Kinetics of deactivation for thermostable DNA polymerase enzymes

Summary The kinetics of thermal deactivation for thermostable DNA polymerase enzymes were investigated by using the experimental data published elsewhere (Nielson et al. 1996. Strategies. 9, 7–8). The order of deactivation (a) and the deactivation rate constants (k _d) were determined for different Taq DNA polymerase enzymes and were found to be of first order.     

HIV-1 Vpr Modulates Macrophage Metabolic Pathways: A SILAC-Based Quantitative Analysis

Human immunodeficiency virus type 1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Furthermore, these macrophages are resistant to cell death and are viral reservoir. However, the impact of Vpr on the macrophage proteome is yet to be comprehended. The goal of the present study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. Cultured human monocytic cells, U937, were differentiated into macrophages and transduced with adenovirus construct harboring the Vpr gene. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. Quantified proteins were selected and clustered by biological functions, pathway and network analysis using Ingenuity computational pathway analysis. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. In addition, the proteomic data demonstrate down regulation of some key mitochondrial enzymes such as glutamate dehydrogenase 2 (GLUD2), adenylate kinase 2 (AK2) and transketolase (TKT). Based on these observations we postulate that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehyrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. Furthermore, dysregulation of mitochondrial glutamate metabolism in macrophages can contribute to neurodegeneration via neuroexcitotoxic mechanisms in the context of NeuroAIDS.     

Effects of starvation,chilling, and injury on endocrine gland function in Galleria mellonella

Starvation, chilling, and injury of last instar Galleria mellonella larvae typically elicit extra larval molts or a delay in pupation. The primary sites of action and the nature of the signals by which these treatments affect development are not known. However, since the connections of the brain to the nerve cord are crucial for the effects of starvation and chilling, these signals apparently affect the brain-centered program of developmental regulation via the nerve cord. Chilling, and occasionally starvation, cause extra larval molts in last instar larvae treated prior to the nervous inhibition of their corpora allata; release of a cerebral allatotropin, which stimulates the production of juvenile hormone, appears to be involved in this effect. After this time, a delay in pupation is the principal effect of starvation and chilling, and is apparently due to a temporal inhibition of the release of the prothoracicotropic hormone. Chilling also appears to inhibit unstimulated ecdysteroid production by the prothoracic glands. The effect of injury is not mediated by the nerve cord, but appears to involve an inhibitory humoral factor that affects either the brain or the prothoracic glands themselves. Injury also stimulates juvenile hormone production, an effect which is enhanced when the brain is separated from the nerve cord and which is evidenced by a delay of ecdysis and the occasional retention of some larval features in the ecdysed insects. None of the effects of these various treatments on the brain and the endocrine glands persist when the brains or glands are implanted into untreated hosts.     

The effect of oxidants on biomembranes and cellular metabolism

During the reductive process in the tissues, the aerobes generate a number of oxidants. Unless these oxidants are reduced, oxidative damage and cell death would occur. Oxidation of plasma membrane lipids leads to autocatalytic chain reactions which eventually alter the permeability of the cell. The role of oxidative damage in the pathophysiology of diabetic complications and ischemic reperfusion injury of myocardium, especially the changes in the channel activity which may lead to arrhythmia have been studied. Hyperglycemia activates aldose reductase which could efficiently reduce glucose to sorbitol in the presence of NADPH. Since NADPH is also aldose required by glutathione reductase for reducing oxidants, its diversion would lead to membrane lipid oxidation and permeability changes which are probably responsible for diabetic complications such as cataractogenesis, retinopathy, neuropathy etc. Antioxidants such as butylated hydroxy toluene (BHT) and also reductase inhibitors prevent or delay some of these complications. By using patch-clamp technique in isolated frog myocytes, we have shown that hydroxy radicals generated by ferrous sulfate and ascorbate as well as lipid peroxides such as t-butyl hydroperoxide facilitate the entry of Na⁺ by oxidizing Na⁺-channels. Increased intracellular Na⁺ leads to an increase in Na⁺/Ca²⁺ exchange. The increased Na⁺ concentration by itself may produce electrical disturbance which would result in arrhythmia. Increased Ca²⁺ may affect proteases and may help in the conversion of xanthine dehydrogenase to xanthine oxidase, consequently increased production of super oxide radicals. Increased membrane lipid peroxidation and other oxygen free-radical associated membrane damage in myocytes has been demonstrated.     

