Oligomer formation of the gB glycoprotein of herpes simplex virus type 1.

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1 was studied by sedimentation analysis of radioactively labeled infected cell and virion lysates. Fractions from sucrose gradients were precipitated with a pool of gB-specific monoclonal antibodies and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pulse-labeled gB from infected cell was synthesized as monomers and converted to oligomers posttranslationally. The oligomers from infected cells and from virions sedimented as dimers, and there was no evidence of higher-molecular-weight forms. To identify amino acid sequences of gB that contribute to oligomer formation, pairs of mutant plasmids were transfected into Vero cells and superinfected with a gB-null mutant virus to stimulate plasmid-specified gene expression. Radioactively labeled lysates were precipitated with antibodies and examined by SDS-PAGE. Polypeptides from cotransfections were precipitated with an antibody that recognized amino acid sequences present in only one of the two polypeptides. A coprecipitated polypeptide lacking the antibody target epitope was presumed to contain the sequences necessary for oligomer formation. Using this technique, two noncontiguous sites for oligomer formation were detected. An upstream site was localized between residues 93 and 282, and a downstream site was localized between residues 596 and 711. Oligomer formation resulted from molecular interactions between two upstream sites, between two downstream sites, and between an upstream and a downstream site. A schematic diagram of a gB oligomer is presented that is consistent with these data.     

The immunological characterization of several human ribonucleases by using primary binding tests.

RNAases (ribonucleases), purified from four human tissues, as well as bovine pancreatic RNAase (RNAase A), were studied by immunodiffusion methods and by two different primary binding tests. The enzymes fell into two groups immunologically, those purified from plasma and pancreas in one and those from spleen and liver in the other. No antigenic cross-reaction between the two groups was detected by any of the immunoassays used. There was a slight antigenic cross-reaction between the human and bovine pancreatic RNAases. The liver and spleen RNAases were immunologically identical by all criteria used, whereas a small but consistent antigenic difference between the human plasma and human pancreas enzymes was detected. The significance of this difference between the human plasma and pancreas RNAases is discussed in relation to similarities and differences in their properties.     

应用real-timePCR法快速定量人类粪便中双歧杆菌的研究

目的建立快速、准确从粪便标本中定量双歧杆菌的RT—PCR技术。方法传统培养定量法,普通PCR定量法,real—timePCR比较测量。结果（I）粪便标本前处理采取简单的离心和清洗、稀释步骤能去除粪便标本中的抑制物,实现不提取DNA直接进行PCR、real—time定量粪便中双歧杆菌。（2）本实验建立的PCR方法直接半定量粪便双歧杆菌技术在双歧杆菌值介于10^3～10^7CFU／ml时具有较好的分辨率,粪便标本普通PCR得理论菌数与培养得菌数值之间差异无显著性（P〉0．05）;real-timePCR直接定量双歧杆菌技术在双歧杆菌值介于10^1-10^7CFU／ml时具有较好的分辨率,粪便标本RT—PCR得理论菌数与培养得菌数值之间差异无显著性（P〉0．05）。结论利用PCR、real—timePCR直接半定量和定量粪便中的双歧杆菌可行。     

Force velocity relations of single cardiac muscle cells: calcium dependency

Cellular cardiac preparations in which spontaneous activity was suppressed by EGTA buffering were isolated by microdissection. Uniform and reproducible contractions were induced by iontophoretically released calcium ions. No effects of a diffusional barrier to calcium ions between the micropipette and the contractile system were detected since the sensitivity of the mechanical performance for calcium was the same regardless of whether a constant amount of calcium ions was released from a single micropipette or from two micropipettes positioned at different sites along the longitudinal axis of the preparation. Force development, muscle length, and shortening velocity of eitherisometric or isotopic contractions were measured simultaneously. Initial length, and hence preload of the preparation were established by means of an electronic stop and any additional load was sensed as afterload. Mechanical performance was derived from force velocity relations and from the interrelationship between simultaneously measured force, length, and shortening velocity. From phase plane analysis of shortening velocity vs, instantaneous length during shortening and from load clamp experiments, the interrelationship between force, shortening, and velocity was shown to be independent of time during the major portion of shortening. Moreover, peak force, shortening, and velocity of shortening depended on the amount of calcium ions in the medium at low and high ionic strength.     

