兔子宫内膜蛋白等电聚焦电泳载体两性电解质pH范围及比例的选择

兔子宫内膜蛋白是研究早期胚胎发育过程中胚泡附植机制的关键因素之一．为了对子宫内膜蛋白进行有效的分离，本研究探讨了用于等电聚焦电泳的载体两性电解质pH范围及其比例．研究结果表明：在只有pH3～9．5的情况下，蛋白质谱带主要分布于胶条碱性端1／3区域，酸性端无谱带；在载体两性电解质pH3-9．5：pH4～6=1：4的情况下，蛋白质谱带主要分布于胶条碱性端2／3部分；在载体两性电解质以pH3-9．5：pH4～6：pH6—9=1：4：1时，蛋白质谱带分布比较均匀，酸性端也有谱带分布．这说明在作子宫内膜蛋白的等电聚焦电泳时采用pH3～9．5：pH4～6：pH6～9=1：4：1比较理想．     

绵农小麦种子巯基蛋白酶抑制剂的纯化及性质研究

绵农11号小麦种子磨粉后的胚和麸皮，经溶液浸取和硫酸铵分级沉淀获得巯基蛋白酶抑制剂（CPI）粗品，再经木瓜蛋白酶－Sepharose4B亲和层析，DEAE－Sepharose离子交换层析和SephadexG－100分子筛层析，可获得两种CPI．通过SDS－PAGE或凝胶过滤法测得其Mr分别为18800［简称CPI（H）］和10500［简称CPI（L）］．它们在PAGE，SDS－PAGE和HPLC上均为单一蛋白带．CPI（L）和CPI（H）对木瓜白酶的抑制活性（ID50）分别为7．8×10－6mol／L和3．40×10－6mol／L．对无花果蛋白酶的抑制活性分别为9．5×10－5mol／L和5．8×10－6mol／L；CPI（H）对菠萝蛋白酶有弱抑制作用，但CPI（L）则无抑制作用；无论是CPI（L）或CPI（H）对胰蛋白酶、胰凝乳蛋白酶和胃蛋白酶均无抑制作用．CPI（H）的N－末端为Ile．CPI（H）和CPI（L）在pH2．0～11．0范围内和90℃处理5min，两种CPI的抑制活性均无变化；CPI（H）和CPI（L）对木爪蛋白酶的抑制曲线经Dixon作图法，得出CPI（H）为反竞争性抑制类型，而CPI（L）为竞争性抑制类型，其Ki值为8．32×10－6mol／L．     

Origin and nature of spermatophoric mass of the spiny lobster Panulirus homarus

In the spiny lobster Panulirus homarus (Linnaeus) the spermatophoric mass consists of a highly convoluted tube, containing the sperm mass and a gelatinous matrix. The spermatophoric components have their origin in the proximal vas deferens and the completed spermatophoric mass is stored within the distal dilated part of the vas deferens. In the proximal vas deferens the inner glandular epithelial cells give rise to leaf-like typhlosoles which gradually diminish in size and finally disappear in the distal vas deferens. The secretions originating from the typhlosole form the outer gelatinous matrix of the spermatophoric mass. Histochemical observations reveal that the wall of the spermatophore tube consists of neutral mucopolysaccharide, whereas the sperm mass and the gelatinous matrix are rich in acidic mucopolysaccharides. Both polyacrylamide gel electrophoresis and agarose gel electrophoresis were used to separate the acidic mucopolysaccharide (AMPS) complexed with the proteins and the simple proteins. In addition, AMPS have been isolated following the method of Rahemtulla and Lovtrup (1974) and used for electrophoresis. In both the polyacrylamide gel electrophoresis and agarose gel electrophoresis, the extracted acid mucopolysaccharides give only a single fraction which, in comparison with the standard acid mucopolysaccharides, corresponds to chondroitin sulphate. The functional significance of the chondroitin sulphate and neutral mucopolysaccharides in the spermatophoric components of P. homarus is discussed in relation to their functional role in spermatophore hardening and protection of the delicate spermatozoa during their prolonged storage on the sternum of the female.     

