. 8 (April 2012): 1227–35. http://dx.doi.org/10.1002/elps.201100622.

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Zhu, Fanyi, and Mark A. Hayes. "Simulation and experiment of asymmetric electrode placement for electrophoretic exclusion in a microdevice." ELECTROPHORESIS 40, no. 2 (November 6, 2018): 304–14. http://dx.doi.org/10.1002/elps.201700497.

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Kazmierczak, S. C., F. Van Lente, A. M. McHugh, and W. E. Katzin. "Macroamylasemia with a markedly increased amylase clearance ratio in a patient with renal cell carcinoma." Clinical Chemistry 34, no. 2 (February 1, 1988): 435–38. http://dx.doi.org/10.1093/clinchem/34.2.435.

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Abstract We report hyperamylasemia, macroamylasemia, and a markedly increased amylase clearance/creatinine clearance ratio in a patient with renal cell carcinoma. Serum amylase activity was characterized as macroamylase by gel exclusion chromatography. Electrophoretic separation revealed an atypical band of amylase, migrating anodal to the S2 control fraction. Electrophoresis of urine revealed the presence of both S1 and S2 fractions, but not the atypical band found in serum. Quantification of the salivary- and pancreatic-type amylase fractions showed amylase in urine to be 100% salivary. Immunofixation disclosed the macroamylase to consist of an immune complex between amylase and IgA-lambda antibody. Binding-capacity studies showed that the serum immunoglobulin was present in excess and could bind 46% and 49% additional S-type amylase activity derived from saliva and the patient's urine, respectively. The amylase clearance/creatinine clearance ratio was markedly supranormal (0.134), unexpected in a patient with macroamylasemia. A biopsy specimen of the renal cell tumor was found to contain significant salivary-type amylase activity. These results suggest production of amylase by tumor tissue in the renal carcinoma and secretion of S-type amylase into the patient's urine. Evidently, macroamylase should be confirmed by gel exclusion chromatography.

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Duggleby, Ronald G. "Calculation of the molecular weight of proteins from electrophoretic and gel exclusion chromatographic experiments." Bioinformatics 10, no. 2 (1994): 133–35. http://dx.doi.org/10.1093/bioinformatics/10.2.133.

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Sherwood, J. D., and S. Ghosal. "Nonlinear electrophoresis of a tightly fitting sphere in a cylindrical tube." Journal of Fluid Mechanics 843 (March 28, 2018): 847–71. http://dx.doi.org/10.1017/jfm.2018.212.

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We investigate electrophoresis of a tightly fitting sphere of radius $R-h_{0}$ on the axis of a circular tube of radius $R$, using lubrication theory and ideas due to Schnitzer & Yariv (Phys. Fluids, vol. 26, 2014, 122002). The electrical charge clouds on both the cylindrical wall and the surface of the sphere are assumed thin compared to the gap between the sphere and cylinder, so that charge clouds do not overlap and ion exclusion effects are minimal. Nevertheless, non-uniform pumping of counter-ions within the charge clouds leads to a change in the ionic concentration outside the charge clouds in the narrow gap between sphere and cylinder. The electro-osmotic slip velocities at the two surfaces are modified, leading to a decrease in the electrophoretic velocity of the sphere at low Péclet numbers and an increase in the velocity at high Péclet numbers. When the field strength $E_{0}$ is low, it is known that the electrophoretic velocity $U_{0}$ is proportional to $E_{0}(\unicode[STIX]{x1D701}_{s}-\unicode[STIX]{x1D701}_{c})$ which is zero when the zeta potential $\unicode[STIX]{x1D701}_{s}$ on the sphere surface is equal to the zeta potential $\unicode[STIX]{x1D701}_{c}$ on the cylinder. The perturbation to the above low field strength electrophoretic velocity (at high Péclet number) is predicted to be proportional to $E_{0}^{3}(\unicode[STIX]{x1D701}_{s}+\unicode[STIX]{x1D701}_{c})^{2}(\unicode[STIX]{x1D70E}_{s}+\unicode[STIX]{x1D70E}_{c})$, where $\unicode[STIX]{x1D70E}_{s}$ and $\unicode[STIX]{x1D70E}_{c}$ are the surface charge densities on the sphere and cylinder. The choice of materials with similar or identical zeta potentials (and surface charge densities) for the cylinder and sphere should therefore facilitate the observation of velocities nonlinear in the field strength $E_{0}$, since the reference linear electrophoretic velocity will be small.

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Hill, Terry W. "Electrophoretic characterization of endo-(1,4)-β-glucanases secreted during assimilative growth and antheridiol-induced branching inAchlya ambisexualis." Canadian Journal of Microbiology 42, no. 6 (June 1, 1996): 557–61. http://dx.doi.org/10.1139/m96-075.

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Secreted endo-(1,4)-β-glucanases ("cellulases") of Achlya ambisexualis were analyzed by a technique that permits visualization of enzyme activity in situ after electrophoresis in gels containing sodium dodecyl sulfate. Catalytic polypeptides with molecular masses of about 97, 74, 36, 29, and 25 kDa were observed in media from young cultures, though progressively fewer bands were observed as cultures aged. Based on size estimations of native enzymes with gel exclusion chromatography, the 97- and 36-kDa polypeptides were concluded to be subunits of a 245-kDa holoenzyme and the 25-kDa polypeptides were concluded to be subunits of a second holoenzyme of about 92 kDa. The data were insufficient to allow similar assignments for the more ephemeral 74- and 29-kDa polypeptides. The endoglucanases secreted during branch induction by antheridiol or 0.2% peptone comigrated in electrophoretic gels with enzymes secreted during normal assimilative growth. No endoglucanases specific to induced branching were observed.Key words: oomycetes, cell walls, endoglucanases, cellulases, antheridiol.

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Larroque, Oscar R., Maria C. Gianibelli, Ian L. Batey, and Fin Macritchie. "Electrophoretic characterisation of fractions collected from gluten protein extracts subjected to size-exclusion high-performance liquid chromatography." Electrophoresis 18, no. 7 (1997): 1064–67. http://dx.doi.org/10.1002/elps.1150180706.

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13

Varlet-Marie, Emmanuelle, Michael Ashenden, Françoise Lasne, Marie-Therese Sicart, Benedicte Marion, Jacques de Ceaurriz, and Michel Audran. "Detection of Hemoglobin-Based Oxygen Carriers in Human Serum for Doping Analysis: Confirmation by Size-Exclusion HPLC." Clinical Chemistry 50, no. 4 (April 1, 2004): 723–31. http://dx.doi.org/10.1373/clinchem.2003.026591.

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Abstract Background: Hemoglobin-based oxygen carriers (HBOCs) are being developed as potential substitutes for the oxygen-carrying functions of erythrocytes, but athletes may obtain and experiment with HBOCs as an illicit means of enhancing oxygen transport. An electrophoretic technique has been developed to screen for the presence of HBOCs in blood samples (Lasne et al. Clin Chem 2004;50:410–5). Interest has focused on complementary methods that can provide legally defensible scientific evidence for the presence of HBOCs in blood samples collected for doping control. Methods: The aim of this research was to develop a size-exclusion SEC-HPLC technique to identify in plasma or serum samples the presence of HBOCs that are currently under development. This method was also used to detect a polymerized bovine hemoglobin (Hemopure®) after infusion in 12 healthy males. Results: The chromatograms of all HBOCs tested were clearly separated from the 54-min peak associated with human hemoglobin dimers. It was possible to differentiate between the different HBOC products based solely on their chromatographic profiles, provided they were at high concentrations. Differences were discernible not only based on the presence (or absence) of peaks, but also the separation between respective peaks. The profiles for serum samples collected from the men immediately after infusion of Hemopure showed a distinctive profile. The shape of the chromatographic profile remained consistent for at least 48 h. Conclusions: Under the analytical conditions reported here, SEC-HPLC was able to separate native hemoglobin from the modified hemoglobin molecules present in each of the HBOC products studied. In tandem with electrophoretic screening, SEC-HPLC provides evidence of the presence of HBOCs and can therefore be regarded as a method that satisfies the criteria for use in an antidoping control setting.

