Degradation of apolipoprotein B-100 in human chylomicrons

The purpose of this study was to investigate the molecular forms of apolipoprotein B (ApoB) in human chylomicrons under well-preserved conditions. To this end, plasma and serum were collected from the same normal subjects after ingestion of a fatty meal. The samples were divided into three or four aliquots before the addition of various preservative mixtures, including antibiotics, antioxidants and proteinase inhibitors. The chylomicrons were isolated immediately, and all steps were carried out at or below 4 degrees C. Changes in the molecular weight of ApoB in chylomicrons were followed by a time study using 3.3% polyacrylamide gel electrophoresis containing SDS. ApoB from chylomicrons analyzed within 5 h of blood collection showed a single band with mobility identical to that of ApoB (ApoB-100) in low-density lipoproteins. When analyzed after 1-2 days, satellite bands smaller than ApoB-100 were observed, and a very faint band with Mr 200,000 appeared, which comigrated with intestinal ApoB (ApoB-48). Upon storage, the molecular weight of ApoB was smaller in chylomicrons subjected to a higher number of reflotations than those in chylomicrons washed less frequently, suggesting that purified chylomicrons degrade faster. A longer storage time at 4 degrees C (i.e., 7 or 14 days) revealed a stepwise degradation of ApoB, yielding Mr 200,000 band as the prominent form. The degradation of ApoB-100 was slower when both proteinase inhibitors, leupeptin and epsilon-amino caproic acid, were employed, and the appearance of Mr 200,000 band was quicker when the chylomicrons were processed at higher temperature (15-25 degrees C) in the absence of a proteinase inhibitor. Immunoblotting shows that the segment removed from ApoB-100 was the carboxyl-terminal portion. These results suggest the possible presence of a proteinase(s), which copurified with chylomicrons, and which converts ApoB-100 from a large to a smaller molecular form. Although the stop codon has been discovered recently in intestinal ApoB mRNA, which explains the mechanism for direct synthesis of ApoB-48, apparently ApoB-100 is also synthesized in the intestine of all eight subjects studied here, and the ApoB-100 degrades to a form which is ApoB-48-like.     

Plant water relations as selection criteria for drought tolerance in mustard

Genotypes of mustard (B. juncea) were evaluated for concurrent changes in leaf water potential (Ψ), leaf osmotic potential (π), leaf turgor potential (P) and leaf relative water content (RWC) during moisture stress at reproductive stage of growth. The slope ‘b’ in the regression between Ψ and π varied from 0.43 to 0.97 and was positively correlated with P and RWC. The genotypes with ‘b’ around 0.7 were able to maintain P of about 0.5 MPa at Ψ of − 2.5 MPa and thus such value of ‘b’ seems to provide enough degree of tolerance against drought.     

Cell cycle dependent variation of calmodulin in Tetrahymena

The level of calmodulin (CaM), a ubiquitous calcium-binding protein of eukaryotic cells was determined at different phases of the cell cycle in a synchronized Tetrahymena population. It was found that the concentration of CaM at G1 was approximately half of the concentration of S and this 2 x G1 level of CaM was maintained through the G2 and M stages of the cell cycle. To ascertain the role of CaM in the initiation of DNA synthesis, the cells were treated with trifluoperazine (TFP), a CaM antagonist, and EGTA (Ca2+-chelator) at the G1/S boundary. It was found that DNA synthesis was inhibited in these drug-treated cells. The uptake of the nucleotide precursor was not affected in TFP and EGTA treated cells, thus excluding the possibility of alteration in the membrane transport properties. Treatment with TFP failed to inhibit the synchronous mitotic division in Tetrahymena. The existence of a variable content of CaM through the cell cycle of Tetrahymena was demonstrated, suggesting the possible involvement of this Ca2+-binding protein in the nuclear DNA replication process.     

The inhibitory effects of some adenosine analogues on transmitter release at the mammalian neuromuscular junction

An electrophysiological study was made of the effects of four adenosine analogues, 2-chloroadenosine (2-CIA), 5'-N-ethylcarboxamidoadenosine (NECA), L-N6-phenylisopropyladenosine (L-PIA), and 2-(p-methoxyphenyl)-adenosine (CV-1674) on neurotransmitter release in the mouse phrenic nerve - hemidiaphragm preparation. All four drugs decreased miniature end-plate potential frequency in a dose-dependent manner. Evoked transmitter release in the cut diaphragm preparation was depressed by 2-CIA and CV-1674 to a similar extent. The ability of theophylline to antagonize the inhibitory effect of CV-1674 on spontaneous transmitter release was also established. On the basis of these results, the rank order of potencies was: L-PIA greater than NECA greater than 2-CIA greater than CV-1674. A clear classification of receptor type could not be made, since the ratio of potencies of L-PIA and NECA was narrow. Different slopes of the concentration-effect curves for 2-CIA and CV-1674 compared with L-PIA and NECA suggest an additional component to simple agonist action in their overall effects.     

