Immunological properties of the β-subunit of human chorionic gonadotropin

The β-subunit of human chorionic gonadotropin (hCG-β) has been modified to a varying degree by the cleavage of the disulfide bonds by reduction with dithioerythritol followed by S-carboxamidomethylation. The resulting derivatives of hCG-β show a preferential loss in their immunological cross-reactivity with human luteinizing hormone (hLH). The immunological specificity of the partially reduced and S-alkylated derivatives can be further enhanced by conjugation with tetanous toxoid using glutaraldehyde. Neither conjugation of hCG-β with the toxoid nor its treatment with anti-hLH immunoadsorbent affects its cross-reactivity with hLH.     

The basis of differential staining of ascospores and vegetative cells of yeasts

Summary An investigation of the staining reactions of ascospores from 10 species of yeasts with 19 different dyes, showed that differentiation of spores from vegetative cells by staining depends on their relative permeability properties. Three possible staining mechanisms are discussed and examined for consistency with the data. It was also discovered that ascospores of Hansenula saturnus, which are not stained by the malachite green method, could be differentiated very satisfactorily from the vegetative cells by staining with hot rose bengal, followed by counter-staining with cold victoria blue B.     

Correlation of egg counts of Clonorchis sinensis by three methods of fecal examination

The Kato-Katz (KK) method is a well-known method of fecal examination for helminthiases. Its diagnostic sensitivity was found very high for clonorchiasis. The present study evaluated the correlation of Clonorchis sinensis egg counts by the KK method with those by direct smear and formalin-ether (FE) technique. The egg counts obtained by the KK method (Y) were correlated with the counts by direct smear (X) with the equation of Y = 659.4 + 0.266X (r2 = 0.738), but not with those by the FE method. The present study demonstrated that the KK method and direct smear were useful for both qualitative and quantitative diagnosis of clonorchiasis, especially in the field.     

Acceleration of cleavage of the carbon-cobalt bond of sterically hindered alkylcobalamins by binding to apoprotein of diol dehydrase

Cleavage of the C-Co bond of sterically hindered alkylcobalamins bearing neither an adenine moiety nor functional groups, such as isobutylcobalamin, neopentylcobalamin, and cyclohexylcobalamin, was markedly accelerated by their interaction with apoprotein of diol dehydrase, although these cobalamins do not function as coenzyme. Acceleration of the conversion of alkylcobalamins to enzyme-bound hydroxocobalamin was stoichiometric and obeyed first-order reaction kinetics. These results, together with strong competitive inhibition by these alkylcobalamins with respect to adenosylcobalamin, indicate that acceleration of the C-Co bond cleavage by the apoenzyme is due to labilization of their C-Co bond by binding to the active site of the enzyme. This labilization is considered to be caused by a steric distortion of the corrin ring which is induced by specific tight interaction of the cobalamin moiety with apoprotein. The importance of such a labilizing effect for activation of the C-Co bond of adenosylcobalamin in enzymatic reactions is discussed.     

Synthesis of tetracontapeptide--a hypothetical evolutionary precursor of calcium-binding proteins. II. Condensation of fragments

A tetracontapeptide was obtained by condensation of synthetic fragments (see the preceding paper) with the use of pentafluorophenyl esters as well as with carbodiimide and hydroxybenzotriazole. Racemization during fragment condensation was 1-2 per cent. Deblocking of the protected tetracontapeptide was carried out by treatment with trifluoroacetic acid and hydrogen fluoride with thioanisole. The obtained peptide was purified by gel-chromatography and HPLC.     

Oriented reconstitution of red cell membrane proteins and assessment of their transmembrane disposition by immunoquenching of fluorescence