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Aji, Kaiser, Zhenjiang You, Pavel Bedrikovetsky, Alexander Badalyan, and Themis Carageorgos. "Experimental investigation for colloid migration in porous media under particle-rock repulsion." APPEA Journal 53, no. 2 (2013): 488. http://dx.doi.org/10.1071/aj12099.

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Electrophoretic mobilities of carboxylate-modified latex microspheres and zeta potential of soda-lime glass beads were measured at zero ionic strength and high pH. Surface potential for latex microspheres was calculated using Ohshima’s theory for soft particles. Total potential of interaction between colloidal particles and engineered porous medium evaluated according to DLVO theory showed negligibly small secondary minimum, a high potential barrier, and a primary minimum. Such conditions favour particle-bead repulsion resulting in size exclusion being a major particle retaining mechanism. Normalised outlet concentration is inverse function of particle concentration. Concentration of captured particles reduced along the length of the column in downstream direction. An analytical model for deep bed filtration of suspension in porous media and straining under size exclusion capture mechanism is developed and validated by laboratory tests on suspension flow in engineered media. The model is successfully matched with the results from column experiments, predicting the suspended particle concentrations at the outlet.

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15

Owens, R. A., M. Blackburn, and B. Ding. "Possible Involvement of the Phloem Lectin in Long-Distance Viroid Movement." Molecular Plant-Microbe Interactions® 14, no. 7 (July 2001): 905–9. http://dx.doi.org/10.1094/mpmi.2001.14.7.905.

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Incubation with cucumber phloem exudate in vitro results in a dramatic decrease in the electrophoretic mobility of Hop stunt viroid. UV cross-linking and a combination of size exclusion and ion exchange chromatography indicate that this phenomenon reflects a previously unsuspected ability of phloem protein 2, a dimeric lectin and the most abundant component of phloem exudate, to interact with RNA. In light of its demonstrated ability to move from cell to cell via plasmodesmata as well as long distances in the phloem, our results suggest that phloem protein 2 may facilitate the systemic movement of viroids and, possibly, other RNAs in vivo.

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Chakraborty, R., T. R. Meagher, and P. E. Smouse. "Parentage analysis with genetic markers in natural populations. I. The expected proportion of offspring with unambiguous paternity." Genetics 118, no. 3 (March 1, 1988): 527–36. http://dx.doi.org/10.1093/genetics/118.3.527.

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Abstract Recent studies indicate that polymorphic genetic markers are potentially helpful in resolving genealogical relationships among individuals in a natural population. Genetic data provide opportunities for paternity exclusion when genotypic incompatibilities are observed among individuals, and the present investigation examines the resolving power of genetic markers in unambiguous positive determination of paternity. Under the assumption that the mother for each offspring in a population is unambiguously known, an analytical expression for the fraction of males excluded from paternity is derived for the case where males and females may be derived from two different gene pools. This theoretical formulation can also be used to predict the fraction of births for each of which all but one male can be excluded from paternity. We show that even when the average probability of exclusion approaches unity, a substantial fraction of births yield equivocal mother-father-offspring determinations. The number of loci needed to increase the frequency of unambiguous determinations to a high level is beyond the scope of current electrophoretic studies in most species. Applications of this theory to electrophoretic data on Chamaelirium luteum (L.) shows that in 2255 offspring derived from 273 males and 70 females, only 57 triplets could be unequivocally determined with eight polymorphic protein loci, even though the average combined exclusionary power of these loci was 73%. The distribution of potentially compatible male parents, based on multilocus genotypes, was reasonably well predicted from the allele frequency data available for these loci. We demonstrate that genetic paternity analysis in natural populations cannot be reliably based on exclusionary principles alone. In order to measure the reproductive contributions of individuals in natural populations, more elaborate likelihood principles must be deployed.

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Knowles, G., S. J. Black, and D. D. Whitelaw. "Peptidase in the plasma of mice infected withTrypanosoma brucei brucei." Parasitology 95, no. 2 (October 1987): 291–300. http://dx.doi.org/10.1017/s0031182000057747.

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SUMMARYThe plasma of mice infected with pleomorphicTrypanosoma brucei bruceicontains a peptidase which has the same electrophoretic mobility on starch gels as a parasite peptidase. An enzyme with this electrophoretic mobility was not detected in the plasma of uninfected mice. The molecular weight of this enzyme in either parasite lysate or plasma from infected mice was approximately 40000 Da when assayed on a size exclusion column using high-performance liquid chromatography. The enzyme can cleave the dipeptides leu-ala, val-leu and pro-leu, but not the dipeptide phe-ala. The enzyme also cleaved the tripeptides tyr-tyr-tyr and leu-gly-gly. Another parasite peptidase which migrates on starch gels to a different position than the above-mentioned peptidase cleaved the dipeptides leu-ala, val-leu and pro-leu but could not cleave the tripeptides tyr-tyr-tyr or leu-gly-gly. Furthermore, incubation of this parasite peptidase with normal mouse plasma at 37 °C resulted in an apparent loss of detectable activity. It is postulated that the plasma of mice modifies either the charge or enzymic activity of this peptidase. We speculate that the parasite peptidase present in the plasma of mice infected withT. bruceicould contribute to pathogenesis.

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Trubetskoy, O. A., and O. E. Trubetskaya. "Analysis of fluorophores of organic substances dissolved in Suvani river water using reversed-phase liquid chromatography." Водные ресурсы 46, no. 4 (August 21, 2019): 428–37. http://dx.doi.org/10.31857/s0321-0596464428-437.

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Reversed-phase high-efficiency liquid chromatography was used in combination with detection by multiwave fluorescence for analysis of organic substances dissolved in natural water of the Suwannee River. Also analyzed were the stable electrophoretic fractions А, В, and C+D, obtained by a combination of preparative size-exclusion chromatography and analytical electrophoresis in a polyacrylamide gel. Fraction А has the largest molecular size, and fraction C+D, the smallest. Using 3D fluorescent analysis, humic-like fluorescence was detected both in the original sample and in all fractions; protein-like fluorescence is almost fully localized in fractions А and В of the largest and middle molecular sizes. The wide peak of humic-like fluorescence is split into several groups of fluorophores with different emissions maxima (435, 455, 460, and 465 nm) and degrees of hydrophobicity. The obtained results were analyzed in relation to contemporary theories of formation of humic-like fluorescence of dissolved organic substances. The low-molecular free aromatic amino acids tyrosine and tryptophan were identified in fractions А and В of the highest molecular size and constitute >50% of the protein-like fluorescence of the organic substances dissolved in the Suwannee River water. The data obtained ensure better understanding of the molecular nature of protein-like and humic-like fluorescence of organic substance dissolved in natural water.

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Hosseeini, Soudabeh, Ebrahim Kalantari, Akbar Dorgalaleh, Taregh Bamedi, Massomeh Farzi, and Dorgalele Saeed. "Thalassemia and Hemoglobinopathy Screening By HPLC Method and Comparison With Conventional Methods." Blood 122, no. 21 (November 15, 2013): 4709. http://dx.doi.org/10.1182/blood.v122.21.4709.4709.

