Plasmodium gallinaceum: critical role for microtubules in the transformation of zygotes into Ookinetes

The role of microtubules and microfilaments in the transformation of spherical zygotes of Plasmodium gallinaceum (avian malaria parasite) into vermiform ookinetes has been studied by using specific drugs (taxol, colchicine, and cytochalasin-B). Both taxol and colchicine completely abolished the transformation of zygotes into ookinetes. The inhibitory effect was seen only if the drugs were added during the initial 6 hr of total time (20-24 hr) required for complete transformation; the addition of drugs after 6-8 hr of initiation of transformation had no effect. Electron microscopy revealed that microtubules were depolymerized by colchicine treatment, whereas in taxol-treated cells there was an extensive array of cytoplasmic and nuclear microtubules which appeared to be clumped in bundles. In contrast to the effects of taxol and colchicine, cytochalasin-B, which affects the microfilament system, had no effect on the transformation. Protein synthesis and expression of two ookinete-specific surface proteins were not affected in the drug-inhibited parasites. Zygotes treated with taxol for 4 hr at room temperature failed to develop into oocysts when they were subsequently fed to mosquitoes. These studies demonstrate a critical role for microtubules in the initial stages of transformation of zygotes into ookinetes.     

c-Ha-ras-1 proto-oncogene amplification and overexpression during the limited replicative life span of normal human fibroblasts

The cellular proto-oncogene c-Ha-ras-1 undergoes up to 4-fold amplification during the limited replicative life span of normal human diploid fibroblasts in vitro. Levels of c-Ha-ras-1 messenger RNA and its p21 protein product are correspondingly elevated. Cellular proto-oncogene amplification and overexpression, although frequently associated with tumorigenesis, may thus occur during normal cellular growth.     

Synthesis and biological activity of 3 beta-fluorovitamin D3: comparison of the biological activity of 3 beta-fluorovitamin D3 and 3-deoxyvitamin D3

The alteration in the biologic activity of the vitamin D3 molecule resulting from the replacement of a hydrogen atom with a fluorine atom is a subject of fundamental interest. To investigate this problem we synthesized 3 beta-fluorovitamin D3 6 and its hydrogen analog, 3-deoxyvitamin D3 7, and tested the biologic activity of each by in vitro and in vivo methods. Contrary to previous reports which showed that 3 beta-fluorovitamin D3 was as active as vitamin D3 in vivo, we found that the fluoro-analog was less active than vitamin D3. With regard to stimulation of intestinal calcium transport and bone calcium mobilization in the D-deficient hypocalcemic rat, 3 beta-fluorovitamin D3 showed significantly greater biologic activity than its hydrogen analog, 3-deoxyvitamin D3. In the organ-cultured, embryonic chick duodenum, 3 beta-fluorovitamin D3 was approx 1/1000th as active as the native hormone, 1,25-dihydroxyvitamin D3, while 3-deoxyvitamin D3 was inactive even at microM concentrations, in the induction of the vitamin D-dependent, calcium-binding protein. With regard to in vitro activity in displacing radiolabeled 25-hydroxyvitamin D3 from vitamin D binding protein and radiolabelled 1,25-dihydroxyvitamin D3 from a chick intestinal cytosol receptor, 3 beta-fluorovitamin D3 and 3 beta-deoxyvitamin D3 both showed very poor binding efficiencies when compared with vitamin D3. Our results show that the substitution of a fluorine atom for a hydrogen atom at the C-3 position of the vitamin D3 molecule results in a fluorovitamin 6 with significantly more biological activity than its hydrogen analog, 3-deoxyvitamin D3 7.     

Synthesis and biologic activity of 3 beta-thiovitamin D3

We synthesized 3 beta-thiovitamin D3 from 7-dehydrocholesterol and tested its biological activity and protein binding properties. The thiovitamin was found to be a weak vitamin D agonist at high doses in vivo. It was poorly bound by both vitamin D-binding protein as well as by the intestinal cytosol receptor for 1,25-dihydroxyvitamin D. It did not increase the synthesis of calcium binding protein in the chick embryonic duodenum and did not block the activity of 1,25-dihydroxyvitamin D3 in this system. We conclude that 3 beta-thiovitamin D3 is a weak vitamin D agonist in vivo with no agonist activity or antagonist activity to 1,25-dihydroxyvitamin D3 in the chick embryonic duodenum.     

A physical and genetic map of the IncFI plasmid ColV2-K94

A restriction enzyme map of the IncFI plasmid ColV2-K94 was generated using EcoRI, BamHI, HindIII, and XhoI; the genetic features of this element were then mapped from previous heteroduplex studies.     

A method for the controlled cleavage of disulfide bonds in proteins in the absence of denaturants

A simple method was developed for the controlled cleavage of protein disulfide bonds and the simultaneous blockage of the free sulfhydryl groups in the absence of a denaturant. The disulfide bonds of bovine serum albumin were cleaved unsymmetrically at pH 7.0 using 0.1 M sulfite in 0.1 M phosphate buffer and the free sulfhydryl groups formed were sulfonated in an oxidation-reduction cycle using molecular oxygen and 400 microM cupric sulfate as a catalyst. The reaction was affected by cupric ion concentration, sulfite concentration, reaction pH and temperature. The standardized method was successfully used to cleave the disulfide bonds of other proteins pepsin, trypsin, and chymotrypsin. The method is reliable and can be used for achieving progressive cleavage of disulfide bonds in proteins without employing a denaturant.     

Carbohydrates,lipids and lipoproteins and islet changes in diabetes with superimposed myocardial infarction

Short-term metabolic and concomitant morphologic effects of streptozotocin diabetes on isoproterenol-induced myocardial infarction was studied in Wistar rats, Of particular significance was the observation that myocardial infarction in concert with diabetes brought about a distinctive exacerbation of the severity and complexity of the histopathological lesions. Of all the biochemical parameters, serum glucose and free fatty acids registered maximum elevation and serum lactate and cardiac glycogen levels a maximum reduction. Among the lipoproteins, an inverse relationship was found between high density lipoproteins and low density and very low density lipoproteins; while high density lipoproteins, ratio of high density lipoprotein to low density lipoprotein and the percentage of high density lipoprotein were decreased, there was a significant increase in low density lipoprotein concentration and percentage values of low density and very low density lipoproteins. In diabetes, the B cell of the endocrine pancreas depicted selective necrosis. Loss of insulin granules and wide-spread necrobiosis of cellular elements of the pancreatic islets were observed, respectively, in myocardial infarction and in diabetes plus myocardial infarction combinations. Pathological evidence of chemical-induced mild toxicity was present in the exocrine parenchyma. Mitotic features and the presence of centroacinar cells in the damaged Langerhans’ islets supposedly formed the basis of regeneration of the tissue in diabetes, with or without vascular complications     

Effect of Centchroman (67/20) and 78/224 on nucleic acid and protein biosynthesis during implantation in rats

The biosynthesis of nucleic acids and proteins was studied in rat uterus by following the incorporation of [³H]-thymidine, [³H]-uridineand[¹⁴C]-leucinein control and pregnant rats in the presence and absence of two anti-implantation drugs. One of the drugs, 78/224 caused a significant increase in incorporation whereas the other drug, Centchroman, caused an inhibition in incorporation of all the three precursors. The implications of these changes in the light of estrogenicity, agonist and antagonist actions of anti-estrogens have been analysed. The importance of homeostatic mechanisms involved in nucleic acids and proteins for the maintenance of constant internal milieu for blastocyst attachment has been discussed.