Results Of Electrophoretic Analysis Research Articles

ОПТИМІЗАЦІЯ МЕТОДОЛОГІЧНИХ ПІДХОДІВ ЩОДО ОДЕРЖАННЯ ЦІЛЬОВИХ БІЛКІВ ІЗ ГІДРОБІОНТІВ АНТАРКТИЧНОГО РЕГІОНУ

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.

Genetic variation within and among populations of an arctic/alpine sweat bee (Hymenoptera: Halictidae)

AbstractWe present the results of electrophoretic analyses of allozymes for eight population samples of the arctic/alpine sweat bee,Lasioglossum(Evylaeus)borealeSvensson, Sakagami and Ebmer. This holarctic species is found at high latitudes and at increasingly high elevations in the mountains of western North America as far south as southern Arizona. Our samples encompass a large proportion of the species' range; three samples are from Arizona, one from Utah, two from northern Canada, one from Mount Washington (the highest point in the eastern United States), and one from Sweden. Most samples had high levels of genetic variation compared with other bees, but the one from Sweden had low heterozygosity, suggesting that this location may have been comparatively recently colonized. The three northern North American samples were genetically similar despite the large geographic distances separating the localities (average > 3000 km). In contrast, the southern United States samples were (with the exception of one pairwise estimate) genetically divergent despite the small geographic distances separating them (average < 500 km). These results are consistent with earlier divergence among the southern populations, which are currently separated by regions of low elevation and inhospitable desert, than among the more northern ones. Although the data are not conclusive, they are suggestive of northward dispersal from refugia south of the ice sheets since the last glaciation.

A new enzymatic variant of Leishmania (Leishmania) forattinii isolated from Proechimys iheringi (Rodentia, Echimydae) in Espírito Santo, Brazil.

Taxonomic studies of leishmanial isolates fromthe New World indicate tremendous diversitywithin this genus (E Cupolillo et al. 1994 Am JTrop Med Hyg 50: 296-311). A number of newLeishmania species from sylvan areas of theNeotropics have been described recently. Some ofthose parasites are associated with disease in hu-mans; others appear to be restricted to lower or-ders of mammals, such as rodents and edentates(G Grimaldi et al. 1989 Am J Trop Med Hyg 41:687-725). However, some of the latter parasitesmay yet be shown capable of causing human dis-ease, particularly in persons with altered cellularimmune responses.During a survey of potential reservoir hosts ofLeishmania done in the State of Espirito Santo, Bra-zil (1982-1985), three leishmanial parasites (des-ignated with stock codes MPRO/BR/82/RV203,MPRO/BR/82/RV228, and MPRO/BR/85/RV260)were isolated from skin lesion samples taken fromspiny-rats, Proechimys iheringi (Rodentia,Echimydae), captured in a secondary forest locatedin the municipality of Viana (A Falqueto et al. 1985Mem Inst Oswaldo Cruz 80: 497). In studies us-ing an indirect radioimmune binding assay (RIA)and a large panel of monoclonal antibodies (Mabs)derived for selected species of Leishmania , the iso-lates were characterized as members of the L.mexicana complex. These strains were differentfrom those species infective to humans, in that theyreacted with either the L.(L.) amazonensis or L.(L.) mexicana specific MAbs. Furthermore, thiswas also the case with cloned organisms from twostocks showing that they were not mixed popula-tions of Leishmania (G Grimaldi et al. 1987 Am JTrop Med Hyg 36: 270-287). This group of dis-tinct parasites presented, however, similarserodeme patterns in relation to the new recentlydescribed species of the Leishmania subgenus, L.(L.) forattinii (E Yoshida et al. 1993 Mem InstOswaldo Cruz 88: 397-406).The latter parasite was isolated from sylvaticreservoir hosts (Didelphis marsupialis aurita andP. iheringi denigratus ) captured on regions endemicfor American cutaneous leishmaniasis in Brazil,respectively in the municipalities of Conchas, SaoPaulo (E Yoshida et al. 1979 Rev Inst Med Trop SPaulo 21: 110-113) and Tres Bracos, Bahia (ABarreto et al. 1985 Rev Soc Bras Med Trop 18:243-246). The general morphology and the growthcharacteristics in vitro of the new species were simi-lar to those of other L. mexicana complex para-sites. Here we have further studied the Espirito Santoisolates using additional techniques for the char-acterization of Leishmania . The molecular proce-dures used for typing the strains (isoenzyme elec-trophoresis, blot enzyme binding assay usingMAbs, restriction endonuclease fragment patternsof k-DNA, and molecular karyotype analysis) havebeen described in detail in previous publications(Yoshida et al. 1979, 1983 loc. cit. , Cupolillo et al.1994 loc. cit., A Franco et al. 1997 Mem InstOswaldo Cruz 92: 63-68).Results of electrophoretic analysis were basedon the following enzymatic loci, namely: glucose-6-phosphate dehydrogenase (G6PDH), malate de-hydrogenase (MDH), isocitrate dehydrogenase(IDHNADP), 6-phosphogluconate dehydrogenase(6PGDH), nucleotidase (NH1 and NH2), glucosephosphate isomerase (GPI), phosphoglucomutase(PGM), proline dipeptidase (PEPD), leucine pep-tidase (PEP2), malic enzyme (ME), mannose phos-phate isomerase (MPI), and aconitate hydratase(ACON). The enzyme profiles of the selected iso-lated signature MPRO/BR/82/RV228, which rep-resents the poorly defined group of parasites from