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Background Thalassemia and hemoglobinopathies are heterogeneous group of inherited disorders that affects men and women equally. HPLC is a valuable method for hemoglobinopathy and/or thalassemia carrier screening. This study evaluate the role of cation exchange HPLC along with adjunctive tests as needed in the diagnosis of thalassaemias/haemoglobinopathies and to see the frequency of these disorders in the Iranian population. Methods This five-year study was conducted on 3780 patients. Initially complete blood count was done by autoanalyzer and then for detection of abnormal hemoglobins HPLC and HB electrophoresis methods was used. In cases with low MCV and MCH indices (MCV<80 fl,MCH<27 pg) and Hb-A2< 3.5% and normal Hb-electrophoresis, α-thalssemia trait(αα/--)was considered in the list of differential diagnosis. In cases with low MCV for exclusion of iron deficiency serum ferritin was meseared. Results Our rerults revealed that 1932 (51.11%) had normal electrophoretic pattern, 781 (20.66%) had β-thallasemia trait and 487(12.84%) had β-thallasemia major or intermedia,328( 8.67% ) had normal electrophoresis along with iron deficiency and 142 ( 3.75%) had normal Hb -electrophoresis and normal iron status but low MCV and MCH indices.We also identified 11(0.29%) with Alpha thalasemia variants Hb-H disease/alpha trait and 22(o.58%)with sickle trait and 18(o.47%)with sickle disease and 9(0.23%) S-Thal double heterozygote and 5(0.13%) with E- trait and 32(0.84%) with Hb-D variant and 1(0.026%) with heterozygote Hb-C variant and 5(0.13%) with Hb-D Iran and 1(0.026%) with Hb-J trait and 1(0.026%) Hb-S/D double heterozygote, and 1(0.026%) with Hb-D/J double heterozygote and 1(0,026%) with Hb-constant spring/ HB-H double heterozygote. Conclusion HPLC is a fast and reliable method in clinical laboratories specially in premarital, and neonatal screening laboratories. Disclosures: No relevant conflicts of interest to declare.

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Bhushan, Ravi, and Rachna Agarwal. "Reversed-phase high-performance liquid chromatographic, gel electrophoretic and size exclusion chromatographic studies of subunit structure of arachin and its molecular species." Biomedical Chromatography 20, no. 6-7 (2006): 561–68. http://dx.doi.org/10.1002/bmc.650.

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Simitsek, Phaedra Dora, Panagiota Giannikopoulou, Haralabos Katsoulas, Efstathios Sianos, George Tsoupras, Maria-Helen Spyridaki, and Costas Georgakopoulos. "Electrophoretic, size-exclusion high-performance liquid chromatography and liquid chromatography–electrospray ionization ion trap mass spectrometric detection of hemoglobin-based oxygen carriers." Analytica Chimica Acta 583, no. 2 (February 2007): 223–30. http://dx.doi.org/10.1016/j.aca.2006.10.017.

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22

Khan, Hidayatullah, Irshad Ali, Arif-ullah Khan, Mushtaq Ahmed, Zamarud Shah, Ahmad Saeed, Rubina Naz, Mohamad Rais Mustafa, and Atiya Abbasi. "Purification and Biochemical Characterization of Alkaline Serine Protease from Caesalpinia bonducella." Natural Product Communications 5, no. 6 (June 2010): 1934578X1000500. http://dx.doi.org/10.1177/1934578x1000500625.

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A high molecular weight serine protease has been purified to electrophoretic homogeneity from the seeds of Caesalpinia bonducella Flem. (Caesalpiniaceae) by the combination of size exclusion and ion exchange chromatography. About 524 fold purification was achieved with an overall recovery of 6.8%. The specific activity was found to be 86 U/mg/min at pH 8.0. The calculated Km and Vmax were 1.66 mg/mL and 496.68 units/min per mg of protein, respectively. The molecular mass was estimated to be about 63 kDa by sodium dodecyl sulfate PAGE. The enzyme showed optimum activity at pH 8.0 and exhibited its highest activity at 40°C. The enzyme was strongly inhibited by 2mM phenylmethylsulfonyl fluoride (PMSF), suggesting the presence of a serine residue at the active site. PMSF showed a pure competitive type of inhibition with the serine protease enzyme. It was observed that enzyme activity was enhanced in the presence of dications and was active against a variety of modified substrates and natural proteins.

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Napthine, Sawsan, Chris H. Hill, Holly C. M. Nugent, and Ian Brierley. "Modulation of Viral Programmed Ribosomal Frameshifting and Stop Codon Readthrough by the Host Restriction Factor Shiftless." Viruses 13, no. 7 (June 25, 2021): 1230. http://dx.doi.org/10.3390/v13071230.

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The product of the interferon-stimulated gene C19orf66, Shiftless (SHFL), restricts human immunodeficiency virus replication through downregulation of the efficiency of the viral gag/pol frameshifting signal. In this study, we demonstrate that bacterially expressed, purified SHFL can decrease the efficiency of programmed ribosomal frameshifting in vitro at a variety of sites, including the RNA pseudoknot-dependent signals of the coronaviruses IBV, SARS-CoV and SARS-CoV-2, and the protein-dependent stimulators of the cardioviruses EMCV and TMEV. SHFL also reduced the efficiency of stop-codon readthrough at the murine leukemia virus gag/pol signal. Using size-exclusion chromatography, we confirm the binding of the purified protein to mammalian ribosomes in vitro. Finally, through electrophoretic mobility shift assays and mutational analysis, we show that expressed SHFL has strong RNA binding activity that is necessary for full activity in the inhibition of frameshifting, but shows no clear specificity for stimulatory RNA structures.

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Stöcker, G., H. E. Meyer, C. Wagener, and H. Greiling. "Purification and N-terminal amino acid sequence of a chondroitin sulphate/dermatan sulphate proteoglycan isolated from intima/media preparations of human aorta." Biochemical Journal 274, no. 2 (March 1, 1991): 415–20. http://dx.doi.org/10.1042/bj2740415.

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A proteoglycan (PG) was purified to homogeneity from intima/media preparations of human aorta specimens by the following chromatographic steps: Sepharose Q anion exchange, Sepharose CL-4B size exclusion, hydroxyapatite, MonoQ anion exchange and TSK G 4000 SW size exclusion. The purity of the preparation was established by SDS/PAGE using direct staining by silver or Dimethylmethylene Blue, as well as by Western blots of biotin-labelled samples. The electrophoretic mobility of the native PG was less than that of a 200,000-Mr standard protein. After treatment with chondroitin sulphate lyase ABC, a core protein of Mr 15,000 was revealed. The Mr of the glycosaminoglycan (GAG) peptides was less than 24,000, by comparison with a keratan sulphate peptide. The composition of the GAG chains was determined by differential digestion of the PG by chondroitin sulphate lyases AC/ABC or chondroitin sulphate lyase AC alone followed by anion-exchange chromatography of the resulting disaccharides. The GAG chains are composed of approximately one-third of dermatan sulphate and two-thirds chondroitin sulphate disaccharide units. The sequence of the 20 N-terminal amino acids is identical with the sequence previously reported for PG I isolated from human developing bone [Fisher, Termine & Young (1989) J. Biol. Chem. 264, 4571-4576]. The assignment of glycosylation sites to the serine residues in positions 5 and 10 was confirmed. The findings indicate that the chondroitin sulphate/dermatan sulphate PG is a major PG in intima/media preparations of human aorta and represents a biglycan-type PG.

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LaCrosse, G. L., and F. P. Doerder. "A temperature-sensitive mutation of the temperature-regulated SerH3 i-antigen gene of Tetrahymena thermophila: implications for regulation of mutual exclusion." Genetics 138, no. 2 (October 1, 1994): 297–301. http://dx.doi.org/10.1093/genetics/138.2.297.

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Abstract The Ser genes of Tetrahymena thermophila specify alternative forms of a major cell surface glycoprotein, the immobilization or i-antigen (i-ag). Regulation of i-ag expression assures that at least one i-ag gene is expressed at all times. To learn more about the regulatory system and the possible role of i-ag itself, we studied SerH3-ts1, a temperature-sensitive allele of the temperature-regulated SerH3 gene normally expressed from 20-36 degrees. In homozygotes grown at the nonpermissive temperature (> 32 degrees), H3 is not present on the cell surface, but the gene continues to be transcribed until its 36 degrees cutoff. H3 formed at the permissive temperature is stable at nonpermissive temperatures, indicating that SerH3-ts1 is temperature-sensitive for synthesis rather than function. At nonpermissive temperatures, the S i-ag is expressed in place of H3. This result suggests that normal H protein may play a role in regulating S expression. SerH3-ts1 was isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Sequencing of SerH3-ts1 revealed a single A --> G transition at nucleotide 473, resulting in the substitution of glycine for aspartate. The affected residue is conserved in the internal repeats comprising the H protein, and the charge difference correlates with changes in electrophoretic mobility of the H3 protein.

