Cellular stabilization of the melatonin rhythm enzyme induced by nonhydrolyzable phosphonate incorporation

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) controls daily changes in the production and circulating levels of melatonin. Here, the significance of the phosphorylation of AANAT was studied using a semisynthetic enzyme in which a nonhydrolyzable phosphoserine/threonine mimetic, phosphonomethylenealanine (Pma), was incorporated at position 31 (AANAT-Pma31). The results of studies in which AANAT-Pma31 and related analogs were injected into cells provide the first direct evidence that Thr31 phosphorylation controls AANAT stability in the context of the intact cells by binding to 14-3-3 protein. These findings establish Thr31 phosphorylation as an essential element in the intracellular regulation of melatonin production. The application of Pma in protein semisynthesis is likely to be broadly useful in the analysis of protein serine/threonine phosphorylation.     

Distribution of cells bearing B-cell alloantigen(s) in North Indian rheumatic fever/rheumatic heart disease patients

Numerous investigators have developed monoclonal antibodies against B-cell alloantigen(s) of rheumatic fever. However, the developed monoclonals do not have the same significance in all the populations. We have developed a battery of monoclonals against B-cell alloantigens of North Indian rheumatic fever patients. In the present study, we have used these monoclonals to examine the frequency of rheumatic antigens in 30 patients with recurrence of rheumatic activity (RRA), 30 of rheumatic heart disease (RHD) patients and 50 controls using alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. These patients were examined at the time of registry and after three months follow up. RRA patients showed higher percentage of lymphocyte positive as compare to RHD and controls. Interestingly, On follow-up RRA patients showed significant decline in positive lymphocyte as compare to first visit whereas no such change was observed in RHD patients. There were 90–93% of RRA and RHD patients positive with these monoclonals. A significant age variation of rheumatic cells was also noticed in all groups of rheumatic patients. We conclude that monoclonals raised from the same ethnic population are highly specific and cost effective to use them to develop an easy field test system such as APAAP, to identify the individual at risk, to develop rheumatic fever. It is also suggested that the alloantigen marker may persist through out life and gets activated after recurrence of the disease.     

Inhibition of Helicobacter pylori adherence by a peptide derived from neuraminyl lactose binding adhesin

Helicobacter pylori, like many other gut colonizing bacteria, binds to sialic acid rich macromolecules present on the gastric epithelium. NLBH (neuraminyl lactose binding haemagglutinin) a 32 kDa adhesin located on the surface of H. pylori has been shown to have specific affinity towards NeuAc2,3Gal1,4Gluc(3SL). This sialic acid moiety is over-expressed in an atrophic stomach undergoing parietal cell depletion. Antibodies against a lysine rich peptide fragment of NLBH inhibit agglutination of human erythrocytes. This lysine rich sequence from NLBH was proposed to be the receptor-binding site. In order to elucidate the binding of NLBH to gastric epithelium, a peptide (D-P-K-R-T-I-Q-K-K-S) was synthesized. A series of experiments were performed involving adherence inhibition assays, 2D-NMR, molecular modelling and measurement of modulation in acid secretion. Results indicated that the peptide fragment could be involved in receptor recognition, which is important for the binding of H. pylori to gastric epithelium. The binding is possibly through hydrogen bonding. Two lysines and a threonine residue seem to be within the hydrogen bonding distance of NeuAc2,3Gal1,4Gluc. Further, in vitro assays were performed to evaluate the role of the peptide on acid secretion by parietal cells isolated from human fundal biopsies. Interestingly, the peptide increases acid secretion only in H. pylori negative and in treated patients but not in H. pylori positive patients. This highlights the role of NLBH in acid secretion and could be of some consequence in the prognosis of the disease.     

Gastropathy and defense mechanisms in common bile duct ligated portal hypertensive rats

Portal hypertensive gastropathy is associated with a broad spectrum of gastric mucosal damage inspite of decreased gastric acid secretion, suggestive of compromised endogenous protective mechanisms. To determine the mechanisms of damage in portal hypertensive gastropathy we measured lipid peroxidation, glutathione, antioxidant and lysosomal enzymes in gastric mucosal homogenates from male Wistar rats with elevated intrasplenic pulp pressure, eighteen days after common bile duct ligation. Thiobarbituric acid-reactive substances and lysosomal enzymes (-glucuronidase and acid phosphatase) were increased in the common bile duct ligated group as compared to the sham-operated group. The levels of antioxidant defense enzymes, superoxide dismutase, glutathione peroxidase, catalase and glutathione were decreased as compared to the sham-operated controls. Pre-operative vitamin E administration decreased mucosal lipid peroxidation increased the levels of antioxidant defense enzymes and lowered the lysosomal enzymes. The plasma vitamin E levels in this group were lower when compared to animals receiving it post-operatively. In conclusion, free radical and lysosomal enzyme mediated damage may play a role in portal hypertensive gastropathy.     