IgA and IgG antibodies to Candida albicans in the genital tract secretions of women with or without vaginal candidosis

Levels of anti-Candida albicans immunoglobulin A (IgA) and IgG were measured by enzyme-linked immunosorbent assay in serum and cervicovaginal secretions from 64 non-pregnant women with vaginal candidosis and 158 uninfected non-pregnant women. Specific IgA and IgG were detected in the serum and secretions of all 222 women. There was no significant difference between the mean levels of specific IgA or IgG in secretions from women with candidosis and those of uninfected women. Neither was there a significant difference between mean levels of specific IgA or IgG when women using oral contraception were compared with others who were not. There was a significant correlation between the levels of IgA and IgG in serum and secretions from women with candidosis and from uninfected women. Blastospore and hyphal forms of C. albicans were seen in vaginal smears from 29 of the 64 women with culture-proven candidosis: in nine, both IgA- and IgG-coated C. albicans cells were recovered from the genital tract; in a tenth, IgG-coated cells were found.     

Evolution under relaxed sexual conflict in the bulb mite Rhizoglyphus robini

The experimental evolution under different levels of sexual conflict have been used to demonstrate antagonistic coevolution in muscids, but among other taxa a similar approach has not been employed. Here, we describe the results of 37 generations of evolution under either experimentally enforced monogamy or polygamy in the bulb mite Rhizoglyphus robini. Three replicates were maintained for each treatment. Monogamy makes male and female interests congruent; thus selection is expected to decrease harmfulness of males to their partners. Our results were consistent with this prediction in that females from monogamous lines achieved lower fecundity when housed with males from polygamous lines. Fecundity of polygamous females was not affected by mating system under which their partners evolved, which suggests that they were more resistant to male-induced harm. As predicted by the antagonistic coevolution hypothesis, the decrease in harmfulness of monogamous males was accompanied by a decline in reproductive competitiveness. In contrast, female fecundity and embryonic viability, which were not expected to be correlated with male harmfulness, did not differ between monogamous and polygamous lines. None of the fitness components assayed differed between individuals obtained from crosses between parents from the same line and those obtained from crosses between parents from different lines within the same mating system. This indicates that inbreeding depression did not confound our results. However, interpretation of our results is complicated by the fact that both males and females from monogamous lines evolved smaller body size compared to individuals from polygamous lines. Although a decrease in reproductive performance of males from monogamous lines was still significant when body size was taken into account, we were not able to separate the effects of male body size and mating system in their influence on fecundity of their female partners.     

Body size, age and reproduction in the leopard toad, Bufo pardalis

Data on the relationships between body size and age were obtained for a sample of leopard toads Bufo pardalis from a breeding population of this species from the Cape Peninsula, South Africa. Age was determined by counting the number of lines of arrested growth in histological sections of a digit clipped from each individual. In males there was a positive, but weak, correlation (explaining only 18% of the variance) between body size and age, and in females no correlation at all existed between these two variables. Males which were successful in obtaining matings were not older than unsuccessful males. Age of males at the breeding site ranged from one to three years, whereas females ranged from two to six years old. This represents both the earliest age of reproduction, as well as the greatest difference in longevity between the sexes, documented for an anuran species.     

Population substructure in Alkanna orientalis (Boraginaceae) in the Sinai Desert, in relation to its pollinator behaviour

Subpopulation genetic structure was studied in a population of the short-lived perennial plant Alkanna orientalis from the Sinai Desert, Egypt. The population investigated was subdivided for sampling into four subpopulations, which were located within three steep-sided wadis and a central plain area. Results from previous studies suggested that bee pollinator behaviour was likely to cause limited gene dispersal within the population and that subpopulations might have diverged from each other genetically. Seven RAPD primers were used to detect polymorphisms in the population. Differences between sub-populations in fragment frequency were found for several of the 45 polymorphic RAPD fragments scored. Population subdivision was evident from cluster analysis, and an analysis of genetic distances showed that there was significant genetic differentiation between all subpopulations. Nevertheless, more extensive gene flow appears to take place within the population than was expected, as demonstrated by a higher level of genetic similarity between subpopulations from two of the narrow wadis and the interconnecting plain. It is suggested that seed transport mediated by periodic flash floods is responsible for this pattern.     