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Bhushan, Ravi, and Upasana Arya. "Reversed-phase high-performance liquid chromatographic, size exclusion chromatographic and polyacrylamide gel electrophoretic studies of glycinin: evidence for molecular species and their association–dissociation." Biomedical Chromatography 21, no. 12 (2007): 1245–51. http://dx.doi.org/10.1002/bmc.876.

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Gupta, R. P., S. E. Patton, A. M. Jetten, and G. E. R. Hook. "Purification, characterization and proteinase-inhibitory activity of a Clara-cell secretory protein from the pulmonary extracellular lining of rabbits." Biochemical Journal 248, no. 2 (December 1, 1987): 337–44. http://dx.doi.org/10.1042/bj2480337.

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A low-Mr Clara-cell secretory protein, CCSP, previously shown to be a major secretory product of Clara cells, was isolated from rabbit lung lavage effluents. CCSP accounted for 4.4 +/- 0.5% of the protein in the soluble phase of cell- and surfactant-free pulmonary lavage effluents (LE). Purification of this protein from LE was achieved in two steps. First, the LE was acidified with HCIO4 and, secondly, CCSP was isolated by gel-exclusion chromatography on Sephadex G-50. Purified CCSP was homogeneous by SDS/polyacrylamide-gel electrophoresis (PAGE), consisting of a single major isoform with a pI of 6.0. The Mr of CCSP was 5800 according to SDS/PAGE under reducing conditions and 12,600 under non-reducing conditions. However, by gel chromatography the Mr of the protein under non-reducing conditions was 12,400 and under reducing conditions it increased to 15,000. The discrepancy obtained by using these two techniques was attributed to anomalous electrophoretic mobilities of the protein in its reduced state. The molecule contained three half-cystine residues, but no free thiol groups, and tryptophan was not detectable. The first seven N-terminal amino acid residues were Gly-Ile-Xaa-Pro-Arg-Phe-Ala-. The third residue was not identified. CCSP showed inhibitory activity against the thiol proteinase papain (50% inhibition at 4 microM-CCSP), but only weak activities against human polymorphonuclear-leucocyte elastase, and bovine trypsin. The molecule was not digested by, and did not complex with, trypsin. CCSP was immunochemically different from surfactant apoprotein B (Mr 10,000) present in rabbit lung surfactant. This study is the first partial characterization of the major secretory protein of rabbit lung Clara cells.

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Blahovec, J., Z. Kostecka, MC Lacroix, L. Cabanie, F. Godeau, J. Mester, and F. Cavaille. "Mitogenic activity of high molecular weight forms of insulin-like growth factor-II in amniotic fluid." Journal of Endocrinology 169, no. 3 (June 1, 2001): 563–72. http://dx.doi.org/10.1677/joe.0.1690563.

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Amniotic fluid (AF) collected from ewes and goats at mid gestation displayed mitogenic activity in mouse fibroblasts. Upon fractionation of this material by size exclusion chromatography, the mitogenic activity was resolved into two peaks, whose activity was inhibited by an anti-IGF type 1 receptor blocking antibody. One of the peaks contained IGF-I and IGF-II (mature form), whereas the other contained high M(r) precursor forms of IGF-II. The presence in this latter fraction of IGF-binding proteins (IGFBP) suggests that the AF IGFBPs do not efficiently inhibit the mitogenic activity of the high M(r) forms of IGF-II. In agreement with this conclusion, exogenous IGFBP-1 failed to affect this activity. Analysis of IGF-II in sheep AF showed that the AF concentrations of both forms of IGF-II increased dramatically from mid pregnancy until 106-120 days of gestation, and fell thereafter. The amniotic IGFBPs followed a similar evolution. High M(r) forms of IGF-II were also found in human AF, with a pattern of electrophoretic migration different from that of sheep. We suggest that the precursor forms of IGF-II may play an important role in foetal development.

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Nanjee, M. Nazeem, and Eliot A. Brinton. "Very Small Apolipoprotein A-I-containing Particles from Human Plasma: Isolation and Quantification by High-Performance Size-Exclusion Chromatography." Clinical Chemistry 46, no. 2 (February 1, 2000): 207–23. http://dx.doi.org/10.1093/clinchem/46.2.207.

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Abstract Background: Very small apolipoprotein (apo) A-I-containing lipoprotein (Sm LpA-I) particles with pre-β electrophoretic mobility may play key roles as “nascent” and/or “senescent” HDL; however, methods for their isolation are difficult and often semiquantitative. Methods: We developed a preparative method for separating Sm LpA-I particles from human plasma by high-performance size-exclusion chromatography (HP-SEC), using two gel permeation columns (Superdex 200 and Superdex 75) in series and measuring apo A-I content in column fractions in 30 subjects with HDL-cholesterol (HDL-C) concentrations of 0.4–3.83 mmol/L. Results: Three major sizes of apo A-I-containing particles were detected: an ∼15-nm diameter (∼700 kDa) species; a 7.5–12 nm (100–450 kDa) species; and a 5.8–6.3 nm species (40–60 kDa, Sm LpA-I particles), containing 0.2–3%, 80–96%, and 2–15% of plasma total apo A-I, respectively. Two subjects with severe HDL deficiency had increased relative apo A-I content in Sm LpA-I: 25% and 37%, respectively. The percentage of apo A-I in Sm LpA-I correlated positively with fasting plasma triglyceride concentrations (r = 0.581; P <0.0005) and inversely with total apo A-I (r = −0.551; P <0.0013) and HDL-C concentrations (r = −0.532; P <0.0017), although the latter two relationships were largely attributable to extremely hypoalphalipoproteinemic subjects. The percentage of apo A-I in Sm LpA-I correlated with that in pre-β-migrating species by crossed immunoelectrophoresis (r = 0.98; P <0.0001; n = 24) and with that in the d >1.21 kg/L fraction by ultracentrifugation (r = 0.86; P <0.001; n = 20). Sm LpA-I particles, on average, appear to contain two apo A-I and four phospholipid molecules but little or no apo A-II, triglyceride, or cholesterol. Conclusions: We present a new HP-SEC method for size separation of native HDL particles from plasma, including Sm Lp A-I, which may play important roles in the metabolism of HDL and in its contribution(s) to protection against atherosclerosis. This method provides a basis for further studies of the structure and function of Sm Lp A-I.

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30

Campos, Evangelina, Laura Baldoma, Juan Aguilar, and Josefa Badia. "Regulation of Expression of the Divergent ulaG and ulaABCDEF Operons Involved in l-Ascorbate Dissimilation in Escherichia coli." Journal of Bacteriology 186, no. 6 (March 15, 2004): 1720–28. http://dx.doi.org/10.1128/jb.186.6.1720-1728.2004.

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ABSTRACT The ula regulon, responsible for the utilization of l-ascorbate in Escherichia coli, is formed by two divergently transcribed operons, ulaG and ulaABCDEF. The regulon is negatively regulated by a repressor of the DeoR family which is encoded by the constitutive gene ulaR located downstream of ulaG. Full repression of the ula regulon requires simultaneous interaction of the repressor with both divergent promoters and seems to be dependent on repressor-mediated DNA loop formation, which is helped by the action of integration host factor. Two operator sites have been identified in each promoter. Lack of either of the two sets of operators partially relieved the repression of the other operon; thus, each promoter is dependent on the UlaR operator sites of the other promoter to enhance repression. Electrophoretic mobility shift assays with purified UlaR protein and promoter deletion analyses revealed a conserved sequence, present in each of the four operators, acting as a UlaR binding site. Glucose represses the ula regulon via at least two mechanisms, one dependent on cyclic AMP (cAMP)-cAMP receptor protein (CRP) and the other (possibly inducer exclusion) independent of it. Glucose effects mediated by other global regulators cannot be ruled out with the present information. Changes in cAMP-CRP levels affected only the expression of the ulaABCDEF operon.