The two major membrane glycoproteins of human red cells, glycophorin and band 3, the anion exchange protein, were isolated from cells exofacially labeled with fluorescein and reconstituted into vesicles with defined transmembrane disposition. Uniform orientation of polypeptides was accomplished by two procedures: Vesicles with single protein units were obtained by a one-step dilution of a protein/detergent suspension with a vast excess of phospholipid. Vesicles with uniform orientation of protein were selected by affinity chromatography on derivatized Sepharoses (organomercurial, wheat germ agglutinin, aminoethyl or diethylaminoethyl). Vesicles with multiple protein units with uniform orientation were generated by vectorial immobilization of detergent solubilized proteins on the above affinity matrices and in situ formation of proteoliposomes by detergent substitution for phospholipid. The proteoliposomes were released from the column by addition of excess free ligand. The orientation of band 3 and glycophorin in the reconstituted vesicles was first assessed by immunofluorescence quenching, using anti-fluorescein antibodies, to quantitatively quench fluorescein residues exposed on the outer surface of vesicles. Further assessment was achieved by chromatographing the vesicles through various affinity and ionic matrices. Vesicle populations of higher than 90% homogeneity in protein orientation (right-side-out or inside-out) were obtained with both procedures. The above methods provide a convenient experimental tool for the oriented reconstitution of proteins and the evaluation of their transmembrane disposition.     

Facile synthesis of arabinomannose penta- and decasaccharide fragments of the lipoarabinomannan of the equine pathogen, Rhodococcus equi

Pentasaccharide repeating unit 20 of the lipoarabinomannan from the equine pathogen, Rhodococcus equi, and its dimer 31, were synthesized. The pentasaccharide was obtained by assembling a benzoylated 2,6-branched mannosyl trisaccharide acceptor 13 with a free hydroxyl group at C-2' of the mannose residue attached to the core mannose residue by (1 --> 6)-linkage, followed by coupling with 2,3,5-tri-O-benzoyl-alpha-D-arabinofuranosyl-(1 --> 2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (18), and by deacylation. Meanwhile, the decamer 31 was obtained by firstly preparing a benzoylated mannose (1 --> 6)-linked tetrasaccharide backbone 26 with 2-, 2"-O-ClAc, and 2'-, 2'-O-Ac groups, respectively, then by dechloroacetylation and subsequent condensation with perbenzoylated trichloroacetimidate, and then by deacetylation and subsequent coupling with 18, and finally, by deacylation.     

Synthesis of a fluorescent analogue of phosphatidylcholine

A fluorescent analogue of phosphatidylcholine was synthesized by acylation of 1-oleoyl-sn-glycero-3-phosphocholine with 6-N-(tert-butyloxycarbonyl)aminocaproic acid anhydride employing the catalyst 4-pyrrolidinopyridine. Removal of the protective group by treatment with HCl in chloroform was followed by subsequent reaction with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) to form the fluorescent analogue of phosphatidylcholine, 1-oleoyl-2-(NBD)aminocaproyl-sn-glycero-3-phosphocholine, in good yield and with high isomeric purity.     

Aggregation of fragmented chromatin associated with the synthesis of products of its treatment with nuclease

Isolated cell nuclei were incubated with nucleases followed by extraction of chromatin with a low salt buffer. With an increase of nuclear chromatin degradation with DNAse I or micrococcal nuclease, solubilization of deoxyribonucleoprotein (DNP) by a low salt buffer increases, reaching a maximum upon hydrolysis with 2-4% nuclear DNA and then decreases appreciably after extensive treatment with nucleases. Soluble fragmented chromatin aggregates in the course of treatment with DNAase. I. Addition to gel chromatin preparations of exogenous products of nuclease treatment of isolated nuclei leads to its aggregation. Pretreatment of nuclear chromatin with RNAase prevents solubilization of DNP by low ionic strength solutions. Some experimental data obtained with the use of severe nuclease treatment are discussed; for a correct interpretation of these data the aggregation of fragmented chromatin by products of its nuclease degradation should be taken into consideration.     

Flow cytometric analysis of oxidative burst of phagocytes with small amount of peripheral blood

We have developed a simple method for assessing the oxidative metabolic burst of peripheral blood leukocytes with a minute amount of whole peripheral blood by flow cytometry according to the method of Bass et al. with some modification. By this method, we can measure the H2O2 production by both granulocytes and monocytes in the same blood sample. The oxidative product formation by peripheral blood neutrophils can be monitored sequentially in the same mouse infected with E. coli. The mice infected intravenously with 0.1 LD50 of the bacteria showed increased basal activities from an early stage of infection; those infected intraperitoneally with the same dose of the bacteria showed a delayed enhancement. In case of infection with 0.01 LD50, the enhanced basal activities lasted for only a short period of time. The H2O2 production was correlated well with the clearance of the infected bacteria. These results demonstrated that the oxidative-product formation by peripheral blood neutrophils is affected by both the route and the dose of infection.     