Influence of prolactin on neural and glial cellular adenosine triphosphatases in immature male bonnet monkeys (Macaca radiata, Geoffroy)

The effect of prolactin on specific activities of adenosine triphosphatases (ATPases) in neural and gliar cells of cerebral cortex, cerebellum and pons-medulla of immature male bonnet monkeys was studied. Na+, K+ dependent ATPase was stimulated, while Mg2+ and Ca2+ dependent ATPase activities showed reduction in neural as well as glial cells of cerebral cortex and cerebellum. However, in pons-medulla, Na+, K+ and Mg2+ dependent ATPases showed the same trend in neural and glial cells, respectively, as in the other two regions. The data obtained reveal that prolactin has specific effect on different ATPases, in different regions of the brain.     

Quantitative estimation of the pathways followed in the conversion to glycogen of glucose administered to the fasted rat

When [6-3H,6-14C]glucose was given in glucose loads to fasted rats, the average 3H/14C ratios in the glycogens deposited in their livers, relative to that in the glucoses administered, were 0.85 and 0.88. When [3-3H,3-14C]lactate was given in trace quantity along with unlabeled glucose loads, the average 3H/14C ratio in the glycogens deposited was 0.08. This indicates that a major fraction of the carbons of the glucose loads was converted to liver glycogen without first being converted to lactate. When [3-3H,6-14C]glucose was given in glucose loads, the 3H/14C ratios in the glycogens deposited averaged 0.44. This indicates that a significant amount of H bound to carbon 3, but not carbon 6, of glucose is removed within liver in the conversion of the carbons of the glucose to glycogen. This can occur in the pentose cycle and by cycling of glucose-6-P via triose phosphates: glucose----glucose-6-P----triose phosphates----glucose-6-P----glycogen. The contributions of these pathways were estimated by giving glucose loads labeled with [1-14C]glucose, [2-14C]glucose, [5-14C]glucose, and [6-14C]glucose and degrading the glucoses obtained by hydrolyzing the glycogens that deposited. Only a few per cent of the glucose carbons deposited in glycogen were deposited in liver via glucose-6-P conversion to triose phosphates. Between 4 and 9% of the glucose utilized by the liver was utilized in the pentose cycle. While these are relatively small percentages, since three NADP3H molecules are formed from each molecule of [3-3H]glucose-6-P utilized in the cycle, a major portion of the difference between the ratios obtained with [3-3H]glucose and with [6-3H]glucose is attributable to metabolism in the pentose cycle. Because 3H of [3-3H]glucose is extensively removed during the conversion of the glucose to glycogen within liver the extent of incorporation of the 3H into liver glycogen is not the measure of glucose's metabolism in other tissues before its carbons are deposited in liver glycogen. The distributions of 14C from the 14C-labeled glucoses into the carbons of the liver glycogens mean that at a minimum about 30% of the carbons of the glucose deposited in the glycogen were first converted to lactate or its metabolic equivalent.     

A study of diabetes in relation to blood groups and cholesterol levels

Summary Four hundred and sixteen patients suffering from diabetes were studied for AB0 Rhesus blood groups, to find out whether there is any association between blood group and diabetes. Though the relative incidence was found to be higher in blood group 0 and B, the difference was not statistically significant. This study confirms the earlier findings of high cholesterol levels in diabetes, particularly in patients with blood groups A and AB, and the overall mean cholesterol level as 253 mg/100 ml. This study also shows that diabetes with a cholesterol level of more than 300 mg/100 ml is a coronary risk factor.

