Biomedical subjects
Publications and source records attributed to R Rai.
This report documents an unusal case of extranodal sinus histiocytosis (Rosai-Dorfman disease) which produced a solitary intracranial mass lesion that was diagnosed clinically and radiologically as a meningioma. Morphological features have been described and literature extensively reviewed.
UASNTR, the UAS responsible for nitrogen catabolite repression-sensitive transcriptional activation of many nitrogen catabolic genes in Saccharomyces cerevisiae, has been previously thought to operate only as a pair of closely related dodecanucleotide sites each containing the sequence GATAA at its core. Here we show that a single UASNTR the unrelated cis-acting element was TTTGTTTAC situated upstream of GLN1, while in another the cis-acting element was the one previously shown to bind the PUT3 protein. When a UASNTR site functions in combination with an unrelated site, the regulatory responses observed are a hybrid consisting of characteristics derived from both the UASNTR site and the unrelated site as well. These observations resolve several significant inconsistencies that have plagued studies focused on elucidation of the mechanisms involved in the global regulation of nitrogen catabolism.
The expression of many nitrogen catabolic genes decreases to low levels when readily used nitrogen sources (e.g., asparagine and glutamine) are provided in the growth medium; this physiological response is termed nitrogen catabolite repression (NCR). Transcriptional activation of these genes is mediated by the cis-acting element UASNTR and the trans-acting factor Gln3p. A second protein encoded by URE2 possesses the genetic characteristics of a negative regulator of nitrogen catabolic gene expression. A third locus, DAL80, encodes a repressor that binds to sequences required for Gln3p-dependent transcription and may compete with Gln3p for binding to them. These observations are consistent with an NCR regulatory pathway with the structure environmental signal-->Ure2p-->(Gln3p/Dal80p)-->UASNTR operation-->NCR-sensitive gene expression. If NCR-sensitive gene expression occurs exclusively by this pathway, as has been thought to be the case, then the NCR sensitivity of a gene's expression should be abolished by a ure2 delta mutation. This expectation was not realized experimentally; the responses of highly NCR-sensitive genes to ure2 delta mutations varied widely. This suggested that NCR was not mediated exclusively through Ure2p and Gln3p. We tested this idea by assaying GAP1, CAN1, DAL5, PUT1, UGA1, and GLN1 expression in single, double, and triple mutants lacking Gln3p, Dal80p, and/or Ure2p. All of these genes were expressed in the triple mutant, and this expression was NCR sensitive for four of the six genes. These results indicate that the NCR regulatory network consists of multiple branches, with the Ure2p-Gln3p-UASNTR pathway representing only one of them.
NIT2, a positive-acting regulatory protein in Neurospora crassa, activates the expression of a series of unlinked structural genes that encode nitrogen catabolic enzymes. NIT2 binding sites in the promoter regions of nit3, alc and lao have at least two GATA sequence elements. We have examined the binding affinity of the NIT2 protein for the yeast DAL5 wild-type upstream activation sequence UASNTR, which contains two GATA elements, and for a series of mutated binding sites, each differing from the wild-type site by a single base. Substitution for individual nucleotides within 5' or 3' sequences that flank the GATA elements had only modest effects upon NIT2 binding. In contrast, nearly all substitutions within the GATA elements almost completely eliminated NIT2 binding, demonstrating the importance of the GATA sequence for NIT2 binding. Four high-affinity binding sites for the NIT2 protein were found within a central region of the nit-2 gene itself.
Long-term potentiation (LTP) in the CA1 region of the hippocampus is widely believed to occur through a strengthening of efficacy of excitatory synapses between afferent fibers and pyramidal cells. An alternative mechanism of LTP, reduction of efficacy of synaptic inhibition, was examined in the present report. The present study demonstrates that the maintenance of LTP in the CA1 hippocampal subfield of guinea pigs is accompanied by impairment of type A gamma-aminobutyric acid (GABA) receptor function, particularly at apical dendritic sites of CA1 pyramidal cells. Enhanced excitability of GABAergic interneurons during LTP represents a strengthening of inhibitory efficacy. The net effect of opposite modifications of synaptic inhibition during LTP of CA1 pyramidal cells is an overall impairment of the strength of GABAergic inhibition, and disinhibition could contribute importantly to CA1 pyramidal cell LTP.
