Effects of prostaglandin E1, isoproterenol and forskolin on cyclic AMP levels and tension in rabbit aortic rings

The role of cyclic AMP in the control of vascular smooth muscle tone was studied by monitoring the effects of prostaglandin E1 (PGE1), isoproterenol and forskolin on cyclic AMP levels and tension in rabbit aortic rings. PGE1, isoproterenol and forskolin all increased cyclic AMP levels in rabbit aortic rings. Isoproterenol and forskolin relaxed phenylephrine-contracted aortic rings, but PGE1 contracted the rings in the presence or absence of phenylephrine. Isoproterenol relaxed these PGE1-contracted aortic rings without further change in total cyclic AMP levels, which were already elevated by the PGE1 alone. Pretreatment with forskolin potentiated the effects of PGE1 on cyclic AMP levels. PGE1 caused contractions in muscles partially relaxed by forskolin, even though very large increases in cyclic AMP levels (30 fold) were produced by PGE1 in the presence of forskolin. Isoproterenol was able to relax these forskolin-treated, PGE1-contracted muscles with no further increase in cyclic AMP levels. Thus, there does not appear to be a good correlation between total tissue levels of cyclic AMP and tension in these experiments. Our results suggest that, if cyclic AMP is responsible for relaxation of smooth muscle, some form of functional compartmentalization of cyclic AMP must exist in this tissue.     

Irreversible platelet aggregation does not depend on lipoxygenase metabolites

Previous investigations in our laboratory demonstrated the existence of an intrinsic mechanism, termed membrane modulation, capable of restoring sensitivity to aspirin treated platelets, resulting in irreversible aggregation in response to arachidonic acid (AA). The mechanism underlying correction of aspirin induced inhibition of platelet function, however, was not clear. In the present study we have evaluated the role of lipoxygenase (LO) metabolites of AA in securing irreversible aggregation of drug induced cyclooxygenase (CO) deficient platelets. Platelets treated with aspirin or Ibuprofen did not convert radiolabeled AA to thromboxane, but generated significant quantities of hydroxy acids via the LO pathway. However, drug exposed platelets, when stirred with epinephrine first and then challenged with AA, aggregated irreversibly. Eicosatetraynoic acid (ETYA 1, U53119) inhibited AA conversion by the LO pathway, whereas 5,8,11,14-eicosatetraynoic acid (ETYA 2) inhibited AA conversion by both CO and LO enzymes. Yet, at the inhibitory concentration these fatty acids failed to prevent AA induced irreversible aggregation of CO deficient, alpha adrenergic receptor stimulated platelets. Results of four studies show that the generation of LO metabolites of AA are not essential for securing irreversible aggregation of platelets.     

Effects of isoproterenol and forskolin on tension, cyclic AMP levels, and cyclic AMP dependent protein kinase activity in bovine coronary artery

The effects of isoproterenol and forskolin on tension, cyclic AMP levels, and cyclic AMP dependent protein kinase activity were compared in helical strips of bovine coronary artery. Elevation of cyclic AMP and activation of the protein kinase appeared to be well correlated with relaxation of potassium-contracted arteries by isoproterenol. Forskolin, at 1 microM or higher concentrations, also markedly elevated cyclic AMP levels, activated the kinase, and relaxed the arteries. However, a lower concentration of forskolin (0.1 microM) caused significant increases in both cyclic AMP levels and cyclic AMP dependent protein kinase activity, but did not relax the muscles. Relaxation caused by isoproterenol was accompanied by an apparent translocation of cyclic AMP dependent protein kinase activity from the soluble to the particulate fraction in these preparations. A similar shift in the distribution of the kinase was caused by various concentrations of forskolin, irrespective of whether the arteries were relaxed or not. In contrast to previous results in other tissues, low concentrations of forskolin (less than or equal to 1 microM), which themselves markedly elevated cyclic AMP levels in the arteries, did not potentiate the effects of isoproterenol on cyclic AMP levels or tension in these preparations. These results suggest that either cyclic AMP is not solely responsible for the relaxation caused by these agents, or some form of functional compartmentalization of cyclic AMP and cyclic AMP dependent protein kinase exists in this tissue.     

Oxidative stress and the development of diabetic complications - antioxidants and lipid peroxidation in erythrocytes and cell membrane.

Erythrocytes isolated from 131 cases of Non-Insulin Dependent Diabetes Mellitus (NIDDM) were studied for lipid peroxidation, antioxidant defences, and the maximum peroxidisable substrate in the cell membrane. Antioxidant defences are lowered in NIDDM, followed by significant rise in lipid peroxidation products. However, in the erythrocyte membrane, the total polyunsaturated peroxidisable lipids are lower than in normal erythrocytes which may be a causative factor affecting the survival of the cells.     

In vitro effect of prostaglandins,prostaglandin endoperoxides and thromboxane on rat intestinal alkaline phosphatase and (Ca2+-Mg2+) adenosine triphosphatase

The activity of alkaline phosphate and²⁺-Mg²⁺ adenosine triphosphatase, two of the enzymes involved in limpid and calcium uptake across the intestinal membrane, were increased in experimental atherosclerosis. Administration ofAnnapavala sindhooram, an antiatherosclerotic drug, lowers these enzyme levels to near normal values. Prostaglandin E2 stimulated the enzyme activitiesin vitro, while prostaglandin endoperoxide inhibited the activity. Thromboxane and other prostaglandins had no effect on the enzyme activities. Addition of the antiatherosclerotic drug to thein vitro assay system reversed the effect of both prostaglandin E₂ and prostaglandin endoperoxide.     