Unique DNA binding mode of the N-terminal zinc finger of transcription factor Sp1.

Transcription factor Sp1 has three tandem repeats of a Cys2His2-type zinc finger motif and specifically binds to GC box DNA. Although the DNA binding mode of the three zinc fingers of Sp1 is predicted to be similar to that of Zif268, this model does not explain the DNA binding property of the N-terminal zinc finger (finger 1). To understand the DNA recognition mode of Sp1, we have performed detailed analyses for the contribution of finger 1 to the high-affinity binding to the GC box DNA and for the interaction mechanism between finger 1 and DNA. Results of electrophoretic analyses using finger-deleted mutants of Sp1 and GC box mutants in the finger-contacting subsite demonstrate that the contribution of finger 1 to the total DNA binding affinity is lower than that of the C-terminal finger 3 but is dispensable for the high-affinity binding. The DNA sequence selectivity of finger 1 at the 3'-portion of the GC box is lower than that of fingers 2 and 3 at the 5'-portion. Alanine scanning mutagenesis in the alpha-helix of finger 1 reveals that Lys-1 immediately preceding the helix is important for the recognition of the two guanine bases, but other putative key amino acids do not affect the DNA binding. These results demonstrate that (1) the contribution of finger 1 to the DNA binding affinity and the sequence selectivity of Sp1 is smaller than that of fingers 2 and 3 and (2) the interaction mechanism between finger 1 and DNA is different from the Zif268 model. DNA interaction of Sp1 finger 1 has also been discussed in connection with that of TFIIIA or WT1.

Isolation and characterization of phytokinin-binding proteins from the cotton plant

A highly purified site of a cytokinin-binding protein with a KD value for3H-BAP of 4.3 nM has been isolated from cotton-plant seedlings by the methods of biospecific and hydrophobic chromatography. According to the results of electrophoretic analysis under denaturing conditions its molecular mass is 43 kDa.

Isozyme Polymorphisms in Boll Weevils1 and Thurberia Weevils from Arizona2

Results of electrophoretic analyses for isozyme gene frequency differences showed high heterozygosity and high levels of polymorphism at isozyme loci in samples from three populations of insects as follows: (1) thurberia weevils, Anthonomus grandis thurberiae Pierce, from bolls of a wild cotton, Gossypium thurberi Todaro, collected in the Santa Rita Mountains of Arizona; (2) boll weevils, A. grandis grandis Boheman, from bolls of Pima cotton, G. barbadense L. collected at Three Points, Ariz.; and (3) boll weevils from pheromone traps positioned around a field of short staple stub cotton, G. hirsutum L., at Theba, Ariz. The three collection points were separated by distances of 50 to 210 km. Of the loci, 60% were significantly different in genotypic distributions in the three collections. Analysis of allelic frequency differences showed that these populations are not from the same random mating population. Calculated genetic distances between the three collections ranged from a value of 0.068 between the thurberia weevils and boll weevils from short-staple stub cotton up to 0.206 between thurberia weevils and the boll weevils from Pima cotton. These genetic distance values are higher than similar values found between pheromone races of insects but lower than genetic distances measured between sibling species of insects and may indicate that host races of boll weevils exist in Arizona.