A total of 500 consecutive women (mean age 32.9 years; SD 5 years) presenting with a history of recurrent miscarriages (median 4; range 3-17) were investigated for the presence of antiphospholipid antibodies (APA), polycystic ovaries (PCO), hypersecretion of luteinizing hormone (LH) and chromosome abnormalities in order to detect an underlying cause of their pregnancy losses. All women had details of their previous reproductive history, investigations and treatment documented: 76% of the women had experienced only early pregnancy losses (miscarriage < 13 weeks gestation); 32% had a history of subfertility; and significant parental chromosome rearrangements were present in 3.6% of couples. An ultrasound diagnosis of PCO was made in 56% of women, 58% of whom were demonstrated to hypersecrete LH, based on early morning urinary LH analysis. Circulating APA were found in 14% of women. An underlying cause of recurrent miscarriage--genetic, endocrine or autoimmune--was found in > 50% of couples. Women in the latter two groups are being recruited to randomized treatment trials which are discussed.
Bleeding from duodenal varices, although rare, is often massive and life threatening. Duodenal varices are more common in extrahepatic portal venous obstruction. We report a patient with recurrent bleeding from duodenal varices, secondary to thrombosed portal vein, splenic vein, and mesocaval shunt, who was successfully managed by injection of thrombin.
In organotypic hippocampal slice cultures, kainate (KA) specifically induces cell loss in the CA3 region while N-methyl-D-aspartate induces cell loss in the CA1 region. The sensitivity of slice cultures to KA toxicity appears only after 2 weeks in vitro which parallels the appearance of mossy fibers. KA toxicity is potentiated by co-application with the GABA-A antagonist, picrotoxin. These data suggest that the excitotoxicity of KA in slice cultures is modulated by both excitatory and inhibitory synapses.
We demonstrate that expression of the UGA1, CAN1, GAP1, PUT1, PUT2, PUT4, and DAL4 genes is sensitive to nitrogen catabolite repression. The expression of all these genes, with the exception of UGA1 and PUT2, also required a functional GLN3 protein. In addition, GLN3 protein was required for expression of the DAL1, DAL2, DAL7, GDH1, and GDH2 genes. The UGA1, CAN1, GAP1, and DAL4 genes markedly increased their expression when the DAL80 locus, encoding a negative regulatory element, was disrupted. Expression of the GDH1, PUT1, PUT2, and PUT4 genes also responded to DAL80 disruption, but much more modestly. Expression of GLN1 and GDH2 exhibited parallel responses to the provision of asparagine and glutamine as nitrogen sources but did not follow the regulatory responses noted above for the nitrogen catabolic genes such as DAL5. Steady-state mRNA levels of both genes did not significantly decrease when glutamine was provided as nitrogen source but were lowered by the provision of asparagine. They also did not respond to disruption of DAL80.