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Grau, Florian C., Jeannine Jaeger, Florian Groher, Beatrix Suess, and Yves A. Muller. "The complex formed between a synthetic RNA aptamer and the transcription repressor TetR is a structural and functional twin of the operator DNA–TetR regulator complex." Nucleic Acids Research 48, no. 6 (February 13, 2020): 3366–78. http://dx.doi.org/10.1093/nar/gkaa083.

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Abstract RNAs play major roles in the regulation of gene expression. Hence, designer RNA molecules are increasingly explored as regulatory switches in synthetic biology. Among these, the TetR-binding RNA aptamer was selected by its ability to compete with operator DNA for binding to the bacterial repressor TetR. A fortuitous finding was that induction of TetR by tetracycline abolishes both RNA aptamer and operator DNA binding in TetR. This enabled numerous applications exploiting both the specificity of the RNA aptamer and the efficient gene repressor properties of TetR. Here, we present the crystal structure of the TetR-RNA aptamer complex at 2.7 Å resolution together with a comprehensive characterization of the TetR–RNA aptamer versus TetR–operator DNA interaction using site-directed mutagenesis, size exclusion chromatography, electrophoretic mobility shift assays and isothermal titration calorimetry. The fold of the RNA aptamer bears no resemblance to regular B-DNA, and neither does the thermodynamic characterization of the complex formation reaction. Nevertheless, the functional aptamer-binding epitope of TetR is fully contained within its DNA-binding epitope. In the RNA aptamer complex, TetR adopts the well-characterized DNA-binding-competent conformation of TetR, thus revealing how the synthetic TetR-binding aptamer strikes the chords of the bimodal allosteric behaviour of TetR to function as a synthetic regulator.

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32

Dufort, D., and A. Nepveu. "The human cut homeodomain protein represses transcription from the c-myc promoter." Molecular and Cellular Biology 14, no. 6 (June 1994): 4251–57. http://dx.doi.org/10.1128/mcb.14.6.4251.

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Studies of the c-myc promoter have shown that efficient transcription initiation at the P2 start site as well as the block to elongation of transcription require the presence of the ME1a1 protein binding site upstream of the P2 TATA box. Following fractionation by size exclusion chromatography, three protein-ME1a1 DNA complexes, a, b, and c, were detected by electrophoretic mobility shift assay. A cDNA encoding a protein present in complex c was isolated by screening of an expression library with an ME1a1 DNA probe. This cDNA was found to encode the human homolog of the Drosophila Cut homeodomain protein. The bacterially expressed human Cut (hu-Cut) protein bound to the ME1a1 site, and antibodies against hu-Cut inhibited the ME1a1 binding activity c in nuclear extracts. In cotransfection experiments, the hu-Cut protein repressed transcription from the c-myc promoter, and this repression was shown to be dependent on the presence of the ME1a1 site. Using a reporter construct with a heterologous promoter, we found that c-myc exon 1 sequences were also necessary, in addition to the ME1a1 site, for repression by Cut. Taken together, these results suggest that the human homolog of the Drosophila Cut homeodomain protein is involved in regulation of the c-myc gene.

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33

Odin, P., A. Tingström, and B. Obrink. "Chemical characterization of cell-CAM 105, a cell-adhesion molecule isolated from rat liver membranes." Biochemical Journal 236, no. 2 (June 1, 1986): 559–68. http://dx.doi.org/10.1042/bj2360559.

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Cell-CAM 105, a glycoprotein that is involved in recognition and adhesion between isolated rat hepatocytes in vitro, was purified to homogeneity by a combination of immunoaffinity chromatography, gel-exclusion chromatography and ion-exchange chromatography. Electrophoretic, compositional and enzymic analyses were performed and the glycoprotein was shown to consist of two peptide chains, of apparent Mr 110,000 and 105,000 respectively, that are glycosylated to similar extents. Carbohydrate analyses demonstrated the presence of sialic acid, galactose, mannose, fucose and glucosamine, but no galactosamine, indicating that only N-linked oligosaccharides occurred. The total content of carbohydrate amounted to 33%. Peptide mapping indicated that the two peptide chains were structurally very similar. After incubation of cultured hepatocytes with [32P]Pi, phosphorylated cell-CAM 105 could be isolated. Both peptide chains were labelled and phospho-amino-acid analysis demonstrated that serine residues had become phosphorylated. A significant feature of cell-CAM 105 was a susceptibility to autolytic degradation that was difficult to inhibit. The major degradation products had apparent Mr 90,000 and 70,000, respectively. The effect of purified cell-CAM 105 on cell-cell adhesion of re-aggregating hepatocytes was studied. A significant inhibition was observed, indicating that the protein is directly involved in intercellular adhesion of these cells.

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34

Dufort, D., and A. Nepveu. "The human cut homeodomain protein represses transcription from the c-myc promoter." Molecular and Cellular Biology 14, no. 6 (June 1994): 4251–57. http://dx.doi.org/10.1128/mcb.14.6.4251-4257.1994.

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Studies of the c-myc promoter have shown that efficient transcription initiation at the P2 start site as well as the block to elongation of transcription require the presence of the ME1a1 protein binding site upstream of the P2 TATA box. Following fractionation by size exclusion chromatography, three protein-ME1a1 DNA complexes, a, b, and c, were detected by electrophoretic mobility shift assay. A cDNA encoding a protein present in complex c was isolated by screening of an expression library with an ME1a1 DNA probe. This cDNA was found to encode the human homolog of the Drosophila Cut homeodomain protein. The bacterially expressed human Cut (hu-Cut) protein bound to the ME1a1 site, and antibodies against hu-Cut inhibited the ME1a1 binding activity c in nuclear extracts. In cotransfection experiments, the hu-Cut protein repressed transcription from the c-myc promoter, and this repression was shown to be dependent on the presence of the ME1a1 site. Using a reporter construct with a heterologous promoter, we found that c-myc exon 1 sequences were also necessary, in addition to the ME1a1 site, for repression by Cut. Taken together, these results suggest that the human homolog of the Drosophila Cut homeodomain protein is involved in regulation of the c-myc gene.

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35

Cruz Rodríguez, Alberto, Fabiola Anaid Sánchez Esperanza, Eduardo Pérez-Campos, María Teresa Hernández-Huerta, Laura Pérez-Campos Mayoral, Carlos Alberto Matias-Cervantes, Alexis Martínez Barras, et al. "Aggregation and Molecular Properties of β-Glucosidase Isoform II in Chayote (Sechium edule)." Molecules 25, no. 7 (April 8, 2020): 1699. http://dx.doi.org/10.3390/molecules25071699.

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The presence of isoforms of β-glucosidase has been reported in some grasses such as sorghum, rice and maize. This work aims to extract and characterize isoform II in β-glucosidase from S. edule. A crude extract was prepared without buffer solution and adjusted to pH 4.6. Contaminating proteins were precipitated at 4 °C for 24 h. The supernatant was purified by chromatography on carboxymethyl cellulose (CMC) column, molecular exclusion on Sephacryl S-200HR, and exchange anionic on QFF column. Electrophoretic analyzes revealed a purified enzyme with aggregating molecular complex on SDS-PAGE, Native-PAGE, and AU-PAGE. Twelve peptides fragments were identified by nano liquid chromatography-tandem mass spectrometry (nano LC-ESI-MS/MS), which presented as 61% identical to Cucurbita moschata β-glucosidase and 55.74% identical to β-glucosidase from Cucumis sativus, another Cucurbitaceous member. The relative masses which contained 39% hydrophobic amino acids ranged from 982.49 to 2,781.26. The enzyme showed a specificity to β-d-glucose with a Km of 4.59 mM, a Vmax value of 104.3 μM∙min−1 and a kcat of 10,087 μM∙min−1 using p-nitrophenyl-β-D-glucopyranoside. The presence of molecular aggregates can be attributed to non-polar amino acids. This property is not mediated by a β-glucosidase aggregating factor (BGAF) as in grasses (maize and sorghum). The role of these aggregates is discussed.