Triterpenoid saponins from the roots of tea plant (Camellia sinensis var. assamica)

Three olean-12-ene type triterpenoid saponins, named TR-saponins A, B and C, were isolated as methyl esters from tea roots (Camellia sinesis var. assamica) after treatment with diazomethane. Their structures were established as the methyl esters of 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-21, 22-di-O-angeloyl-R1-barrigenol-23-oic acid, 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-21-O-angeloyl-22-O-2-me thylbutanoyl-R1- barrigenol-23-oic acid and 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-16 alpha-O-acetyl-21-O-angeloyl-22-O-2-methylbutanoyl-R1-bar rigenol-23-oic acid, by extensive 1D and 2D-NMR as well as FABMS and HR-MS analyses.     

Sleep-Dependent Reactivation of Ensembles in Motor Cortex Promotes Skill Consolidation

Despite many prior studies demonstrating offline behavioral gains in motor skills after sleep, the underlying neural mechanisms remain poorly understood. To investigate the neurophysiological basis for offline gains, we performed single-unit recordings in motor cortex as rats learned a skilled upper-limb task. We found that sleep improved movement speed with preservation of accuracy. These offline improvements were linked to both replay of task-related ensembles during non-rapid eye movement (NREM) sleep and temporal shifts that more tightly bound motor cortical ensembles to movements; such offline gains and temporal shifts were not evident with sleep restriction. Interestingly, replay was linked to the coincidence of slow-wave events and bursts of spindle activity. Neurons that experienced the most consistent replay also underwent the most significant temporal shift and binding to the motor task. Significantly, replay and the associated performance gains after sleep only occurred when animals first learned the skill; continued practice during later stages of learning (i.e., after motor kinematics had stabilized) did not show evidence of replay. Our results highlight how replay of synchronous neural activity during sleep mediates large-scale neural plasticity and stabilizes kinematics during early motor learning.     

Mutation Spectrum of RB1 Gene in Unilateral Retinoblastoma Cases from Tunisia and Correlations with Clinical Features

Retinoblastoma, an embryonic neoplasm of retinal origin, is the most common primary intraocular malignancy in children. Somatic inactivation of both alleles of the RB1 tumor suppressor gene in a retinal progenitor cell through diverse mechanisms including genetic and epigenetic modifications, is the crucial event in initiation of tumorigenesis in most cases of isolated unilateral retinoblastoma. We analyzed DNA from tumor tissue and from peripheral blood to determine the RB1 mutation status and seek correlations with clinical features of 37 unrelated cases of Tunisian origin with sporadic retinoblastoma. All cases were unilateral except one who presented with bilateral disease, in whom no germline coding sequence alteration was identified. A multi-step mutation scanning protocol identified bi-allelic inactivation of RB1 gene in 30 (81%) of the samples tested. A total of 7 novel mutations were identified. There were three tumors without any detectable mutation while a subset contained multiple mutations in RB1 gene. The latter group included tumors collected after treatment with chemotherapy. There were seven individuals with germline mutations and all presented with advanced stage of tumor. There was no difference in age of onset of RB based on the germline mutation status. Thus 20% of the individuals with sporadic unilateral RB in this series carried germline mutations and indicate the importance of genetic testing all children with sporadic retinoblastoma. These findings help to characterize the spectrum of mutations present in the Tunisian population and can improve genetic diagnosis of retinoblastoma.     

A density functional study towards substituent effects on anion sensing with urea receptors

Effects of substituents on anion binding in different urea based receptors have been examined using density functional (B3LYP/6-311+G**) level of theory. The complexes formed by a variety of substituted urea with a halide anion (fluoride) and an oxy-anion (acetate) have been calculated. The stronger complexes were predicted for receptors with fluoride ion than that of acetate ion, however, in water the preference was found to be reversed. The pK _a calculations showed the preferred sites of deprotonation for positional isomers, while interacting with anions. The position of the substituent in the receptor, however, could change the preferred sites of deprotonation compared to the site predicted with pK _a values.