Previously, we have reported that some guanidino-substituted alpha- and beta-aryl enol lactones I and II behaved as selective, mechanism-based inhibitors of some trypsin-like proteases (Rai, R.; Katzenellenbogen, J.A. J. Med. Chem., submitted). In this study, we describe the synthesis and kinetic evaluation of some related, guanidino-substituted enol lactones having greater conformational mobility and affording additional hydrogen-bonding sites at the active site. The alpha-aryl-substituted lactones 1 and 2, which have greater conformational mobility in the guanidinoaryl linkage than I, selectively inhibited the trypsin-like enzymes, and they were relatively poor inactivators of alpha-chymotrypsin and human neutrophil elastase (HNE). The iodo enol lactone 2 permanently inactivated trypsin, urokinase, tissue plasminogen activator, and plasmin, showing exceptionally high specificity in its interaction with trypsin and urokinase. The selectivity pattern exhibited by the closely related, conformationally less mobile alpha-aryl-substituted iodo lactone Ib, which was previously shown to be a selective suicide substrate of urokinase and plasmin, provides an interesting comparison. The alpha-benzamido-substituted lactones 3 and 4, which afford an additional site for active-site hydrogen bonding, were found to be very potent alternate substrate inhibitors of trypsin and urokinase. In addition, the iodo lactone 4 permanently inactivated alpha-chymotrypsin. The importance of secondary interactions in increasing the specificities in the case of alpha-chymotrypsin is discussed.
We have synthesized four guanidinophenyl-substituted protio enol and iodo enol lactones (3-(4-guanidinophenyl)-6-methylidenetetrahydro-2-pyranone (1), 3-(4-guanidinophenyl)-6-(E)-(iodomethylidene)tetrahydro-2-pyran one (2), 4-(4-guanidinophenyl)-6-methylidenetetrahydro-2-pyranone+ ++ (3), and 4-(4-guanidinophenyl)-6-(E)-(iodomethylidene)tetrahydro-2-pyran one (4)) and tested them for inhibitory activity against some trypsin-like enzymes, namely trypsin, urokinase, tissue plasminogen activator (t-PA), plasmin, and thrombin, as well as alpha-chymotrypsin and human neutrophil elastase (HNE). The beta-aryl-substituted protio lactone 3 was a potent alternate substrate inhibitor of trypsin and urokinase. The alpha-aryl-substituted iodo lactone 2 was a permanent inactivator of urokinase, plasmin, t-PA, thrombin, and alpha-chymotrypsin, exhibiting a relatively high specificity for the former two enzymes. In general, these compounds showed a preference for inactivating trypsin-like enzymes over alpha-chymotrypsin and HNE. Also, within the class of trypsin-like enzymes, there was generally good selectivity of inhibition.
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Expression of the DAL2, DAL4, DAL7, DUR1,2, and DUR3 genes in Saccharomyces cerevisiae is induced by the presence of allophanate, the last intermediate of the allantoin degradative pathway. Analysis of the DAL7 5'-flanking region identified an element, designated the DAL upstream induction sequence (DAL UIS), required for response to inducer. The operation of this cis-acting element requires functional DAL81 and DAL82 gene products. We determined the DAL UIS structure by using saturation mutagenesis. A specific dodecanucleotide sequence is the minimum required for response of reporter gene transcription to inducer. There are two copies of the sequence in the 5'-flanking region of the DAL7 gene. There are one or more copies of the sequence upstream of each allantoin pathway gene that responds to inducer. The sequence is also found 5' of the allophanate-inducible CAR2 gene as well. No such sequences were detected upstream of allantoin pathway genes that do not respond to the presence of inducer. We also demonstrated that the presence of a UIS element adjacent to the nitrogen-regulated upstream activation sequence significantly enhances its operation.