Specific antigen of granular cells of the human endometrium

Rabbits were immunized by homogenates of endometrium obtained from women during 10-12 weeks of gestation. A specific antiserum was obtained by absorption of the crude antiserum by blood cells and plasma proteins of men with different kinds of ABO and Rh antigens, till disappearance of positive reaction with men's serum protein in the Ouchterlony test. Such an adsorbed specific antiserum continued to react with the sera of pregnant women. Two antigens, numbers 1 and 2, respectively, were determined by the Ouchterlony test. Another group of rabbits was immunized by antigens detected in the precipitation test. A monospecific antidecidual antiserum (ADS 1092) was obtained against number 2 antigens. This antiserum revealed only one antigen in sera of women with gestation and did not react with sera of non-pregnant women. In the slides of endometrium of pregnant women of 10-12 weeks of gestation ADS 1092 had a strong positive reactive with the cytoplasm of one type of endometrium cells. The immunomorphological analysis by the non-direct Coons test and the PAP-test permits to identify cells with the positive reaction as granular cells. It is concluded that the granular cells may be a source of one of the serum antigens detected in women with gestation.     

Mode of action of nitrofurazone

When Escherichia coli strain B/r is exposed to 10 to 20 mug of nitrofurazone per ml, mutants with roughly threefold resistance are obtained. Treatment of these mutants with higher concentrations of nitrofurazone yields strains with six- to seven-fold resistance over strain B/r. Each of these steps toward nitrofurazone resistance is accompanied by loss of soluble nitrofurazone reductase activity. When sensitive bacteria are exposed to labeled nitrofurazone or labeled 2-nitrofuran, a considerable amount of radioactivity becomes bound to the cold trichloroacetic acid-insoluble fraction. Very little activity becomes bound in the mutants with six- to seven-fold resistance; mutants with intermediate resistance show intermediate levels of binding. Partially purified nitrofurazone reductase preparations catalyze the conversion of nitrofurazone to compounds which bind to protein and are not removed by prolonged dialysis against 8 m urea or by cold acid. Nitrofurazone reduced by xanthine oxidase or electrolytically reduced also yields compounds which react with protein to form stable derivatives.     

Heterogeneity of reducing terminals of urinary chondroitin sulfates

Human urinary chondroitin sulfates were isolated by precipitation with cetylpyridinium chloride of the non-dialyzable fraction of pooled urine, followed by ethanol fractionation and successive enzymic digestions with neuraminidase and mucopolysaccharides. Further purification was achieved by Dowex-1 chromatography with stepwise elution by increasing the concentration of NaCl at intervals of 0.25 M from 0.75 M to 1.5 M. The chondroitin sulfates thus obtained were characterized by the analysis and quantification on of carbohydrate, amino acid and sulfate, and by electrophoresis on cellulose acetate membrane. Then reducing terminals were identified by gas liquid chromatographic analyses of the acetyl and butaneboronate derivatives of hydrolysates, after reduction of the reducing terminals with sodium borohydride. About 22.8% of the urinary chondroitin sulfate in the 1.5 M fraction was peptide-bound, and the remainder was peptide-free, with xylose (29.8%), galactose (23.6%) and glucuronic acid (18.7%) at the reducing terminal. The amount of peptide-free chondroitin sulfate with xylose and galactose at its reducing terminals in the 0.75 M-, 1.0 M-, 1.25 M- and 1.5 M-fractions increased in the order described in parallel with the increase of sulfation and the decrease of peptide content. It was thus suggested that the endo-types of beta-xylosidase, beta-galactosidase and beta-glucuronidase acted on the carbohydrate-peptide linkage region of proteo-chondroitin sulfate in the tissues and produced various types of urinary chondroitin sulfate with heterogeneity at reducing terminals.     