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36

Manolaridis, Ioannis, Eleni Mumtsidu, Peter Konarev, Alexander M. Makhov, Stephen W. Fullerton, Andrea Sinz, Stefan Kalkhof, et al. "Structural and Biophysical Characterization of the Proteins Interacting with the Herpes Simplex Virus 1 Origin of Replication." Journal of Biological Chemistry 284, no. 24 (March 27, 2009): 16343–53. http://dx.doi.org/10.1074/jbc.m806134200.

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The C terminus of the herpes simplex virus type 1 origin-binding protein, UL9ct, interacts directly with the viral single-stranded DNA-binding protein ICP8. We show that a 60-amino acid C-terminal deletion mutant of ICP8 (ICP8ΔC) also binds very strongly to UL9ct. Using small angle x-ray scattering, the low resolution solution structures of UL9ct alone, in complex with ICP8ΔC, and in complex with a 15-mer double-stranded DNA containing Box I of the origin of replication are described. Size exclusion chromatography, analytical ultracentrifugation, and electrophoretic mobility shift assays, backed up by isothermal titration calorimetry measurements, are used to show that the stoichiometry of the UL9ct-dsDNA15-mer complex is 2:1 at micromolar protein concentrations. The reaction occurs in two steps with initial binding of UL9ct to DNA (Kd ∼ 6 nm) followed by a second binding event (Kd ∼ 0.8 nm). It is also shown that the stoichiometry of the ternary UL9ct-ICP8ΔC-dsDNA15-mer complex is 2:1:1, at the concentrations used in the different assays. Electron microscopy indicates that the complex assembled on the extended origin, oriS, rather than Box I alone, is much larger. The results are consistent with a simple model whereby a conformational switch of the UL9 DNA-binding domain upon binding to Box I allows the recruitment of a UL9-ICP8 complex by interaction between the UL9 DNA-binding domains.

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37

Martínez-Carranza, Markel, Venkateswara Rao Jonna, Daniel Lundin, Margareta Sahlin, Lars-Anders Carlson, Newal Jemal, Martin Högbom, Britt-Marie Sjöberg, Pål Stenmark, and Anders Hofer. "A ribonucleotide reductase from Clostridium botulinum reveals distinct evolutionary pathways to regulation via the overall activity site." Journal of Biological Chemistry 295, no. 46 (September 3, 2020): 15576–87. http://dx.doi.org/10.1074/jbc.ra120.014895.

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Ribonucleotide reductase (RNR) is a central enzyme for the synthesis of DNA building blocks. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1–R2 octamers in Escherichia coli. To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size-exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, EM, X-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1–R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1–R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.

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38

Jiz, Mario, Hai-Wei Wu, Rui Meng, Sunthorn Pond-Tor, Mindy Reynolds, Jennifer F. Friedman, Remigio Olveda, Luz Acosta, and Jonathan D. Kurtis. "Pilot-Scale Production and Characterization of Paramyosin, a Vaccine Candidate for Schistosomiasis Japonica." Infection and Immunity 76, no. 7 (April 21, 2008): 3164–69. http://dx.doi.org/10.1128/iai.00409-08.

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ABSTRACT Despite effective chemotherapy, schistosomiasis remains a major public health problem in the developing world, with at least 200 million active infections resulting in significant morbidity. Rapid reinfection after treatment, accompanied by extensive residual morbidity, mandates alternative control strategies, including vaccine development. Paramyosin, a myofibrillar protein found only in invertebrates, has been widely studied as a vaccine candidate for both Schistosoma mansoni and Schistosoma japonicum. Recently, we demonstrated that Th2-biased immune responses to paramyosin are associated with resistance to reinfection with S. japonicum in humans; however, challenges in the pilot-scale production of schistosome paramyosin have hampered further studies of this promising vaccine candidate. Here we report a method for the pilot-scale expression and purification of recombinant S. japonicum paramyosin (rSj97). rSj97 was extracted from Escherichia coli inclusion bodies and purified with sequential anion-exchange, hydroxyapatite, and size exclusion chromatography. The purified rSj97 was >95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis and was free of significant endotoxin contamination. We demonstrate that, like native paramyosin, rSj97 adopts an alpha-helical coiled-coil tertiary structure and binds immunoglobulin and collagen. Naïve mice infected with S. japonicum produce anti-rSj97 immunoglobulin G (IgG) antibodies as early as 4 weeks postinfection, while sera collected from S. japonicum-infected individuals contain anti-rSj97 IgE antibodies. Our method for pilot-scale production of recombinant full-length paramyosin will facilitate preclinical evaluation of paramyosin as a vaccine for schistosomiasis.

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39

Ding, Haitao, Lili Zhou, Qian Zeng, Yong Yu, and Bo Chen. "Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides." Marine Drugs 16, no. 11 (October 30, 2018): 415. http://dx.doi.org/10.3390/md16110415.

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A thermostable β-1,3-galactosidase from Marinomonas sp. BSi20414 was successfully heterologously expressed in Escherichia coli BL21 (DE3), with optimum over-expression conditions as follows: the recombinant cells were induced by adding 0.1 mM of IPTG to the medium when the OD600 of the culture reached between 0.6 and 0.9, followed by 22 h incubation at 20 °C. The recombinant enzyme β-1,3-galactosidase (rMaBGA) was further purified to electrophoretic purity by immobilized metal affinity chromatography and size exclusion chromatography. The specific activity of the purified enzyme was 126.4 U mg−1 at 37 °C using ONPG (o-nitrophenyl-β-galactoside) as a substrate. The optimum temperature and pH of rMaBGA were determined as 60 °C and 6.0, respectively, resembling with its wild-type counterpart, wild type (wt)MaBGA. However, rMaBGA and wtMaBGA displayed different thermal stability and steady-state kinetics, although they share identical primary structures. It is postulated that the stability of the enzyme was altered by heterologous expression with the absence of post-translational modifications such as glycosylation, as well as the steady-state kinetics. To evaluate the potential of the enzyme in synthesis of galactooligosaccharides (GOS), the purified recombinant enzyme was employed to catalyze the transgalactosylation reaction at the lab scale. One of the transgalactosylation products was resolved as 3′-galactosyl-lactose, which had been proven to be a better bifidogenic effector than GOS with β-1,4 linkage and β-1,6 linkages. The results indicated that the recombinant enzyme would be a promising alternative for biosynthesis of GOS mainly with β-1,3 linkage.

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Abramova, M., and N. Raksha. "Development of methodological approaches to the purification of target proteins from marine hydrobionts of Antarctic region." Bulletin of Taras Shevchenko National University of Kyiv. Series: Biology 78, no. 2 (2019): 7–13. http://dx.doi.org/10.17721/1728_2748.2019.78.7-13.

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Enzymes from organisms that are adapted to the existence at low temperatures attract significant attention of scientists as a perspective objects not only on a practical point of view, but also as a valuable tools for conducting basic research. This is due to unusual environmental conditions (low temperature, high hydrostatic pressure, low illumination), as well as a significant level of economic profitability due to the widespread of marine hydrobionts and high efficiency of psychrophilic enzymes in comparison with their mesophilic and thermophilic analogues. The expediency of using the hydrobionts of the Antarctic region Parborlasia corrugatus and Sterechinus neumayeri as a source for producing individual enzymes was indicated by the results of electrophoretic analysis of enzymes from hydrobionts tissues extract, so it was concluded that the total extract contained a number of enzymes that were active with gelatin and fibrinogen. As a result of a combination of several chromatographic stages, which included affinity chromatography on Blue Sepharose 6 FF column and size exclusion chromatography on Superdex 75 PG and Superdex 200 PG columns, from the total hydrobiont tissue extract were obtained fractions of hydrolytic enzymes. From the total tissues extract of both hydrobionts which was explored were isolated 4 fractions which showed gelatinase activity. Also from the tissues of Sterechinus neumayeri a fraction containing high-molecular enzymes capable of cleaving fibrinogen was isolated. The developed method of two-stage chromatography can be used further as a basis for optimizing the obtaining of enzymes of a similar spectrum of action from other objects.

