Prostatic acid phosphomonoesterase of the Japanese monkey (Macaca f. fuscata)

Possible immunological differences between monkey and human prostate gland proteins and also between seminal vesicle proteins of these species were investigated by agarose gel electrophoresis, agarose gel immunoelectrophoresis and by the agar gel immunodiffusion method, using anti-sera against human plasma, human seminal plasma and human prostatic acid phosphomonoesterase (PMEase). At the same time, the electrophoretic mobility of these prostatic acid PMEases was compared by means of starch gel electrophoresis.Each of these two tissues, monkey and human, was found to contain antigenic proteins with immunological identity. Though antigenic similarity of monkey and human prostatic acid PMEase was demonstrated by immunological methods, a clear difference was observed in the electrophoretic mobility of these enzymes when examined by starch gel electrophoresis.     

Water buffalo (Bubalus bubalis) hemoglobins: an electrophoretic and chromatographic study

1. Hemoglobins from three phenotypes of Italian water buffalo (Bubalus bubalis), named AA, AB and BB, were selected by starch gel electrophoresis at alkaline pH and analyzed using polyacrylamide gel isoelectric focusing and subsequent analysis of titration curves to reveal differences between two types of hemoglobin identified as Hb fast and Hb slow. 2. Globins from Hb fast and Hb slow were purified by fast protein liquid chromatography (FPLC). Electrophoretic differences were found in the respective alpha-chains using polyacrylamide gel disc-electrophoresis at acid pH, polyacrylamide gel isoelectric focusing and by subsequently analyzing titration curves. 3. The results suggest that the alpha chains of Hb fast and Hb slow, called I alpha and II alpha, respectively, differ in at least two aminoacid residues. Subsequently, these amino acids were identified as lysine and cysteine.     

Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata)

The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit.     

Genetic polymorphism of horse serum protein 3 (SP3)

Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of horse serum samples, followed by general protein staining, revealed genetic polymorphism of an unidentified protein tentatively designated serum protein 3 (SP3). The SP3 fractions appeared distinctly when a 14% concentration of acrylamide was used in the separation gels. The 2-D mobilities of SP3 fractions were quite similar to that of albumin. Family data were consistent with the hypothesis that the observed SP3 phenotypes were controlled by four co-dominant, autosomal alleles (D, F, I, S). Evidence was provided that the F allele can be further divided into two alleles (F1 and F2); the mobilities of F1 and F2 variants were very similar. Each of the SP3 alleles gave rise to one fraction and each of the heterozygous types showed two fractions. More than 600 horses representing five different breeds (Swedish Trotter, North-Swedish Trotter, Thoroughbred, Arab and Polish Tarpan) were typed for SP3, and allele frequency estimates were calculated. SP3 was highly polymorphic in all breeds studied.     

Modification of the isoinhibitors of human serum alpha 1-proteinase inhibitor (alpha 1-antitrypsin) by pancreatic proteases

Due to the action of a serum protease, the two most cathodal isoinhibitors of the alpha 1-proteinase inhibitor (alpha 1-PI) are cleaved at the Gly5-Asp6 bond and lack two negative charges. In spite of this, these can bind trypsin and chymotrypsin, showing that the N-terminal pentapeptide is not indispensable for inhibition function. Pancreatic proteases also cleave a bond near the N-terminus in alpha 1-PI, resulting in a loss of two negative charges and a corresponding cathodal shift in the electrofocusing behavior of the isoinhibitors. Trypsin cleaves isoinhibitors near the N-terminus at a large inhibitor excess and unless an additional cleavage takes place, at least two of the new isoinhibitors remain active. An additional cleavage(s), most likely at a distance of 30-40 residues from the C-terminus results in a corresponding decrease of the molecular mass and a loss of inhibition function. Although the C-terminal cleavage peptide does separate from the protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it remains associated with it under conditions of polyacrylamide gel isoelectric focusing. Chymotrypsin also cleaved alpha 1-PI near the N-terminus but this could be observed only at protease excess and the modified isoinhibitors did not form complexes with chymotrypsin. The molecular polymorphism of alpha 1-PI is partly explained by the absence of the N-terminal pentapeptide from some of the isoinhibitors.     