Genetic variation versus recombination rate in a structured population of mice

The correlation between genetic variation and recombination rate was investigated in a structured mouse population. Nucleotide sequence data from 19 autosomal DNA loci from eight inbred strains of mouse (Mus musculus) sampled from three major subspecies were analyzed. The recombination rate was estimated from the comparison of genetic and physical map distances between markers flanking a 10-cM region of each locus. The strains were categorized into four groups (subpopulations) based on geography. By partitioning the genetic diversity into within-group and among-group variation, we detected a positive correlation between the recombination rate and nucleotide diversity within groups. The level of nucleotide differentiation among groups (G(ST)) showed a negative correlation with the rate of recombination. There was no significant correlation between recombination rate and nucleotide diversity when data from different subpopulations were pooled. No correlation was detected between recombination rate and nucleotide divergence of M. musculus and M. spicilegus. These patterns deviate from the strict neutral expectation under the constant nucleotide substitution rate, and they are likely to have been formed either by a hitchhiking effect of positively selected mutants or by background selection of deleterious mutants occurring in a subdivided population. Our series of comparisons show that because a real population always has some structure, incorporation of its information is important in detecting non-neutral evolution.     

Molecular genetic diversity of the French-American grapevine hybrids cultivated in North America.

French-American hybrid grapevines are most popular in eastern and mid-western North America: they are hardy cultivars derived from crosses between the European Vitis vinifera and American wild vines. The aim of this study was to characterize their genetic background using 6 microsatellite (SSR) markers and a set of 33 diagnostic RAPD markers. The latter were reproducible with different PCR thermal cyclers. Two SSR loci were found to be synonymous, VrZAG47 and VVMD27. The DNA profile frequencies estimated for each cultivar were much lower with multi-locus SSR data than that obtained from multi-fragment RAPD data. There was no significant correlation between the multi-locus DNA profile frequencies derived from SSRs and those from RAPDs. Estimates of genetic diversity derived from SSRs were generally higher and the average similarity between cultivars was generally lower than values reported for subgroups of V. vinifera, in accordance with expectations for hybrid cultivars. The phenetic relationships depicted by UPGMA (unweighted pair-group method with arithmetic averaging) and neighbor-joining analyses of microsatellite data were congruent and, to a large extent, in agreement with the known pedigree or history of each cultivar. A major dichotomy was observed between one group where the known genetic background was dominated by the North American Vitis riparia and Vitis labrusca, and another one where the genetic background was dominated by the European V. vinifera. Two Kulhmann varieties thought to be synonymous were found to be different, though closely related.     

Plasma cholesterol,coronary heart disease,and cancer in the Renfrew and Paisley survey.

The relation between plasma cholesterol concentration and mortality from coronary heart disease, incidence of and mortality from cancer, and all cause mortality was studied in a general population aged 45-64 living in the west of Scotland. Seven thousand men (yielding 653 deaths from coronary heart disease, 630 new cases of cancer, and 463 deaths from cancer) and 8262 women (322 deaths from coronary heart disease, 554 new cases of cancer, and 395 deaths from cancer) were examined initially in 1972-6 and followed up for an average of 12 years. All cause mortality was not related to plasma cholesterol concentration. This was largely a consequence of a positive relation between cholesterol values and mortality from coronary heart disease being balanced by inverse relations between cholesterol and cancer and between cholesterol and other causes of death. These changes were highly significant for coronary heart disease and cancer in men and significant for coronary heart disease and other causes of death in women. The inverse association between cholesterol concentration and cancer in men was strongest for lung cancer, was not merely a function of the age at which a subject died, was present for the incidence of cancer as well as mortality from cancer, and persisted when new cases or deaths occurring within the first four years of follow up were excluded from the analysis.     

Bitter taste thresholds, numbers and diameters of circumvallate papillae and their relation with age in a Turkish population

Objectives: This study analyses the relationship between the sense of bitter taste and age. The relationships between these and the numbers and diameters of Circumvallate Papillae (CP) are also analysed. Subjects: Twenty-four elderly subjects (from 65 to 85 years) and 30 young subjects (from 17 to 25 years) were studied. Method: Bitter taste thresholds were determined by the three drop method with an ascending series of concentrations. The numbers and diameters of CP were observed by direct naked eye observation using a wooden tongue depressor, a gauge and a light source. Results: The bitter taste acuity was significantly poorer in the older sample. There was no relationship between the bitter taste acuity and number of papillae but an inverse relationship was observed between the bitter taste acuity and diameters of papillae, thus lower acuity was associated with larger papillae. It was also observed that the diameters of papillae were inversely related to the numbers of CP. The distribution of numbers and diameters of CP were not significantly different between these samples with age.     