A study of the effect of solvent composition and viscosity on the molecular dynamics of human serum albumin using Rayleigh scattering of M?ssbauer radiation

By means of RSMR changes of human serum albumen intramolecular mobility by addition of 1.5% and 7.5% of glutar dialdehyde (GD) in concentrated protein solution, heat denaturation of a protein or substitution of water by water-glycerol solvent with amount of water to glycerol: 1 to 2 were studied. It is shown that the elastic fraction for HSA is changed much less addition of GD or by heat denaturation than by substitution of water solution by water-glycerol. It seems that the observed strong influence of glycerol on intramolecular mobility of HSA is connected mostly with effective dehydration of protein (by substitution of the part of a water solvent by glycerol) and with a small volume decrease of protein (due to preference hydration effect) rather than with the increase of the solvent viscosity.     

Purification to homogeneity of extracellular polygalacturonase and isoenzymes of pectate lyase of Erwinia carotovora subsp. atroseptica by column chromatography

Extracellular polygalacturonase (PG) and two pectate lyase isoenzymes (PLI and PLII) produced by a 48 h culture of Erwinia carotovora subsp. atroseptica in pectate-based medium were purified 2027, 2036 and 2374-fold respectively to homogeneity with corresponding 59%, 61% and 32% recovery. This was achieved first by ion exchange chromatography on a S-Sepharose fast flow column with 20 mmol/1 Tris at pH 8.0 followed by elution of bound proteins with a 1 mol/1 NaCl gradient which separated PG from PL. The two enzymes were then further purified to homogeneity (assessed by SDS-PAGE) by selective adsorption chromatography on a hydroxyapatite column equilibrated with distilled water; PG was eluted with a 3 mol/1 KCl gradient and PLI with a 3 mol/1 KCl gradient followed by a 1.2 mol/1 PO₄ buffer pH 6.5 gradient to elute PLII. The Mr of the three enzymes determined by SDS-PAGE was 39 kDa and the pI values for PG, PLI and PII were 10.3, 10.3 and 10.0 respectively as determined by isoelectric focusing (IEF)-gel electrophoresis followed by activity staining.     

Kinetic studies of fructokinase I of pea seeds

Fructokinase I of pea seeds has been purified to homogeneity and the enzyme shown to be monomeric, with a molecular weight of 72,000 +/- 4000. The reaction mechanism was investigated by means of initial velocity studies. Both substrates inhibited the enzyme; the inhibition caused by MgATP was linear-uncompetitive with respect to fructose whereas that caused by D-fructose was hyperbolic-noncompetitive against MgATP. The product D-fructose 6-phosphate caused hyperbolic-noncompetitive inhibition with respect to both substrates. MgADP caused noncompetitive inhibition, which gave intercept and slope replots that were linear with D-fructose but hyperbolic with MgATP. Free Mg2+ caused linear-uncompetitive inhibition when either substrate was varied. L-Sorbose and beta, gamma-methyleneadenosine 5'-triphosphate were used as analogs of D-fructose and MgATP, respectively. Inhibition experiments using these compounds indicated that substrate addition was steady-state ordered, with MgATP adding first. The product inhibition experiments were found to be consistent with a steady-state random release of products. The substrate inhibition caused by MgATP was most likely due to the formation of an enzyme-MgATP-product dead-end complex, whereas that caused by D-fructose was due to alternative pathways in the reaction mechanism. The inhibition caused by Mg2+ can be explained in terms of a dead-end complex with either a central complex or an enzyme-product complex.     

亚致死剂量杀虫剂对异色瓢虫繁殖力的影响

为协调害虫的化学防治与生物防治,测定了吡虫啉、鱼藤酮、氰戊菊酯、阿维菌素、抗蚜威和印楝素6种杀虫剂亚致死剂量对异色瓢虫成虫繁殖力的影响．结果表明,各处理异色瓢虫每雌每日产卵量为40～55粒,吡虫啉接触法和饲喂法预处理及鱼藤酮和印楝素饲喂预处理后,均显著低于对照．所有处理间每雌累积产卵量差异不显著．6种药剂2种方式处理异色瓢虫后,其所产卵的孵化率均低于对照,从卵发育至蛹期的累积存活率均显著降低．吡虫啉两种方式处理和氰戊菊酯接触处理,从卵至蛹期的总发育历期显著延长,鱼藤酮两种方式处理和氰戊菊酯、阿维菌素、抗蚜威、印楝素饲喂法处理的总发育历期明显比对照缩短,而鱼藤酮和阿维菌素接触处理的与对照差异不显著．药剂直接浸渍处理异色瓢虫卵后,各处理卵的孵化率均较对照降低．氰戊菊酯处理的l龄幼虫存活率也明显较对照低,2龄至蛹期的存活率也有所下降．鱼藤酮和氰戊菊酯处理的总发育历期显著延长．这些研究结果显示,低剂量的杀虫剂对异色瓢虫繁殖力的影响很复杂,药剂的类型、作用方式、毒性高低以及给药途径等均影响到试验结果．     