The isoinhibitors of chymotrypsin/elastase from Ascaris lumbricoides: isolation by affinity chromatography and association with the enzymes

Five isoinhibitors, proteins that inactivate chymotrypsin and elastase, were isolated from aqueous extracts of the intestinal parasite Ascaris lumbricoides var. suum by affinity chromatography. They were named in the order that they eluted from a CM-Sephadex C-25 column at pH 8.6 using a salt gradient. Isoinhibitor 1, first reported in this paper, is anionic on polyacrylamide gel electrophoresis at pH 9.3. The other four isoinhibitors are cationic on electrophoresis at pH 9.3, separable from each other, and identical with those reported previously [R.J. Peanasky and G. M. Abu-Erreish (1971) in Proceedings International Research Conference on Proteinase Inhibitors (Fritz, H., and Tschesche, H., eds.), pp. 281-293, de Gruyter, New York]. Amino acid compositions show differences between the isoinhibitors. Antibody to isoinhibitor 1 reacts with its self-antigen only. Antibody to isoinhibitor 5 reacts with isoinhibitors 2-5 but not with isoinhibitor 1. Association equilibrium constants show that each of the isoinhibitors interacts most avidly with alpha-chymotrypsin. For isoinhibitor 1, the K alpha for alpha-chymotrypsin was 2.6 X 10(11) M-1, for porcine elastase I 1.6 X 10(10) M-1, and for Subtilisin Carlsberg 3.3 X 10(7) M-1. For isoinhibitors 2-5, the K alpha ranges were 7.1 X 10(10) to 1.3 X 10(11) M-1 for alpha-chymotrypsin, 1.0 X 10(9) to 5.6 X 10(9) M-1 for porcine elastase I, and 6.0 X 10(8) to 1.3 X 10(9) M-1 for subtilisin Carlsberg. Because of the strong affinity of these inhibitors for alpha-chymotrypsin and elastase, two proteins in the normal environment of the nematode, the name isoinhibitors of chymotrypsin/elastase is suggested for these proteins.     

An electrophoretic characterization of serum proteins of the collared peccary (Tayassu tajacu)

Serum proteins of the collared peccary (Tayassu tajacu) were analyzed by agarose gel electrophoresis for wild adult males and females, nursing young, and reproductively-active females in captivity. Electrophoretic profiles of the adult peccary showed at least six distinct protein bands corresponding to the fractions: albumin, alpha-1, alpha-2, beta-1, beta-2, and gamma globulin. Globulin fractions of the peccary had different mobilities from the domestic swine. The only sexual dimorphisms were associated with the beta globulin:albumin ratio and the albumin:globulin ratio. Ingestion of colostrum in 1-day-old neonates was marked by a very large increase in gamma globulins. The only significant difference between pregnant and lactating females was in the alpha globulin:beta globulin ratio. Lactating females had higher concentrations of alpha-2 globulin than non-pregnant females.     

对5个S180克隆细胞株RAPD特征的研究

目的运用随机扩增多态性DNA(RAPD)技术建立S180的5个克隆细胞株的特异性图谱。方法运用23条引物对S180-S2D9、S180-S2D7、S180-S1F11、S180-S1H10、S180-S1B115个克隆细胞株的基因组DNA进行RAPD-PCR扩增,以琼脂糖电泳观察结果。结果筛选出3条在各克隆株扩增产物间有差异的引物。结论5个S180克隆细胞株的RAPD特征有明显区别。     

Genetic polymorphism of horse serum protein 3 (SP3)

Summary. Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of horse serum samples, followed by general protein staining, revealed genetic polymorphism of an unidentified protein tentatively designated serum protein 3 (SP3). The SP3 fractions appeared distinctly when a 14% concentration of acrylamide was used in the separation gels. The 2-D mobilities of SP3 fractions were quite similar to that of albumin. Family data were consistent with the hypothesis that the observed SP3 phenotypes were controlled by four co-dominant, autosomal alleles ( D,F,I,S ). Evidence was provided that the F allele can be further divided into two alleles ( F ₁ and F ₂); the mobilities of F₁ and F₂ variants were very similar. Each of the SP3 alleles gave rise to one fraction and each of the heterozygous types showed two fractions. More than 600 horses representing five different breeds (Swedish Trotter, North-Swedish Trotter, Thoroughbred, Arab and Polish Tarpan) were typed for SP3, and allele frequency estimates were calculated. SP3 was highly polymorphic in all breeds studied.     

Isolation and partial characterization of a alpha 2-beta 1-glycoprotein from horse plasma

A alpha 2-beta 1-glycoprotein was isolated from horse plasma by classical methods. The final product appeared homogeneous by agarose gel and pore limit SDS polyacrylamide gel electrophoresis, immunoelectrophoresis and crossed immunoelectrophoresis. The protein moved in agarose gel electrophoresis just above the beta 1 region and seemed composed of a single polypeptide chain. A highly heterogenic banding pattern, focused between pH 5.1 and 6.5 was revealed by isoelectric focusing. The molecular weights determined by gel filtration on Sephadex G100 and by a pore limit polyacrylamide gel electrophoresis in presence of SDS were 65,000 and 82,300 dalton, respectively. No serological relation was found between the horse alpha 2-beta 1-glycoprotein and human and bovine plasma proteins.     