A probabilist model of the relations between pollen and climate and its application to 80 European annual spectra

The relation between pollen and climate is commonly computed by regressing the climatical factor. The disadvantage of this method is that it does not respect the ordinal and intermittent nature of field data. This paper overcomes the artefacts created by this problem by using a probabilist calibration, that quantifies the ecological linkage between a taxon T and a factor with a general parameter, the probability PROX for an abundance A being confined near the rank F of the factor. Confining simulates the effect of the factor upon the concentration of presences and ordering of abundances, and calibrates the climatical behaviour of a taxon with the set of PROX for all possible pairs (A,F). It summarizes a behaviour with the probable position of each abundance A in the range of the factor. Calibration was applied to 130 pollen taxa observed in a network of 80 standardized annual aeropollinic spectra. Spectra were mostly from France, the rest being from a transect stretching from Sweden to Algeria. Spectra were characterized by the values of 10 climatic factors, as well as the presence and abundance of 130 pollen taxa. The influence of geographical climate differences upon pollen content in the atmosphere was quantified by comparing the spectra. Pairs from different localities but the same year were compared. The reliability of indicator taxa was tested by estimating the climate in the 80 spectra using calibration. For all the taxa observed in a spectrum, the envelope of confinings generally followed an unimodal gradient, whose mode was the probable position of the spectrum. Reliability of the estimate was measured by its accuracy, being the agreement between estimates and measures; and by its stability, being the agreement between two estimates from the same climate according to two different flora (the two halves of a spectrum for instance). Average accuracy was 72%, and average stability 87%.     

Evaluation of intrinsic spectroscopic properties of chromophore assemblies by shielding with cyclohexyl base pairs within a DNA duplex

Here, we investigated spectroscopic behaviors of tetramethylrhodamine (TMR) homo- and hetero-dimers within DNA duplex. In order to shield the chromophores from natural base pairs, we used cyclohexyl base pairs as ‘insulators’; these pairs were inserted between the chromophores and nucleobases. When a single TMR moiety was sandwiched between cyclohexyl base pairs, the emission intensity increased by fivefold relative to a TMR between natural base pairs, because electron transfer from nucleobases was suppressed. Next, we inserted two TMRs between the cyclohexyl base pairs and found that they facilitated H-dimer formation of TMR; a distinct hypsochromic shift was induced only when cyclohexyl base pairs were inserted. We further examined quenching behavior of a TMR paired with a quencher dye between cyclohexyl base pairs. Interestingly, fluorescence from TMR was quenched by nitro methyl red more efficiently in the presence of cyclohexyl base pairs than in their absence. This suggests that neighboring natural base pairs disturbed electron or hole transfer between the fluorophore and the quencher. The cyclohexyl base pairs shielded the chromophore pair from the natural base pairs and allowed intrinsic electron transfer.     

Transition metal and organic radical components of carp liver tissue observed by electron paramagnetic resonance spectroscopy

Paramagnetic transition metal centers and organic radicals in liver from wild-type carp (Cyprinus carpio) were characterized by electron paramagnetic resonance (EPR) spectroscopy. Approximately twelve EPR signals were observed at 77 K with resonance positions between g=1.8 and g=2.5. Identification was facilitated by a study of the variation in signal intensity with microwave power (microwave power saturation) for each signal. Many were organic radical or iron signals from typical liver enzymes, including cytochrome P450, coenzyme Q₁₀, NADH dehydrogenase, and succinate dehydrogenase, cytochrome c oxidase and/or catalase. Of special interest were two signals that are not normally found in mammalian liver. The first was a six-line signal from divalent manganese, which was evident in the spectra in quantities suggestive of a functional role. The second was probably a signal from nitrosylated non-heme iron and may be related to the presence of nitrogen-containing compounds produced by nitrifying bacteria in the aquatic environment. These notable differences between the EPR spectra of fish and mammalian liver suggest major metabolic differences between the two systems.     

THE DNA BASE COMPOSITION OF INDIVIDUAL CHROMOSOMES AND CHROMOSOME SEGMENTS FROM CHIRONOMUS TENTANS

The base composition of DNA was determined for individual chromosomes from the dipteran Chironomus tentans and for each one of six different segments of one of the chromosomes. The isolations were carried out by micromanipulation and the DNA purines were first extracted from the isolated components and afterwards separated by means of microelectrophoresis on a cellulose fiber. It was found that DNA from this material has an unusual composition corresponding to a guanine + cytosine content of about 30%. This composition was not a function of the polytenic condition but was also found for DNA from testis tissue. Furthermore Drosophila has a more traditional base composition for the bulk of DNA. Statistically significant variations in base data were found between whole chromosomes as well as between the segments from one of the chromosomes.     