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Maillard, Julien, Wolfram Schumacher, Francisco Vazquez, Christophe Regeard, Wilfred R. Hagen, and Christof Holliger. "Characterization of the Corrinoid Iron-Sulfur Protein Tetrachloroethene Reductive Dehalogenase of Dehalobacter restrictus." Applied and Environmental Microbiology 69, no. 8 (August 2003): 4628–38. http://dx.doi.org/10.1128/aem.69.8.4628-4638.2003.

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ABSTRACT The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 ± 1 kDa, whereas the native molecular mass was 71± 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-β-d-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0± 0.1 mol of cobalamin, 0.6 ± 0.02 mol of cobalt, 7.1± 0.6 mol of iron, and 5.8 ± 0.5 mol of acid-labile sulfur. Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with a specific activity of 250 ± 12 nkat/mg of protein. In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA. The Km values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4± 3.2, 23.7 ± 5.2, and 47 ± 10 μM, respectively. The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air. Based on the almost identical N-terminal amino acid sequences of PceA of Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced. Together with the pceA genes of Desulfitobacterium hafniense strains PCE-S and Y51, the pceA genes of Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the pceB genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of Dehalobacter restrictus and the three desulfitobacteria had identical sequences. Whereas the cprB (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of cprA, all pceB genes known so far are located downstream of pceA. The possible consequences of this feature for the annotation of putative reductive dehalogenase genes are discussed, as are the sequence around the iron-sulfur cluster binding motifs and the type of iron-sulfur clusters of the reductive dehalogenases of Dehalobacter restrictus and Desulfitobacterium dehalogenans identified by electron paramagnetic resonance spectroscopy.

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Ong, Alan Ann Lerk, Jiazi Tan, Malini Bhadra, Clément Dezanet, Kiran M. Patil, Mei Sian Chong, Ryszard Kierzek, Jean-Luc Decout, Xavier Roca, and Gang Chen. "RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing." Molecules 24, no. 16 (August 20, 2019): 3020. http://dx.doi.org/10.3390/molecules24163020.

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Alternative splicing of tau pre-mRNA is regulated by a 5′ splice site (5′ss) hairpin present at the exon 10–intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant splicing of exon 10. The hairpin structure contains about seven stably formed base pairs and thus may be suitable for targeting through antisense strands. Here, we used antisense peptide nucleic acids (asPNAs) to probe and target the tau pre-mRNA exon 10 5′ss hairpin structure through strand invasion. We characterized by electrophoretic mobility shift assay the binding of the designed asPNAs to model tau splice site hairpins. The relatively short (10–15 mer) asPNAs showed nanomolar binding to wild-type hairpins as well as a disease-causing mutant hairpin C+19G, albeit with reduced binding strength. Thus, the structural stabilizing effect of C+19G mutation could be revealed by asPNA binding. In addition, our cell culture minigene splicing assay data revealed that application of an asPNA targeting the 3′ arm of the hairpin resulted in an increased exon 10 inclusion level for the disease-associated mutant C+19G, probably by exposing the 5′ss as well as inhibiting the binding of protein factors to the intronic spicing silencer. On the contrary, the application of asPNAs targeting the 5′ arm of the hairpin caused an increased exon 10 exclusion for a disease-associated mutant C+14U, mainly by blocking the 5′ss. PNAs could enter cells through conjugation with amino sugar neamine or by cotransfection with minigene plasmids using a commercially available transfection reagent.

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43

VENTER, Henrietta, Alison E. ASHCROFT, Jeffrey N. KEEN, Peter J. F. HENDERSON, and Richard B. HERBERT. "Molecular dissection of membrane-transport proteins: mass spectrometry and sequence determination of the galactose–H+ symport protein, GalP, of Escherichia coli and quantitative assay of the incorporation of [ring-2-13C]histidine and 15NH3." Biochemical Journal 363, no. 2 (April 8, 2002): 243–52. http://dx.doi.org/10.1042/bj3630243.

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The molecular mass of the galactose—H+ symport protein GalP, as its histidine-tagged derivative GalP(His)6, has been determined by electrospray MS (ESI-MS) with an error of <0.02%. One methionine residue, predicted to be present from the DNA sequence, was deduced to be absent. This is a significant advance on the estimation of the molecular masses of membrane-transport proteins by SDS/PAGE, where there is a consistent under-estimation of the true molecular mass due to anomalous electrophoretic migration. Addition of a size-exclusion chromatography step after Ni2+-nitrilotriacetate affinity purification was essential to obtain GalP(His)6 suitable for ESI-MS. Controlled trypsin, trypsin+chymotrypsin and CNBr digestion of the protein yielded peptide fragments suitable for ESI-MS and tandem MS analysis, and accurate mass determination of the derived fragments resulted in identification of 82% of the GalP(His)6 protein. Tandem MS analysis of selected peptides then afforded 49% of the actual amino acid sequence of the protein; the absence of the N-terminal methionine was confirmed. Matrix-assisted laser-desorption ionization MS allowed identification of one peptide that was not detected by ESI-MS. All the protein/peptide mass and sequence determinations were in accord with the predictions of amino acid sequence deduced from the DNA sequence of the galP gene. [ring-2-13C]Histidine was incorporated into GalP(His)6in vivo, and ESI-MS analysis enabled the measurement of a high (80%) and specific incorporation of label into the histidine residues in the protein. MS could also be used to confirm the labelling of the protein by 15NH3 (93% enrichment) and [19F]tryptophan (83% enrichment). Such MS measurements will serve in the future analysis of the structures of membrane-transport proteins by NMR, and of their topology by indirect techniques.

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Secades, P., B. Alvarez, and J. A. Guijarro. "Purification and Characterization of a Psychrophilic, Calcium-Induced, Growth-Phase-Dependent Metalloprotease from the Fish Pathogen Flavobacterium psychrophilum." Applied and Environmental Microbiology 67, no. 6 (June 1, 2001): 2436–44. http://dx.doi.org/10.1128/aem.67.6.2436-2444.2001.

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ABSTRACT Flavobacterium psychrophilum is a fish pathogen that commonly affects salmonids. This bacterium produced an extracellular protease with an estimated molecular mass of 55 kDa. This enzyme, designated Fpp1 ( F . p sychrophilum p rotease 1), was purified to electrophoretic homogeneity from the culture supernatant by using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, and size exclusion chromatography. On the basis of its biochemical characteristics, Fpp1 can be included in the group of metalloproteases that have an optimum pH for activity of 6.5 and are inhibited by 1,10-phenanthroline, EDTA, or EGTA but not by phenylmethylsulfonyl fluoride. Fpp1 activity was dependent on calcium ions not only for its activity but also for its thermal stability. In addition to calcium, strontium and barium can activate the protein. The enzyme showed typical psychrophilic behavior; it had an activation energy of 5.58 kcal/mol and was more active at temperatures between 25 and 40°C, and its activity decreased rapidly at 45°C. Fpp1 cleaved gelatin, laminin, fibronectin, fibrinogen, collagen type IV, and, to a lesser extent, collagen types I and II. Fpp1 also degraded actin and myosin, basic elements of the fish muscular system. The presence of this enzyme in culture media was specifically dependent on the calcium concentration. Fpp1 production started early in the exponential growth phase and reached a maximum during this period. Addition of calcium during the stationary phase did not induce Fpp1 production at all. Besides calcium and the growth phase, temperature also seems to play a role in production of Fpp1. In this study we found that production of Fpp1 depends on factors such as calcium concentration, growth phase of the culture, and temperature. The combination of these parameters corresponds to the combination in the natural host during outbreaks of disease caused by F. psychrophilum. Consequently, we suggest that environmental host factors govern Fpp1 production.