Tyrosinase maturation and pigment expression in B16 melanoma: Relation to theophylline treatment and intracellular cyclic AMP

We have studied the effects of theophylline treatment on pigmentation characteristics and growth of two B16 melanoma cell lines, HFH-18 and P/140. Cell counts of control and theophylline-treated cultures confirmed that the drug inhibits cell growth. Light and electron microscope cytochemistry with the L-dopa reaction indicated that the two cell lines differ in their ability to transfer Golgi-associated tyrosinase to developing premelanosomes. The results of these experiments, considered with results of electrophoretic analyses and activity measurements by the Pomerantz method, also provide evidence that increased tyrosinase synthesis occurs in response to theophylline treatment. In addition, results indicate that theophylline induces changes in the rate of synthetic or degradative posttranslational modification of tyrosinase. Measurements of intracellular cyclic AMP levels by radioimmunoassay in control cultures and in theophylline- and alpha-MSH-treated cultures were made. Although the hormone induced spectacular increases in cyclic AMP levels, theophylline produced no detectable change. These results indicate that theophylline differs from alpha-MSH because theophylline-induced changes in pigmentation may not require the participation of intracellular cyclic AMP.

Types and molecular weights of subunits of arachin-P6.

Abstract Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that arachin-P6 contained nine types of subunits. The subunits were designated K15 (15, 069), K17 (17, 334), K20 (20, 000), K22 (21, 750), K24 (23, 557), K29 (29, 019), K37 (37, 161), K41 (41, 326) and K43 (43, 104) and molecular weight of each is given in parenthesis following its name. This observation was qualitatively comparable to the results of electrophoretic analysis of arachin-P6 conducted in the presence of urea.

Thyroxine-Protein Interactions: V. ISOLATION AND CHARACTERIZATION OF A THYROXINE-BINDING GLOBULIN FROM HUMAN PLASMA

A thyroxine-binding globulin has been isolated in highly purified form from human plasma. The purification procedure consisted of the following steps: (a) fractionation of plasma with ammonium sulfate, (b) zone electrophoresis on an LKB Porath cellulose column, (c) re-electrophoresis on the Porath column, (d) chromatography on DEAE-cellulose, (e) gel filtration on Sephadex G-150 or G-200, and (f) preparative electrophoresis in polyacrylamide gel. The isolated thyroxine-binding protein was homogeneous when analyzed by electrophoresis in starch gel, immunoelectrophoresis, and disc electrophoresis. The protein sedimented as a single component in the ultracentrifuge when analyzed by the methods of sedimentation equilibrium and sedimentation velocity. The weight average molecular weight as determined by sedimentation equilibrium is 58,000; the sedimentation constant, s20, w0, is 3.92 × 10−13 sec. The thyroxine-binding globulin is a glycoprotein containing 6.70% neutral hexose, 2.38% sialic acid, 3.31% glucosamine, and 0.70% galactosamine. Based on the results of electrophoretic analysis on paper, the final product of thyroxinebinding globulin exhibited a high affinity for thyroxine, with a binding constant of the order of 109M−1. However, the maximal binding capacity of the protein for thyroxine appears to be reduced to about 25% of the expected value if binding is assumed to take place at a single strong binding site on the protein. The isolated thyroxine-binding protein was indistinguishable from endogenous thyroxine-binding α-globulin of serum when analyzed by electrophoresis in starch gel, in polyacrylamide gel, and on paper.

Electrophoretic Studies on Swine Sera Hyperimmunized with Hog Cholera Virus (HCV)