Changes in carbohydrates and lipids during embryonic development ofAntheraea mylitta (Lepidoptera)

Changes in carbohydrate and lipid metabolism during embryonic development inAntheraea mylitta were studied. While carbohydrates were metabolized during early embryogenesis, lipids were catabolised at the later stages. A significant increase in both total carbohydrates and glycogen on days 5 and 6 suggested the concurrent occurrence of both gluconeogenesis and glycogenesis. As the development of the embryo proceeds, both lipids and carbohydrates were utilised, resulting in the increase in the concentration of citrate, pyruvate and lactate.     

Major vault protein (MVP) gene polymorphisms and drug resistance in mesial temporal lobe epilepsy with hippocampal sclerosis

The human major vault protein (MVP) has been implicated in the development of drug resistance in cancer cells. Over expression of MVP has also been reported in brain tissue samples from antiepileptic drug (AED)-resistant human focal epilepsies. To investigate the relationship between single nucleotide polymorphisms (SNPs) involving the MVP gene and AED-resistance, we compared the distribution of three SNPs in the MVP gene, rs4788187, rs3815824 and rs3815823, among 220 patients with mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (prototype of AED-resistant epilepsy syndrome), 201 patients with juvenile myoclonic epilepsy (JME) (prototype of AED-responsive epilepsy syndrome) and 213 ethnically matched non-epilepsy controls. All the patients and controls were residents of the South Indian state of Kerala for more than three generations. We did not find any significant difference in allele and genotypic frequencies of the studied SNPs between AED-resistant and AED-responsive cohorts, and between AED-resistant and AED-responsive cohorts independently and pooled together when compared with the controls. We conclude that rs4788187, rs3815824, rs3815823 variants of the MVP gene are associated neither with predisposition for epilepsy nor with AED-resistance in the population that we have studied. Our results suggest the need for further research into the link between MVP and AED-resistance.     

Albirhodobacter marinus gen. nov., sp. nov., a member of the family Rhodobacteraceae isolated from sea shore water of Visakhapatnam, India

A novel marine, Gram-negative, rod-shaped bacterium, designated strain N9^T, was isolated from a water sample of the sea shore at Visakhapatnam, Andhra Pradesh (India). Strain N9^T was found to be positive for oxidase and catalase activities. The fatty acids were found to be dominated by C_16:0, C_18:1 ω7c and summed in feature 3 (C_16:1 ω7c and/or C_16:1 ω6c). Strain N9^T was determined to contain Q-10 as the major respiratory quinone and phosphatidylethanolamine, phosphatidylglycerol, two aminophospholipids, two phospholipids and four unidentified lipids as polar lipids. The DNA G+C content of the strain N9^T was found to be 63 mol%. 16S rRNA gene sequence analysis indicated that Rhodobacter sphaeroides, Rhodobacter johrii, Pseudorhodobacter ferrugineus, Rhodobacter azotoformans, Rhodobacter ovatus and Pseudorhodobacter aquimaris were the nearest phylogenetic neighbours, with pair-wise sequence similarities of 95.43, 95.36, 94.24, 95.31, 95.60 and 94.74 %, respectively. Phylogenetic analysis showed that strain N9^T formed a distinct branch within the family Rhodobacteraceae and clustered with the clade comprising species of the genus Pseudorhodobacter, together with species of the genera Roseicitreum, Roseinatronobacter, Roseibaca and Rhodobaca. Species of the genus Pseudorhodobacter are phylogenetically close with a 16S rRNA gene sequence dissimilarity of 5.9–7.3 % (92.7–94.1 % similarity). Based on the above-mentioned phenotypic characteristics and on phylogenetic inference, strain N9^T is proposed as a representative of a new genus and a novel species of the family Rhodobacteraceae as Albirhodobacter marinus gen. nov., sp. nov. The type strain of Albirhodobacter marinus is N9 (= MTCC 11277^T = JCM 17680^T).     

Late‐onset retinal degeneration pathology due to mutations in CTRP5 is mediated through HTRA1

Late‐onset retinal degeneration (L‐ORD) is an autosomal dominant macular degeneration characterized by the formation of sub‐retinal pigment epithelium (RPE) deposits and neuroretinal atrophy. L‐ORD results from mutations in the C1q‐tumor necrosis factor‐5 protein (CTRP5), encoded by the CTRP5/C1QTNF5 gene. To understand the mechanism underlying L‐ORD pathology, we used a human cDNA library yeast two‐hybrid screen to identify interacting partners of CTRP5. Additionally, we analyzed the Bruch's membrane/choroid (BM‐Ch) from wild‐type (Wt), heterozygous S163R Ctrp5 mutation knock‐in (Ctrp5^S163R/wt), and homozygous knock‐in (Ctrp5^S163R/S163R) mice using mass spectrometry. Both approaches showed an association between CTRP5 and HTRA1 via its C‐terminal PDZ‐binding motif, stimulation of the HTRA1 protease activity by CTRP5, and CTRP5 serving as an HTRA1 substrate. The S163R‐CTRP5 protein also binds to HTRA1 but is resistant to HTRA1‐mediated cleavage. Immunohistochemistry and proteomic analysis showed significant accumulation of CTRP5 and HTRA1 in BM‐Ch of Ctrp5^S163R/S163R and Ctrp5^S163R/wt mice compared with Wt. Additional extracellular matrix (ECM) components that are HTRA1 substrates also accumulated in these mice. These results implicate HTRA1 and its interaction with CTRP5 in L‐ORD pathology.