Biomedical subjects
N Prakash
Publications and source records attributed to N Prakash.
Vasopressin mRNA localization in nerve cells: characterization of cis-acting elements and trans-acting factors.
mRNA localization is a complex pathway. Besides mRNA sorting per se, this process includes aspects of regulated translation. It requires protein factors that interact with defined sequences (or sequence motifs) of the transcript, and the protein/RNA complexes are finally guided along the cytoskeleton to their ultimate destinations. The mRNA encoding the vasopressin (VP) precursor protein is localized to the nerve cell processes in vivo and in primary cultured nerve cells. Sorting of VP transcripts to dendrites is mediated by the last 395 nucleotides of the mRNA, the dendritic localizer sequence, and it depends on intact microtubules. In vitro interaction studies with cytosolic extracts demonstrated specific binding of a protein, enriched in nerve cell tissues, to the radiolabeled dendritic localizer sequence probe. Biochemical purification revealed that this protein is the multifunctional poly(A)-binding protein (PABP). It is well known for its ability to bind with high affinity to poly(A) tails of mRNAs, prerequisite for mRNA stabilization and stimulation of translational initiation, respectively. With lower affinities, PABP can also associate with non-poly(A) sequences. The physiological consequences of these PABP/RNA interactions are far from clear but may include functions such as translational silencing. Presumably, the translational state of mRNAs subject to dendritic sorting is influenced by external stimuli. PABP thus could be a component required to regulate local synthesis of the VP precursor and possibly of other proteins.
Identification of biochemical pathways for the metabolism of oxidized low-density lipoprotein derived aldehyde-4-hydroxy trans-2-nonenal in vascular smooth muscle cells.
Oxidation of low-density lipoproteins (LDL) generates high concentrations of unsaturated aldehydes, such as 4-hydroxy trans-2-nonenal (HNE). These aldehydes are mitogenic to vascular smooth muscle cells and sustain a vascular inflammation. Nevertheless, the processes that mediate and regulate the vascular metabolism of these aldehydes have not been examined. In this communication, we report the identification of the major metabolic pathways and products of [(3)H]-HNE in rat aortic smooth muscle cells in culture. High-performance liquid chromatography separation of the radioactivity recovered from these cells revealed that a large (60-65%) proportion of the metabolism was linked to glutathione (GSH). Electrospray mass spectrometry showed that glutathionyl-1,4 dihydroxynonene (GS-DHN) was the major metabolite of HNE in these cells. The formation of GS-DHN appears to be due aldose reductase (AR)-catalyzed reduction of glutathionyl 4-hydroxynonanal (GS-HNE), since inhibitors of AR (tolrestat or sorbinil) prevented GS-DHN formation, and increased the fraction of the glutathione conjugate remaining as GS-HNE. Gas chromatography-chemical ionization mass spectroscopy of the metabolites identified a subsidiary route of HNE metabolism leading to the formation of 4-hydroxynonanoic acid (HNA). Oxidation to HNA accounted for 25-30% of HNE metabolism. The formation of HNA was inhibited by cyanamide, indicating that the acid is derived from an aldehyde dehydrogenase (ALDH)-catalyzed pathway. The overall rate of HNE metabolism was insensitive to inhibition of AR or ALDH, although inhibition of HNA formation by cyanamide led to a corresponding increase in the fraction of HNE metabolized by the GSH-linked pathway, indicating that ALDH-catalyzed oxidation competes with glutathione conjugation. These metabolic pathways may be the key regulators of the vascular effects of HNE and oxidized LDL.
The effects of remifentanil on haemodynamic stability during rigid bronchoscopy.
We examined the effect of remifentanil on the haemodynamic response to rigid bronchoscopy in 22 adult ASA 2-4 patients, randomly allocated to receive 1.0 microg.kg-1 remifentanil over 1 min followed by 0.5 microg.kg-1.min-1 (remifentanil group) or 2.0 microg.kg-1 fentanyl followed by a saline infusion (control group). Following the initial infusion, all patients received a sleep dose of propofol followed by rocuronium 0.6 mg.kg-1 and their lungs were ventilated using a Sander's injector attached to the rigid bronchoscope. Direct arterial blood pressures, heart rate and ST segment changes on the CM5 lead configuration of the electrocardiograph were measured at 1-min intervals from before induction of anaesthesia until the end of bronchoscopy. Arterial pressures and heart rate were similar in the two groups before and after induction of anaesthesia. Remifentanil attenuated the haemodynamic response to bronchoscopy (p < 0.05 for increases in arterial pressures and heart rate). Four patients in the remifentanil group had ST segment depression compared with eight patients in the control group, but this was not statistically significant.