Detection of base mutations in genomic DNA using denaturing gradient gel electrophoresis (DGGE) followed by transfer and hybridization with gene-specific probes

It has been shown that minor differences, such as single-base-pair substitutions between otherwise identical DNA fragments can result in altered melting behavior detectable by denaturing gradient gel electrophoresis (DGGE). Sequence variations in only a small DNA region within one locus can be detected using the previously described procedures. We have developed a method for the efficient Southern transfer of genomic DNA fragments from the denaturing gradient gels in order to be able to analyze larger regions in several loci for variation. The gels were made using polyacrylamide containing 2% low-geling-temperature agarose (LGT). The polyacrylamide gel (PAG) was crosslinked with a reversible crosslinker, and after electrophoresis the crosslinks were cleaved, the structure of the gel being maintained by the agarose. After this treatment of the denaturing gels, more than 90% of the DNA fragments could be transferred to nylon membranes by alkaline transfer, while electroblotting transferred only 10% of the DNA. Hybridization with gene-specific probes was then performed. We have used this technique to identify an RFLP in the COL1A2 gene in a human genomic DNA sample. The transfer technique described should make the use of DGGE more widely applicable since the genomic DNA fragments separated on one gel can be screened with several different probes, both cDNA and genomic probes.     

RAPD及SRAP两种分子标记技术对苦瓜杂交种纯度的鉴定分析

以F1代苦瓜杂交种如玉11号及其亲本为材料,利用RAPD及SRAP两种分子标记技术对这3种苦瓜基因组DNA进行比较分析,以获得该杂交种及其亲本（或母本）差异目的基因片段。经过多次对该3种苦瓜叶片DNA提取,PCR扩增及其PCR产物的琼脂糖凝胶电泳分析,在供试的46个RAPD引物及121对SRAP引物中,筛选出1个RAPD引物及1对SRAP引物能区分该苦瓜杂交种及其母本种子,通过进一步验证分析,证明该两种分子标记的特异引物可作为如玉11号苦瓜杂交种子的纯度鉴定之用。     

Distribution of AMY2 polymorphism in S. Tomé and Príncipe (West Africa)

The genetic polymorphism of AMY2 was studied in the population of S. Tomé and Príncipe (West Africa) using agarose gel electrophoresis. AMY2 frequencies are reported for the first time in a subSaharian population. The gene frequencies found were: AMY2*1=0.948, AMY2*3=0.052 (N=173).     

Human plasma dopamine beta-hydroxylase. Purification and properties

Dopamine beta-hydroxylase was isolated from normal human plasma. The major form of the active enxyme in plasma was purified to apparent homogeneity and is a 300,000-dalton tetramer containing 4 atoms of tightly bound copper. About 20% of the enzyme activity in plasma was isolated as a dimeric form of this enzyme. Sodium dodecyl sulfate gel electrophoresis of the purified form gave a polypeptide subunit molecular weight of 72,000 and disulfide-linked dimers of this component were observed. Both forms of the enzyme are apparently glycoproteins and interact with immobilized concanavalin A. Furthermore, the enzyme is capable of binding to alkyl-substituted agarose by hydrophobic interaction. Advantage was taken of these properties to purify the enzyme. Both purified tetramer and partially purified dimer were further characterized by kinetic analysis and the Stokes radii and S20,W of these species were compared. Rabbit antiserum to the purified tetramer revealed no immunochemical differences between the two enzyme forms by using a method of immunotitration.     

Investigation of the heterogeneity of rhesus monkey alpha 1-antitrypsin

Rhesus monkey alpha 1-antitrypsin (n = 144) was examined for heterogeneity by acid starch gel electrophoresis, isoelectric focusing in agarose and agarose gel electrophoresis. In contrast to other studies, no heterogeneity of Rhesus monkey alpha 1-antitrypsin could be documented using specific antisera. Rhesus monkey alpha 1-antitrypsin contained a reactive thiol. The pIs of the major isoforms of Rhesus monkey alpha 1-antitrypsin were 4.63, 4.69, 4.84 and 4.86 at 4 degrees C. No deficiency state of Rhesus monkey alpha 1-antitrypsin was detected. The six protease inhibitors in Rhesus monkey sera cross-reacted with antisera to the six human protease inhibitors.     

Isolation of acidic acrosin isoinhibitors (BUSI I A, BUSI IB1 and BUSI I B2) from bull seminal plasma.

Three natural proteinase isoinhibitors with low isoelectric points BUSI I A (pI = 3.9), BUSI I B1 (pI = 3.4 and BUSI I B2 (pI = 3.7) were isolated from bull seminal plasma by gel filtration on Sephadex G-50 and ion exchange chromatography on DEAE-Sephadex and SE-Sephadex. Isoinhibitors Bl and B2 have identical amino acid composition. Isoinhibitor A contains six amino acid residues less than isoinhibitors B1 and B2. Since sugars have been detected in the isoinhibitors, heterogeneity may also be due to the sugar component. The isoinhibitors show the same inhibitory properties; all of them inhibit acrosin, trypsin and chymotrypsin. Glandular kallikrein is also inhibited, but to a very low extent only. The molecular weight (Mr approximately 8 900) was determined by gel filtration.     