The electrophoretic analysis was applied to sera of 15 normal swine, those of 9 swine hyperimmunized with hog cholera virus (HCV), and those of various stages of the immunization of four swine hyperimmunized with HCV.Methods of electrophoretic analysisThe electrophoretic analysis was carried out with the “HT-B Hitachi Tiselius” apparatus. Electrophoretic conditions were; 9-11MA, 100-110V., 40-50 minutes, and 15-22°C. The phosphate buffer solution was used. Total serum protein was determined with the “Hitachi Hand Protein Refractometer”. Protein fraction was measured with a ascending pattern by the weight method.Experimental results1) The results of electrophoretic analysis on sera of 15 swine which were considered to be in normal conditions are shown in Table 1; total protein 7.1g/dl, albumin (alb) 46.2%, α-globulin (glob) 19.7%, β-glob 15.7%, γ-glob 18.4%, and A/G 0.877.2) The results of electrophoretic analysis on antisera of 9 swine hyperimmunized with HCV are shown in Table 2; total protein 7.6g/dl, alb 40.8%, α-glob 19.4%, β-glob 14.7%, γ-glob 25.1%, and A/G 0.701.3) Mathews et al. have reported the increase of γ-glob of sera of swine hyperimmunized with HCV. This paper shows that as they reported, the average percent of γ-glob of hyperimmune sera was observed to be higher than that of normal sera, and that the increase of γ-glob was not always observed in all hyperimmune sera (see Table 2).4) The results of electrophoretic analysis on sera of various stages of the immunization of 4 swine hyperimmunized with HCV are shown in Table 3. No remarkable electrophoretic changes of the sera were observed at any stages of the immunization of the cases concerned, and the antiserum which showed a remarkable increase of γ-glob was not observed in any cases.5) The relationship of viral antibody to the protein fraction of swine sera hyperimmunized with HCV is of considerable importance. This paper reports that the relationship was investigated electrophoretically and immunologically.The serum protein fractions were isolated from hyperimmune sera by means of the starch-electrophoresis and the neutralizing antibody titer of each fraction (alb, α-, β-, γ-glob) was detected by the tissue culture method, and the protective antibody was proved by injection of each fraction into swine.From the above experimental results, it has been found that the neutralizing antibody is contained in β-glob and γ-glob fraction of the hyperimmune sera, and that the protective antibody consists mainly in γ-glob fraction of the hyperimmune sera.

Studies on Milk Powders. IV. The Foam and Sediment Fractions of Reconstituted Whole Milk

Summary Whole milk powder was reconstituted and the resultant whole milk was passed through a 200-mesh sieve. This treatment removed virtually all the foam or scum from the surface of the milk. The milk was then centrifuged. The insoluble material (foam fraction) which remained on the sieve, and the insoluble material (sediment fraction) which was recovered by centrifugation, were washed with water and freeze-dried. The dried foam fraction contained about 50% more fat than did the sediment fraction. Melting point and iodine value determinations on the ether-extractable portions of the foam and sediment indicated that the fat of the sediment contained a higher proportion of unsaturated fatty acids than did the fat of the foam. Results of electrophoretic analyses of the ether-insoluble portions of the foam and sediment indicated that the major protein constituent of the sediment was beta-lactoglobulin, and that of the foam was casein.

Paper electrophoresis of the proteins in the cystic fluid contained in central nervous system tumors

Estudo do perfil eletroforético das frações protêicas de líquidos de cistos de tumores do sistema nervoso central (6 astrocitomas, um ependimoma, um meningeoma e dois craniofaringeomas). Os achados foram homogêneos para o grupo de astrocitomas, caracterizando-se por elevado teor albumínimo e pequeno teor de α-globulinas. Em relação às proteínas destes líquidos císticos, que constituem o maior grupo estudado, foi encontrado comportamento variável para os líquidos dos outros tipos de tumor: o perfil eletroforético apresentava caracteres semelhantes no caso de ependimoma e diversos nos demais. Estas diferenças se caracterizam especialmente por um teor albumínico menor, achado mais evidente nos casos de craniofaringeoma. Os achados comprovam o valor do estudo eletroforético das proteínas dos líquidos de cistos de tumores do S.N.C, e apontam a possibilidade de serem obtidos dados para compreender a patogenia dessas coleções líqüidas e para auxiliar no diagnóstico da espécie do tumor.

唾液腺ホルモンの研究 (第15報)

Equine parotic gland is fairly different from bovine parotic gland, both in outward appearance and in properties. The pH 5.4-precipitate obtained from equine gland is larger in yield than that from bovine gland, but its effect is far weaker. The 12% (NH4)2SO4-fraction, obtained by the purification of the equine pH 5.4-precipitate by fractional precipitation with ammonium sulfate, could not be induced to crystallize, remaining granular. The effect of this fraction is much weaker than the 12.1% (NH4)2SO4-fraction from the bovine gland, and was found to be still rather impure from the results of electrophoretic analysis. The mobility of the active fraction obtained from the equine parotic gland is much larger than that of the active fraction from the bovine gland. The presence of a substance in the equine parotic gland which lowers the calcium level of rabbit serum is similar to the case in bovine gland, but its properties other than such physiological action, are slightly different from those of the bovine gland.