Molecular cloning of the cDNA for androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis

cDNA clones coding for two closely related androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis were selected by screening an epididymal cDNA library constructed in lambda gt 11 with affinity-purified antibody directed against the glycoproteins. The largest clone of 956 nucleotides provided coding information for a protein of 246 amino acids of which the first 19 residues comprise a putative signal peptide sequence which when cleaved would produce a mature protein of 227 residues and a molecular mass of 26 kDa. Confirmation of the identity of the clone was provided by a match between the amino acid sequence predicted from the cDNA sequence and the actual amino acid sequence determined for a tryptic peptide fragment of one of the pure glycoproteins. It is probable that the primary amino acid sequence of the two glycoproteins is identical. Northern blot and slot-blot analysis revealed that the mRNA for the glycoproteins is approximately 1250 nucleotides long and that the concentration of the mRNA in the epididymis is androgen-dependent. The glycoproteins and their mRNAs were unique to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from skin, brain, liver, kidney, heart, skeletal muscle and testis. Cross-reacting proteins of slightly smaller apparent molecular mass were detected in extracts of mouse and guinea-pig epididymis, but not rabbit or bull epididymis. Comparison with existing protein data bases revealed that the epididymal glycoproteins display significant sequence homology with yeast carboxypeptidase Y.     

Population genetics of glyoxalase I (GLO) polymorphism in India

Phenotype and gene frequency data are presented on the glyoxalase I (GLO) polymorphism in seven endogamous caste groups: Jat Sikh, Ramdasia Sikh, Ramgarhia Sikh, Khatri, Brahmin and Bania of Patiala district, and Jat Sikh of Faridkot district of Punjab, North-West India. Apparently, there is considerable heterogeneity in the frequency distribution of the GLO1 gene that varies from 0.168 in Bania to 0.287 in Brahmin. However, these differences are not statistically significant, and the overall GLO1 frequency in Punjab is well within the North Indian range.     

Effect of centchroman on tubal transport and preimplantation embryonic development in rats

A single oral administration of centchroman (1.25 mg/kg) to adult female rats within 24 h of mating induced slight acceleration in the rate of transport of embryos through the oviducts. The compound did not seem to produce any deleterious effect on preimplantation embryonic development since well organized and apparently normal embryos were collected from the genital tract up to Day 12 of pregnancy. The recovery rate of embryos from centchroman-treated rats was, however, significantly reduced after Day 4 of pregnancy. There was some stimulation in the rate of cleavage of embryos and morula to blastocyst transformation, but retardation in the shedding of the zona pellucida. The rate of blastocyst formation was not altered when 6-8-cell embryos collected from the oviducts of control rats were transferred to the uteri of control or centchroman-treated females. A delay in zona shedding was observed in the centchroman-treated recipients.     

Protection from quinidine or physostigmine against in vitro inhibition by sarin of acetylcholinesterase activity

We have studied the relative effectiveness of quinidine and physostigmine in protecting against the inhibition of acetylcholinesterase (AChE) by sarin, an organophosphate (OP) compound. The protective effects of these compounds were studied in vitro in both synaptosomal and soluble samples obtained from various regions of sarin-administered or control isolated, perfused canine brain. Although AChE activities in the sarin-administered brain were substantially lower than in the control brain, we observed regional differences in the AChE activity in both. The AChE in the control brain and the AChE remaining in sarin-administered brain had different susceptibilities to inhibition from OP compounds in vitro and, therefore, have different properties. Quinidine partially protected AChE from the inhibitory effects of sarin in vitro possibly by altering the sarin binding sites. Addition of sarin to physostigmine-treated control brain samples allowed partial recovery of the AChE activity. The protective effects of quinidine or physostigmine were lost when samples from sarin-administered brain were treated in vitro with these compounds and then again exposed to sarin. Therefore, both quinidine and physostigmine provided partial protection against the inhibitory effects of sarin in vitro if they were added prior to sarin.