Isolation and identification of the third component of complement of Japanese quails

The identification of the third component of complement (C3) of Japanese quails was attempted by using rabbit antiserum prepared against quail serum-treated zymosan (ZX) as an initial reagent. This antiserum (anti-ZX) had agglutinating activity on rabbit erythrocytes reacted with quail antibody and quail complement (EACq) but not on EAq, and developed two precipitin lines against quail serum at beta- and gamma-regions in crossed immunoelectrophoresis. Subsequently, monospecific antisera to each of these precipitin lines were prepared in rabbits, and quail serum proteins reactive with these antisera were purified by salt precipitation followed by Sephadex gel filtration and DEAE cellulose column chromatography. One protein with a m.w. of 184,000 (184K) resembled mammalian C3 in that: 1) monospecific antiserum (anti-184K protein serum) agglutinated EACq but not EAq; 2) treatment of fresh quail serum with either inulin or zymosan resulted in the conversion of the precipitin line developed against 184K protein from gamma to beta in crossed immunoelectrophoresis; 3) the 184K protein was shown to consist of two polypeptide chains of 110K and 73K linked by disulfide bonds. Furthermore, the 184K protein in serum was cleaved through the incubation with inulin to 174K and 140K proteins that might correspond to C3b and C3c of human complement; 4) the 184K protein bound to zymosan was eluted with hydrazine or methylamine but not with Nonidet P-40, indicating that 184K protein binds to zymosan by a covalent bond but not by a hydrophobic one; and 5) by treatment of fresh quail serum with methylamine, complement reactivity was reduced, although its activity was restored by the addition of purified 184K protein. These results suggest the 184K protein is the quail's equivalent to mammalian C3. When quail serum was reacted with cells that had complement-activating capacity, quail C3 deposited on their membrane as in mammalians; however, no conversion of quail C3 was noted by the reaction with CVF. Antibody to quail C3 failed to cross-react with that in mammals.     

Interaction and combination of separate A and B chains of insulin effects of D-and L-tryptophan in position A1

The S-thiomethyl derivatives of insulin A chain with A1-Gly replaced by D- or L-Trp have been prepared and their respective interaction and combination with the S-thiomethyl B chain studied. The UV difference spectra of the mixed against the separated [Trp1]A chains with the B chain at pH 10.8 are similar to those obtained for the unmodified chains except that the 295-nm-negative peak for ionized Tyr residue appears to be less marked. Fluorescence studies show very little environmental changes at the A1-Trp residues when mixed with the B chain. The intact hormone with A1-Gly replaced by D-Trp is known to be considerably more active than the analog with L-Trp replacement. However, for both derivatives the resynthesis of the whole molecules correctly joined by disulfide bridges starting from the separated reduced chains, gives similar low yields as shown by HPLC analysis and by receptor-binding assay. The replacement of A1-Gly by D-Trp appears to affect the separated A chain more than the intact hormone and replacements at A1 by both D- and L-Trp probably lead to significant conformational changes of the A chain so as to prevent its correct pairing with the B chain.     

类金刚石薄膜的制备及其血液相容性的初步研究

本文以单晶硅为基体,Ar等离子体对基体进行清洗,用法制备类金刚石(DLC)膜和掺氮的DLC膜。沉积DLC膜所用的气体是乙炔和氩气,沉积掺氮的DLC膜用乙炔和氮气。利用衰减全反射红外光谱(ATR-FTIR)和扫描电子显徽镜(SEM)对沉积的DLC膜和掺氮的DLC膜进行结构分析和观察表面形貌。考察了乙炔比例、放电功率等沉积参数对DLC膜的影响。通过接触角和血小板黏附实验,进行了DLC膜的抗凝血性能分析,效果较好。