人白血病细胞癌性促凝物(CP)的分离纯化及其特性的初步研究

用三步纯化法从人M_3型白血病细胞中分离纯化出人类肿瘤癌性促凝物(CP)。促凝活性回收率为24％,CP纯化倍数为2481倍。纯化CP在SDS-PAGE上为单一区带,其理化和酶学特性类似于动物肿瘤CP,分子量约为70 000,PI为4.8,在FVⅡ缺乏血浆中以及在含有组织因子(TF)抑制剂情况下仍能激活FX。CP促凝活性能被半胱氨酸蛋白酶抑制剂HgCl_2抑制,纯化CP能与抗动物肿瘤CP抗体形成免疫沉淀反应。     

cAMP-Dependent protein kinase inhibits the chloride conductance in apical membrane vesicles of hunman placenta

Summary The role of adenosine 3,5-monophosphate (cAMP) dependent protein kinase (PK-A) on the Cl^– conductance has been studied in the apical membrane vesicles purified from the chorionic villi of human placenta. In order to phosphorylate the cytosolic side of the membranes, vesicles have been hypotonically lysed, loaded with 100nm catalytic subunit of PK-A purified from human placenta and 1mm of the phosphatase resistant adenosine 5-thiotriphosphate (ATP-gamma-S) and resealed. Cl^– conductance has been measured by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) at 23°C with membrane potential clamped at 0 mV. The actual volume of the resealed vesicles was measured in each experiment by trapping an impermeable radioactive molecule ([¹⁴C]-sucrose) and included in each Cl^– flux calculation. In 19 independent experiments, the mean Cl^– conductance in placental membranes in the absence of phosphorylation was 3.67±3.18 whereas with the addition of PK-A and ATP-gamma-S it was 1.97±1.75 nmol·sec^–1·(mg protein)^–1 (mean±sd). PK-A dependent phosphorylation reduced the Cl^– conductance in 14/19 experiments. The same protocol applied to the apical membranes of bovine trachea, where PK-A is known to activate the Cl^– channels, confirmed that the PK-A dependent phosphorylation increased the Cl^– conductance in 11/13 experiments, from 1.01±0.61 to 1.85±0.99 nmol·sec^–1·(mg protein)^–1(mean±sd). These studies indicate that the PK-A dependent phosphorylation inhibits one or more Cl^– channel(s) of the apical membranes of human placenta.     

Protein extraction by reverse micelles: a study of the factors affecting the forward and backward transfer of alpha-chymotrypsin and its activity

alpha-chymotrypsin is taken as a model protein to investigate three aspects of the protein extraction by reverse micelles: (1) the comparison between the two forward transfer techniques, i.e., the liquid-liquid and the solid state-liquid transfer; (2)the back-transfer, i.e., the capability of the protein to be recovered from the micellar solution; and (3) the maintainance of the enzyme activity at the end of the extraction cycle. Concerning the forward transfer from the liquid phase, we study first the effect of salt initially present in the aqueous phase on the equilibrium concentration of the extracted species; further, we study the forward protein extraction from the solid state, and the effect of pH, salt, and protein concentration on the transfer efficiency. Concerning the back transfer, we find the somewhat surprising result, that the percentage of protein back-extraction depends on the type and concentration of salt used for the forward transfer. Preliminary data concerning an alternative method for the back-transfer using silica gel to liberate the protein from the micellar environment, are presented. Finally, it is found that the enzyme activity depends again on the type and concentration of salt used for the forward transfer.     

Tunicamycin inhibits the initiation of DNA synthesis stimulated by prostaglandin F2α in Swiss mouse 3T3 cells

Tunicamycin, an inhibitor of the asparagine-linked protein N-glycosylation, blocks the initiation of DNA synthesis in Swiss 3T3 cells stimulated by prostaglandin F_2α alone or with insulin. This effect is exerted only when tunicamycin is added from 0 to 8 h after stimulation and it decreases the rate of entry into S phase. Blocking of labeled sugar incorporation to proteins occurs regardless of the time of PGF_2α stimulation. In contrast tunicamicin does not inhibit protein synthesis. These results suggest that N-glycoprotein synthesis early during the prereplicative phase is an important event controlling the mitogenic action of PGF_2α     

Electron microscopic and biochemical evidence that proline-β-naphthylamidase is composed of three identical subunits

Electron microscopy of pig intestinal proline-β-naphthyltamidase revealed that the enzyme is composed of 3 subunits, which are assembled in a trifoliolate shape, At pH 4.5 and 4°C, the enzyme dissociates reversibly into active subunits in 4h. Dissociation also occurs at higher pHs when the enyzme concentration is very low. The activity per mg protein of the native, trimeric enzyme is about 2.5-fold higher than that of the dissociated enzyme.     

Sequences homologous to Tc(s) transposable elements ofCaenorhabditis elegans are widely distributed in the phylum nematoda

Summary To have a better understanding of the evolutionary history of mobile elements within the nematodes, we examined the distribution and the conservation of homologues to transposable elements fromCaenorhabditis elegans (Tc1, Tc2, Tc3, Tc4, Tc5, and FB1) in 19 nematode species belonging to the class Secernentea. Our results show that Tc1 elements display a distribution restricted to the family Rhabditidae with poor conservation. The Tc2 and FB1 homologous elements have the same patchy distribution within the Rhabditidae. They were only found inCaenorhabditis and inTeratorhabditis. The Tc3 element is widely distributed among nematode species. Tc3 homologous elements are present in the majority of the Rhabditidae but also in two genera within the family Panagrolaimidae, and inBursaphelenchus, which belongs to the order Aphelenchida. Tc4 and Tc5 homologues show the most limited distribution of all tested elements, being strictly limited toC. elegans. These data indicate that in some cases, the distribution of transposable elements in the nematode cannot be explained by strict vertical transmission. The distribution of Tc3, Tc4, and Tc5 suggests that horizontal transmission may have occurred between reproductively isolated species during their evolutionary history.     

Molecular considerations in the evolution of bacterial genes

Summary Synonymous and nonsynonymous substitution rates at the loci encoding glyceraldehyde-3-phosphate dehydrogenase (gap) and outer membrane protein 3A (ompA) were examined in 12 species of enteric bacteria. By examining homologous sequences in species of varying degrees of relatedness and of known phylogenetic relationships, we analyzed the patterns of synonymous and nonsynonymous substitutions within and among these genes. Although both loci accumulate synonymous substitutions at reduced rates due to codon usage bias, portions of thegap andompA reading frames show significant deviation in synonymous substitution rates not attributable to local codon bias. A paucity of synonymous substitutions in portions of theompA gene may reflect selection for a novel mRNA secondary structure. In addition, these studies allow comparisons of homologous protein-coding sequences (gap) in plants, animals, and bacteria, revealing differences in evolutionary constraints on this glycolytic enzyme in these lineages.     

Strand symmetry of mutation rates in theβ-globin region

Summary It has been suggested that there may be inequalities in the types of substitution on the two DNA strands (in particular, in the frequencies of transversions from R to Y and from Y to R) due to a higher error rate on the lagging than the leading strand during replication. Reexamination of 11 kb of the -globin region sequenced in six primates fails to confirm this suggestion. Examination of the 73-kb -globin region sequenced in humans shows that the frequency of pyrimidines in different parts of this region is more variable than expected in a random sequence, but the pattern is more consistent with nonrandomness generated by DNA turnover mechanisms than with strand asymmetry due to a higher error rate on the lagging strand.