A proteomic approach to the study of the marine mussels Mytilus edulis and M. galloprovincialis

In this study, a proteomic approach was applied for the generation of reference maps and subsequently to detect, quantify and compare the global protein expression between two related species of marine mussels, Mytilus edulis and Mytilus galloprovincialis, growing in their own geographical habitats. A comparative study of the protein profiles generated from analytical two-dimensional electrophoresis gels was performed, and changes in protein expression were analysed quantitatively by computer analysis. An average of 1,278 spots per gel was detected in 16 individuals (8 M. edulis and 8 M. galloprovincialis); however, not all spots were included in the study. Expression of 420 spots was compared, and significant differences in the intensity levels were detected in 37 protein spots (8.8%). Fifteen proteins showed higher expression in M. edulis, and 22 proteins, in M. galloprovincialis. The technique of peptide mass fingerprinting using MALDI-TOF (matrix-assisted laser desorption ionisation/time-of-flight) and/or nanoelectrospray double subfragmentation mass spectrometry enabled the unambiguous identification of 15 of these 37 differentially expressed proteins. Most of the identified proteins can be grouped basically into four broad functional classes: cytoskeletal and myofibrillar proteins, proteins associated with stress response, proteins associated with the storage or production of energy, and proteins related to rearrangement in the synthesis of native structures. These results expand our understanding of the molecular differentiation of the two mussel taxa and serve as a useful base for future ecological, physiological and genetic studies.     

Identification of protein biomarkers of mercury toxicity in fish

Bioaccumulative metals such as mercury are found in increasing amounts in fish and their consumers. In the region of the Madeira River, in the Brazilian Amazon, mercury (Hg) is a predominant contaminant in the aquatic ecosystem. There is therefore a need to find specific biomarkers of mercury toxicity in fish to monitor contaminations. Here, mercury-bound proteins were identified in the liver tissues of fishes Mylossoma duriventre and Brachyplatystoma rousseauxii. Mercury was quantified in liver tissue, pellets and protein spots by graphite furnace atomic absorption. Proteins were fractionated by two-dimensional polyacrylamide gel electrophoresis and identified by mass spectrometry with electrospray ionization. We identified nine proteins linked to mercury and that presented biomarker characteristics of mercury. Among the proteins identified, isoforms of parvalbumin, ubiquitin-40S ribosomal protein S27a, brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 2 and betaine–homocysteine S-methyltransferase 1 are notable for having the molecular function of binding to metallic ions.     

Protein expression profile in the testis of rats chronically exposed to low-dose 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin

The chronic effects low-dose 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were examined on protein expression profiles in rat testis, sperm, and serum gonadal hormones. A total of 32 male rats were randomly divided into three TCDD-exposed groups, administered either 140, 350, or 875 ng TCDD/kg/week for 29 weeks, respectively, and one control group receiving only corn oil. The proteins from rat testis were separated and analyzed by two-dimensional gel electrophoresis and mass spectrometry. TCDD induced significant decreases in sperm counts and serum gonadal hormone levels compared with controls. TCDD altered testicular protein expression levels. Several interesting volume-altered proteins that were related to the reproductive toxicities or other toxicities of TCDD were identified. Among these proteins, PERF15 was the only down-regulated protein; sperm protein SSP411, ubiquitin carboxyl-terminal hydrolase L-3, and eukaryotic translation elongation factor 1 gamma were up-regulated by TCDD. The differentially expressed proteins and other data provide further insight into the mechanisms of reproductive toxicity mediated by low-dose TCDD exposure.     

Distribution and binding pattern of benzo(a)pyrene in rat liver,lung and kidney constituents after oral administration

Orally administered ³H‐benzo(a)pyrene (BP) was persistent in protein fraction of liver, lung and kidney. The radioactivity in this fraction increased with time after administration and accounted for about 50%, 40% and 65% of total radioactivity in liver, lung and kidney, respectively at 48 hr. The BP metabolites binding proteins were located in cytosol and had molecular weights of 40,000–60,000 and 80,000–100,000 as determined by gel filtration and polyacrylamide gel disc electrophoresis. In addition, at 48hr after administration, about 80% of radioactivity in high molecular weight protein fraction was found to be precipitated by trichloroacetic acid treatment.

These results suggest that BP metabolites might be transported by and are persistent in these protein fractions of liver, lung and kidney if the intake of BP is continuous. These proteins, therefore, appeared to be closely related to cell toxicity or mutagenicity in these organs as well as DNA.     