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Al-Soud, Waleed Abu, Leif J. Jönsson, and Peter Rådström. "Identification and Characterization of Immunoglobulin G in Blood as a Major Inhibitor of Diagnostic PCR." Journal of Clinical Microbiology 38, no. 1 (January 2000): 345–50. http://dx.doi.org/10.1128/jcm.38.1.345-350.2000.

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ABSTRACT A major inhibitor of diagnostic PCR in human plasma was identified and the mechanism of inhibition was characterized. Human blood was divided by centrifugation into buffy coat, plasma, platelets, and erythrocytes. All these blood fractions were found to be highly inhibitory to a standardized PCR mixture containing the thermostable DNA polymerase Ampli Taq Gold. PCR inhibitors in human plasma were purified by chromatographic procedures and were characterized by a process of elimination, so that the PCR-inhibitory effects of plasma fractions were tested after each purification step. The major inhibitor in human plasma, as determined by size-exclusion chromatography, anion-exchange chromatography, and chromatofocusing, was found to be immunoglobulin G (IgG) on the basis of N-terminal amino acid sequencing and electrophoretic analysis of the purified polypeptide. When different concentrations of purified plasma IgG (PIgG) were added to PCR mixtures containing 11 different thermostable DNA polymerases and 1 ng of Listeria monocytogenes DNA as template DNA, the only polymerase that resisted inhibition was rTth . The inhibitory effect was reduced when PIgG was heated at 95°C before it was added to PCR or after the addition of excess nontarget DNA to the PCR mixture. However, heating of PIgG together with target DNA at 95°C was found to block the amplification. Inhibition by PIgG may be due to an interaction with single-stranded DNA, which makes the target DNA unavailable for 10 of the DNA polymerases tested. The results show the danger of using boiling as a method of sample pretreatment or using a hot start prior to PCR. The effect of plasma PCR inhibition could be removed by mixing plasma with DNA-agarose beads prior to amplification, while plasma PCR inhibitors were found to bind to the DNA-agarose beads.

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Trubetskoj, Oleg A., Olga E. Trubetskaya, Gaida V. Afanasieva, Olga I. Reznikova, Bernardo Hermosin, and Cesareo Saiz-Jimenez. "Tandem size exclusion chromatography-Polyacrylamide gel electrophoresis of humic acids." Zeitschrift für Pflanzenernährung und Bodenkunde 161, no. 6 (December 1998): 619–25. http://dx.doi.org/10.1002/jpln.1998.3581610604.

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Pacáková, Věra, Jitka Pechancová, and Karel Štulík. "Size-exclusion liquid chromatography and capillary electrophoresis of pollen allergens." Journal of Chromatography B: Biomedical Sciences and Applications 681, no. 1 (May 1996): 47–53. http://dx.doi.org/10.1016/0378-4347(95)00488-2.

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Souza, E., and M. E. Sorrells. "Inheritance and distribution of variation at four avenin loci in North American oat germ plasm." Genome 33, no. 3 (June 1, 1990): 416–24. http://dx.doi.org/10.1139/g90-063.

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Avenins (prolamines of the Avena genus) have been shown to be useful in taxonomic studies and cultivar identification; specific allelic identification could assist in these types of studies as well as providing a basis for future linkage and gene mapping studies. The avenin patterns produced by nondenaturing polyacrylamide gel electrophoresis were compared in 70 North American oat cultivars and germ plasms. Populations of F2 progeny were subsequently evaluated to test for allelism of proteins found to be noncoincident in the survey of homozygous cultivars. A minimum of four loci (Av1, Av2, Av3, and Av4) were found to possess alternate alleles with distinctive electrophoretic mobilities. Segregation of 10 alternate alleles were observed in studies of F2 progeny: four for Av1, and two each for the other three loci. Additional variation found among the surveyed cultivars suggested at least two additional electrophoretically variant polypeptides. Several of the alleles were found to be associated with cultivars from specific geographic regions. Two examples were (i) the near exclusive association of the Av10.76 allele with Canadian cultivars and (ii) the high association of the Av10.58 allele with fall-planted cultivars. Fifty percent (SE ± 10.7%) of the fall-planted cultivars have the Av40.58 allele compared with 27.1% (SE ± 8.8%) of spring-planted cultivars.Key words: avenins, prolamines, polyacrylamide gel electrophoresis, linkage.

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Rajora, Om P., and Louis Zsuffa. "Allozyme divergence and evolutionary relationships among Populus deltoides, P. nigra, and P. maximowiczii." Genome 33, no. 1 (February 1, 1990): 44–49. http://dx.doi.org/10.1139/g90-008.

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Horizontal starch gel electrophoresis of enzymes was used to study genetic divergence among Populus deltoides Marsh. (section Aigeiros Duby, Salicaceae), P. nigra L. (section Aigeiros), and P. maximowiczii Henry (section Tacamahaca Spach.) at 37 to 40 allozyme loci coding for 12 enzyme systems in root tips. These three Populus species were genetically distinct from each other. Populus deltoides, P. nigra, and P. maximowiczii had mutually exclusive alleles at two loci, and each of these species had unique alleles at many loci. Certain allozyme loci were detected only in one or two of these species. Frequency distributions of allozyme loci were bimodal with respect to genetic identity for comparisons between any two species. The mean genetic distance was 0.36 ± 0.10 between P. deltoides and P. nigra, 0.39 ± 0.09 between P. deltoides and P. maximowiczii, and 0.34 ± 0.10 between P. nigra and P. maximowiczii. The enzyme electrophoretic evidence indicated a monophyletic origin of the three Populus species.Key words: poplars, genetic identity and divergence, allozymes, molecular evolution, phylogenetics.

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Sacchetti, Lucia, Giuseppe Calcagno, Iolanda Coto, Nadia Tinto, Emilia Vuttariello, and Francesco Salvatore. "Efficiency of Two Different Nine-Loci Short Tandem Repeat Systems for DNA Typing Purposes." Clinical Chemistry 45, no. 2 (February 1, 1999): 178–83. http://dx.doi.org/10.1093/clinchem/45.2.178.

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Abstract Background: Genotyping based on short tandem repeat (STR) regions is widely used in human identification and parentage testing, in gene mapping studies, and as an approach to studies on the etiopathogenesis and diagnosis of hereditary diseases. We wished to study a new analytical approach that uses capillary electrophoresis and multicolor fluorescence in place of slab gel electrophoresis. Methods: We evaluated the efficiency for parentage and forensic purposes of the AmpFLSTR Profiler PlusTM typing kit that is used with the ABI Prism 310 Genetic Analyzer (System-2 STR), and that of a widely used panel of nine STRs analyzed with conventional slab-gel electrophoresis followed by radioactive detection (System-1 STR). System-2 STR, based on automated capillary electrophoresis and automated sizing of the alleles by Genotyper 2.0 software, was used to determine the allele frequency of the nine loci in 157 Caucasian subjects from southern Italy. On the basis of the data obtained, we submitted 40 trios to parentage testing. Results: A higher median probability of paternity attribution and power of exclusion were obtained with System-2 STR vs System-1 STR: respectively, 99.99% and 99.95% (P <0.05) for attribution; and five and four excluding loci (P <0.05) for exclusion. The most informative and highly discriminating loci were D18S51, D21S11, and FGA. The combined probability of matching-by-chance for all nine STRs was 1.36 × 10−12 for System-2 compared with 1.11 × 10−7 obtained with the other system. The internal standard and allelic ladder of the System-2 STR facilitated accurate and precise genotyping; furthermore, System-2 STR and was faster than the conventional System-1 STR. Conclusions: The System-2 STR allows rapid testing with higher probabilities of attribution and a higher power of exclusion than with the comparison method with slab-gel electrophoresis.

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Journal articles on the topic 'Electrophoretic exclusion'

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