Genotypic analysis of families with lactate dehydrogenase A(M) deficiency by selective DNA amplification

Summary Genomic DNA prepared from LDH-A-deficient whole blood was amplified by the polymerase chain reaction technique using two primers specific for the active human LDH-A gene. The amplified fragment was examined by direct agarose gel electrophoresis, and a deletion of 20 base pairs (bp) in exon 6 of the LDH-A gene was found. The results permitted a clear distinction between the homozygous mutant, the heterozygous mutant, and wild-type genotypes. Moreover, HinfI digestion and direct sequencing of the amplified product confirmed the results from direct agarose gel electrophoresis. Four families, including 18 individuals, were shown to contain the same mutation, that is a 20-bp deletion in exon 6. All genotypes were consistent with their biochemical phenotypes as evaluated by the ratio of LDH-B to LDH-A subunits in erythrocytes. Thus, all four known affected families in Japan have been shown to carry the same mutant gene, which may have been derived from a single mutational event.     

Comparison of methods for total community DNA extraction and purification from compost

The differences on DNA yield and purity of three different DNA extraction protocols were compared with regard to the use for PCR and other molecular analyses. Total DNA was extracted from compost by the three protocols, and then was purified by spin-bind cartridges after being precipitated by PEG8000. The detection performed on a nucleic acid and protein analyzer showed that all three methods produced high DNA yields. The agarose gel electrophoresis showed that the fragments of crude and purified DNA had a length of about 23 kb. A eubacterial 16S rRNA gene-targeted primer pair was used for PCR amplification, and full length 16S rDNAs were amplified from all the purified DNA samples. After being digested by restriction endonucleases, the restriction map of amplified rDNA showed identical genetic diversity. The products of PCR using primer pair GC341F and 907R were also used for denaturing gradient gel electrophoresis analysis. The results indicated that high-quality DNA was extracted from compost by the three protocols, and each of the protocols is adapted to extract microbial genome DNA from compost expediently and cheaply.     

Identification of Saint Louis encephalitis virus mRNA.

Saint Louis encephalitis (SLE) virus-specific RNA was recovered from infected HeLa cells by sodium dodecyl sulfate (SDS)-phenol-chloroform extraction, and the molecular species were resolved by SDS-sucrose gradient centrifugation and agarose gel electrophoresis. Sucrose gradient centrifugation revealed the presence of a 45S species, minor 20 to 30S heterogeneous species, and an 8 to 10 S RNA species in the cytoplasmic extract. Analysis of the same samples by electrophoresis on agarose gels, under both nondenaturing and denaturing conditions, revealed only two virus-specific RNA molecules, the 45S genome-sized RNA and an 8 to 10S species. Varying the gel concentration to facilitate analysis of nucleic acids with molecular weights ranging from 25,000 to 25 X 10(6) failed to reveal additional RNA species, although low levels of a putative double-stranded replicative form could conceivably have escaped detection. From our observations it appears that the heterogeneous RNA species and presumably the 20S RNase-resistant species reported in other investigations of flavivirus RNA are degradation products or conformers of the 45S molecule. Polysomes from SLE virus-infected cells were prepared and separated from contaminating nucleocapsid by centrifugation on discontinuous sucrose gradients. RNA extracted from these polysome preparations was analyzed by sucrose gradient centrifugation and agarose gel electrophoresis. The 45S SLE virus genome-size molecule was found to be the only RNA species associated with the polysomes. This molecule was sensitive to RNase digestion and was released from polysomes by EDTA and puromycin treatment. These findings provide direct evidence that the 45 S SLE virus RNA serves as the messenger during virus replication, in contrast to the 26S RNA species which functions as the predominant messenger during alphavirus replication.     

S1核酸酶作用与微环DNA分子的克隆策略

米曲霉来源的S1 核酸酶具有降解单链DNA或RNA的作用。在适当的条件下 ,该酶能将不同的环形DNA分子从超螺旋转变成开环和线形结构 ,对质粒pUC19的实验证明 ,S1 核酸酶的这种转变作用与加入的酶量呈正相关。在 2 5 μL总反应体积中 ,按 10 0ngDNA加入 5u至 17u的S1 核酸酶 ,能获得较高比例的线形DNA。由于微环DNA分子太小 ,单酶切位点的出现率较低 ,很难用常规方式进行克隆 ,以S1 核酸酶进行线形化是微环DNA克隆的途径。pC3是已知最小的真核生物线粒体DNA类质粒 (5 37bp) ,经S1 核酸酶线形化后 ,成功地克隆到pMD18 T载体上。