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Zusammenfassung 416 Patienten mit Diabetes wurden nach AB0 und Rh untersucht, um Assoziationen zwischen Blutgruppe und Diabetes zu finden. Es fand sich ein leichtes, nicht signifikantes Überwiegen von 0 und B. Ein besonders hoher Serum-Cholesteral-Spiegel fand sich beim Diabetes insbesondere bei Patienten der Gruppen A und AB. Die Studie zeigt auch, daß ein Cholesteral-Spiegel von mehr als 300 mg% ein Risikofaktor für Coronarerkrankungen ist.

     

The transport of oxidized glutathione from the erythrocytes of various species in the prescence of chromate

1. Erythrocytes from normal and glucose 6-phosphate dehydrogenase-deficient humans were subjected to hydrogen peroxide diffusion to oxidize the GSH. Studies were carried out in the presence and absence of chromate to inhibit glutathione reductase and with or without the addition of glucose. 2. The GSH content of erythrocytes from other species was oxidized by subjecting them to hydrogen peroxide diffusion in the presence of chromate and glucose. 3. Chromate (1.3mm) inhibited glutathione reductase by about 80%, whereas glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, hexokinase, phosphofructokinase and pyruvate kinase were not inhibited. 4. The GSSG formed was transported from the erythrocytes to the medium. 5. The transport rate of GSSG from glucose 6-phosphate dehydrogenase-deficient erythrocytes subjected to hydrogen peroxide diffusion in the presence of chromate was comparable with that from normal and glucose 6-phosphate dehydrogenase-deficient erythrocytes. 6. The rate of transport of GSSG from erythrocytes of various species studied could be ranked: pigeon>rabbit>rat>donkey>man>dog>horse>sheep>chicken>fish.     

MORPHOLOGICAL AND EMBRYOLOGICAL STUDIES ON THE LEMNACEAE. I. THE FLORAL STRUCTURE AND GAMETOPHYTES OF LEMNA PAUCICOSTATA

Maheshwari, Satish C., and R. N. Kapil. (U. Delhi, Delhi, India.) Morphological and embryological studies on the Lemnaceae. I. The floral structure and gametophytes of Lemna paucicostata. Amer. Jour. Bot. 50(7): 677–686. Illus. 1963.—In Lemna paucicostata, a locally occurring member of the Lemnaceae, the plant body is represented by a frond which is devoid of lignified elements. The root shows a winged root sheath but does not have root hairs. There are no distinctive layers like the endodermis and xylem. The male archesporium is hypodermal and differentiates normally as in other angiosperms into parietal and sporogenous layers. The tapetum is single-layered and plasmodial. The partition walls of the anther are not derived by sterilization of the sporogenous cells as believed earlier. The microspore tetrads are isobilateral and decussate, the meiotic divisions being successive. The pollen grains are shed at the 3-celled stage. The ovary contains a single hemianatropous, bitegminal and crassinucellar ovule. The development of the embryo sac conforms to the Allium type.     

High Frequency Divisions in Leaf Base Protoplasts of Wheat (Triticum aestivum L.)

Protoplasts were isolated from the basal meristematic region of leaves from 6-day-old seedlings of wheat (Triticum aestivum). Protoplasts divided when cultured on MS medium (as agarose beads) in presence of nurse tissue. Donor seedlings when grown on BAP-supplemented MS medium were found to be considerably superior for protoplast isolation and culture than when grown on MS basal medium, in terms of protoplast viability, cell wall formation and cell division frequency. In addition, reduction of ammonium content of the culture medium, together with a dark Incubation, led to a high protoplast division frequency of about 70%. Microcolonies of 10-to 12-celled stages were obtained in Triticum aestivum, varieties HD 2329, HD 2285, Kalyan Sona, Arjun and CPAN 1676.