Six Type 1 (insulin-dependent) diabetic subjects were studied in order to determine the contribution of recycling of glucose carbon to the overproduction of glucose which is characteristic of the fasting hyperglycaemia produced by insulin withdrawal. The subjects were studied on two occasions, once after an overnight insulin infusion and once following 24 h of insulin withdrawal. The difference in turnover rates of 1-14C-glucose and 3-3H-glucose was used as a measure of glucose recycling. Insulin withdrawal caused a marked metabolic derangement with a rise in non-esterified fatty acids from 0.69 +/- 0.23 to 1.11 +/- 0.21 mmol/l (mean +/- SEM, p less than 0.05), total ketones from 0.27 +/- 0.06 to 2.06 +/- 0.51 mmol/l (p less than 0.01), cortisol from 341 +/- 43 to 479 +/- 31 nmol/l (p less than 0.05) and growth hormone from 1.1 +/- 0.3 to 19 +/- 5 mu/l (p less than 0.05). Glucose turnover rose from 13.8 +/- 2.3 mumol.kg-1.min-1 at a glucose of 6.9 +/- 0.7 mmol/l in the insulin infused study to 25.8 +/- 4.4 mumol.kg-1.min-1 (p less than 0.05) at a glucose of 16.4 +/- 0.7 mmol/l in the insulin withdrawn study. Recycling also rose from 3.0 +/- 0.4 mumol.kg-1.min-1 to 9.4 +/- 2.2 mumol.kg-1.min-1 (p less than 0.05) when insulin withdrawn, accounting for 23 +/- 3% and 36 +/- 3% of glucose turnover, respectively. We conclude that in the severely insulin deficient Type 1 diabetic subject recycling of glucose carbon is a major contributor to the excess glucose production.
We show that mutation at the GLN3 locus results in decreased steady-state levels of DAL7, DUR1,2, CAR1, and URA3 mRNAs derived from cultures grown in the presence of inducer. Basal levels of these RNA species, however, were not significantly affected by a gln3 mutation. The GLN3 product appears to affect gene expression in two ways. The pleiotropic requirement of GLN3 for induced gene expression probably derives from the need of the GLN3 product for inducer uptake into the cell and its loss in gln3 mutants. We also demonstrate that transcriptional activation, mediated by the DAL5 and DAL7 upstream activation sequences, requires a functional GLN3 gene product. This observation identified transcriptional activation as the most likely point of GLN3 participation in the expression of allantoin system genes.
The prevalence of lupus anticoagulant (LAC) and its relation with reported clinical associations has been determined in 55 patients with systemic lupus erythematosus (SLE) from northern India who were studied prospectively. Kaolin clotting time was used to screen for LAC, which was detected in seven (13%) of the patients. Significant associations were found between LAC and thrombotic events, onset of disease at an early age, and disease of shorter duration. No statistically significant association could be found between LAC and recurrent abortions, pulmonary hypertension, thrombocytopenia, and neurological manifestations. It is concluded that LAC is a useful marker for a subset of patients with SLE at risk of thromboembolic events.
Synthesis of the transport systems and enzymes mediating uptake and catabolism of nitrogenous compounds is sensitive to nitrogen catabolite repression. In spite of the widespread occurrence of the control process, little is known about its mechanism. We have previously demonstrated that growth of cells on repressive nitrogen sources results in a dramatic decrease in the steady-state levels of mRNA encoded by the allantoin and arginine catabolic pathway genes and of the transport systems associated with allantoin metabolism. The present study identified the upstream activation sequences in the 5'-flanking regions of the allantoin system genes as the cis-acting sites through which nitrogen catabolite repression is exerted.
DAL5 is a constitutively expressed allantoin system gene whose product is required for allantoate transport. Its simple pattern of expression prompted us to use this gene for identifying the element(s) that mediates transcriptional activation of allantoin system genes. Deletion analysis of the DAL5 5'-flanking sequences resulted in identification of two small regions required for DAL5 expression. Analysis of these two regions with synthetic oligonucleotides localized the sequences supporting transcriptional activation to two DNA fragments of 10 to 12 base pairs, each containing one copy of the pentanucleotide 5'-GATAA-3'. The 5'-flanking region of DAL5 contained eight copies of this sequence. Synthetic constructions containing single copies of these fragments were unable to support transcriptional activation, while those containing two or more copies supported high-level activation. The 5'-GATAA-3' sequence was also found beneath the footprint of a DNA-binding protein. These observations are consistent with the suggestion that DNA fragments containing the sequence 5'-GATAA-3' play an important role in DAL5 gene expression, probably representing a portion of the binding site for a transcriptional activation factor.