Opsonin-like molecule found in coelomic fluid of a sea cucumber, Holothuria leucospilota

Opsonins, a group of serum proteins present in vertebrates, are known to bind to the surface of many kinds of pathogens and tag them as targets for phagocytosis. Opsonin-like molecules have been suggested to exist in several invertebrates. In this paper, we report the discovery of a new opsonin-like molecule in the coelomic fluid of the sea cucumber Holothuria leucospilota. Opsonic activity in cell-free coelomic fluid was characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, two-dimensional electrophoresis and gel filtration. Phagocytic uptake of yeast cells and latex beads by amoebocytes was quantified by microscopic analysis. It was found that a 20 kdalton protein bound to and enhanced phagocytosis of heat-killed yeast cells. The results of Western blot analysis suggest that both amoebocytes and spherule cells produce this opsonin-like molecule. Received: 22 October 1998 / Accepted: 4 March 2000     

Transport of metanil yellow in the rat plasma and interaction of its metabolite,p‐aminodiphenylamine with serum proteins

Binding affinity of metanil yellow and its breakdown product p‐aminodiphenylamine to serum proteins has been studied employing chromatographic separation on Sephadex G‐200 and by paper and polyacrylamide gel electrophoresis. Metanil yellow has more affinity towards albumin than to globulins. The complexing is presumably through electrostatic forces. p‐Aminodiphenylamine on the other hand, preferably binds to globulin fractions of serum protein. However, a stable binding with BSA alone was also observed. The binding was quite stable and was accompanied by a shift in absorbance from 430 nm to 500 nm. Aspartic acid moiety of protein was found to be one of the units involved in the binding of p‐ADPA to proteins.     

Variation in populations of the coastal dune building grassLeymus arenarius in Iceland revealed by endospermal prolamins

Variation in the endospermal protein patterns of seeds amongst and between inland and coastal populations of the dune building grassLeymus arenarius was examined in Iceland. Seeds were collected from six coastal populations and five inland populations in Iceland. Endospermal proteins (prolamins) of seeds were extracted with Tris-buffered 2-propanol (50%) and β-mercaptoethanol (0.5% v/v). We used 8% and 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to separate the prolamin protein bands, which were stained with Coomassie Brilliant Blue R. Coastal populations were differentiated from inland populations on the basis of high frequency of high molar weight (HMW) (110–150 kg mol⁻¹) prolamins. Coastal populations had significantly higher proportions of their seed with more than two HMW prolamins, than those of inland populations. Subtle differences were found among the HMW prolamins of coastal and inland populations indicating limited intrapopulation variation. The dynamic environment of sand dunes probably influences the genetic composition of these populations. The results suggest that seeds of the inland and coastal populations should be treated separately in sand dune reclamation and restoration works     

Genetic differentiation of populations of the planktonic copepod Labidocera aestiva

Planktonic populations of the calanoid copepod Labidocera aestiva show significant biochemical genetic heterogeneity along the Atlantic coast of the USA. In summer, 1981, copepods were collected by surface tows at Beaufort Inlet, North Carolina; Fort Pierce Inlet, Florida; and Vineyard Sound, Massachusetts. Genetic variation within each population and genetic differentiation among the three populations were studied by micro-acrylamide gel electrophoresis of six loci encoding four enzymes. All six enzyme loci were polymorphic when all populations were considered together, but the North Carolina population was monomorphic at two loci. High genetic variability was indicated by the following: (1) the number of alleles per locus averaged over all loci was 2.57±0.26 SD; (2) the average proportion of loci for which the frequency of the most common allele was not greater than 0.95 was 0.78±0.10; (3) the frequency of heterozygous individuals was 0.25±0.07. Genetic differentiation among population samples in the three regions was demonstrated in several ways: allele frequencies at one aminopeptidase-I locus, Lap-1, differed significantly among samples of the three populations, and there were unique alleles of high frequency at this locus in two population samples. Values of the statistic of genetic distance (D) averaged 0.20±0.08 for pairwise comparisons between all samples. Compared to expected heterozygosity if individuals across the entire range sampled mated at random, there were highly significant heterozygote deficiencies at five of the six loci. Genetic differentiation of populations of L. aestiva may result from (1) differential selection on populations in the three regions, or (2) restricted gene flow between the populations. Gene flow may be limited by geographic separation or differences in life history, such as seasonal presence in the plankton and diapause egg production.Contribution No. 5810 of Woods Hole Oceanographic Institution