Human prolyl hydroxylase. Purification, partial characterization and preparation of antiserum to the enzyme.

Prolyl hydroxylase was purified from human foetal skin and from a mixture of human foetal tissues by the affinity chromatography procedure using poly(L-proline). The enzyme from both sources was pure, when examined by polyacrylamide gel electrophoresis, as a native protein or in the presence of sodium dodecylsulphate, and enzyme activity recovery varied from 38% to 70% with seven enzyme preparations. The enzyme synthesized from 61.0 mumol to 82.7 mumol hydroxyproline mg protein-1 h-1 degrees C with a saturating concentration of (Pro-Pro-Gly)5 as substrate. The molecular weight of the enzyme was identical with that of the chick prolyl hydroxylase when studied by gel filtration, and the molecular weights of the subunits of the enzyme were about 61000 and 64000 as determined by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The amino acid composition of the human enzyme was very similar to that of the chick prolyl hydroxylase. Antisera to human and chick prolyl hydroxylases were prepared in rabbits. A single precipitin line was seen between the antiserum to human prolyl hydroxylase and the human enzyme in double immunodiffusion, and no cross-reactivity was detected between the human chick enzymes by this technique. However, a distinct cross-reactivity was observed between the human and chick enzymes in inhibition experiments.     

Regional variation in the microhardness and mineralization of vertebrae from brown and rainbow trout

Regional variation in properties of vertebral bone from brown Salmo trutta and rainbow trout Oncorhychus mykiss were explored by using microhardness tests. Statistically‐significant positive correlations were identified between the microhardness of bone and its mineral content. In both brown and rainbow trout, the vertebrae from the caudal region were harder than those of the trunk region. There was a significant difference between the species; microhardness of bone from vertebrae of rainbow trout was greater than those from brown trout.     

Cross-linking of proteins in nuclei and DNA-depleted nuclei from Friend erythroleukemia cells.

Reversible cross-linking of proteins in nuclei and DNA-depleted nuclei from Friend erythroleukemia cells was used as a probe to determine whether the protein structure was preserved following treatment with DNAase I. Interactions between histones were analyzed through cross-linking with 2-iminothiolane or dimethyl 3,3'-dithiobispropionimidate. No alterations in the interactions between intranucleosomal histone proteins resulted from digestion of the nuclear DNA. There was, however, a diminished extent of cross-linking of histone H1 to itself and to the intranucleosomal histones in DNA-depleted nuclei. The interactions of a group of nonhistone proteins with histone H3 could be monitored by cross-linking through the formation of disulfide bonds caused by oxidation of nuclei with H2O2. These interactions were not markedly affected by treatment of the nuclei with DNAase I. However, differences were observed in the extent of cross-linking of some of these proteins when cross-linking in nuclei from undifferentiated cells was compared to that in nuclei from cells which had been induced to differentiate with dimethylsulfoxide.     

Restriction endonuclease DNA analysis of antigenic variants of leptospires selected by monoclonal antibodies

The genome of antigenic variant CV (CT3)-1 derived from Leptospira interrogans serovar canicola was compared by cleavage with restriction endonucleases with the parent and serovar bafani, to which the variant was serologically most closely related. No differences were observed between the parent and variant in DNA restriction endonuclease patterns using eight restriction endonucleases. Serovar bafani was different in the patterns from the parent and antigenic variant CV (CT3)-1. The two antigenic variants derived from serovar hebdomadis, HV (H16)-1 and HV (H19)-1 which belonged serologically to serovars jules and hebdomadis, respectively, were compared by restriction endonuclease DNA analysis with the parent and serovar jules. No differences were observed between the parent and variants in DNA restriction endonuclease patterns using the same enzymes. But some differences were observed in DNA restriction endonuclease patterns between HV (H16)-1 and serovar jules. Thus, the antigenic variant selected from the parent by the anti-parent monoclonal antibody and serologically different from the parent, being identified either as a new serovar or as a known one, was found to be similar to the parent by the restriction endonuclease DNA analysis.