Malformation of the functional organization of somatosensory cortex in adult ephrin-A5 knock-out mice revealed by in vivo functional imaging.
The molecular mechanisms that coordinate the functional organization of the mammalian neocortex are largely unknown. We tested the involvement of a putative guidance label, ephrin-A5, in the functional organization of the somatosensory cortex by quantifying the functional representations of individual whiskers in vivo in adult ephrin-A5 knock-out mice, using intrinsic signal optical imaging. In wild-type mice ephrin-A5 is expressed in a gradient in the somatosensory cortex during development. In adult ephrin-A5 knock-out mice, we found a spatial gradient of change in the amount of cortical territory shared by individual whisker functional representations across the somatosensory cortex, as well as a gradient of change in the distance between the functional representations. Both gradients of change were in correspondence with the developmental expression gradient of ephrin-A5 in wild-type mice. These changes involved malformations of the cortical spacing of the thalamocortical components, without concurrent malformations of the intracortical components of individual whisker functional representations. Overall, these results suggest that a developmental guidance label, such as ephrin-A5, is involved in establishing certain spatial relationships of the functional organization of the adult neocortex, and they underscore the advantage of investigating gene manipulation using in vivo functional imaging.
Visualizing and quantifying evoked cortical activity assessed with intrinsic signal imaging.
Intrinsic signal imaging (ISI) measures changes in light reflectance from the illuminated cortex (intrinsic signals or IS) attributed to various vascular and metabolic sources that, when using illumination in the 600 nm range, appear to co-localize with neuronal activity. Given the multiple sources contributing to the collected IS, the common practice of averaging across an extended post-stimulus time epoch before dividing by baseline data typically visualizes evoked IS overlying both the cortical tissue and the large surface blood vessels. In rat PMBSF, the contribution from these vessels are problematic as they do not co-localize with known PMBSF function. Determining a means for quantifying the evoked IS area poses an additional challenge. Here, we describe how exploiting IS collected shortly after stimulus onset (within 1.5 s), which coincides with fast oxygen consumption of active neurons, visualizes evoked IS overlying the cortical tissue without the large surface vessels. We also describe how the use of absolute thresholds combined with a baseline determined from data collected immediately prior to stimulus onset (within 1 s) targets most precisely a specific evoked IS amplitude, a method that should be especially useful when evoked areas are expected to occupy a substantial portion of the total imaged area and/or when peak activity is expected to differ between subjects.
A mapping label required for normal scale of body representation in the cortex.
The neocortical primary somatosensory area (S1) consists of a map of the body surface. The cortical area devoted to different regions, such as parts of the face or hands, reflects their functional importance. Here we investigated the role of genetically determined positional labels in neocortical mapping. Ephrin-A5 was expressed in a medial > lateral gradient across S1, whereas its receptor EphA4 was in a matching gradient across the thalamic ventrobasal (VB) complex, which provides S1 input. Ephrin-A5 had topographically specific effects on VB axon guidance in vitro. Ephrin-A5 gene disruption caused graded, topographically specific distortion in the S1 body map, with medial regions contracted and lateral regions expanded, changing relative areas up to 50% in developing and adult mice. These results provide evidence for within-area thalamocortical mapping labels and show that a genetic difference can cause a lasting change in relative scale of different regions within a topographic map.
Cardiac arrest after video-assisted thoracoscopic drainage of pleural effusion.
Explore the source record for details and available documents.
Identification of both general and region-specific embryonic CNS enhancer elements in the nestin promoter.
In this report, we investigate how nestin expression is controlled in neural progenitor cells of the embryonic CNS. A 374-bp region in the second intron of the human nestin gene is sufficient, and a 120-bp sequence in this region is required, to express the lacZ reporter gene throughout the developing CNS of E9.5-10.5 transgenic mouse embryos. The 120-bp element region contains putative binding sites for nuclear hormone receptors and we show that TRs, RXR, RAR, and COUP-TF bind to these motifs. A separate enhancer, located most probably 5' to the 120-bp sequence in the second intron, controls midbrain expression at E10.5. In conclusion, our data show that the nestin enhancer in the second intron contains elements both for general and for region-specific CNS progenitor cell expression and suggest that nuclear hormone receptors play a role in the regulation of nestin expression in the early CNS.
Dendritic localization of rat vasopressin mRNA: ultrastructural analysis and mapping of targeting elements.
Transcripts encoding the vasopressin precursor are located in axons and dendrites of rat hypothalamic magnocellular neurons. While the axonal vasopressin mRNA has been extensively characterized both at the biochemical and morphological level, little is known about those transcripts residing in dendrites of magnocellular neurons. As revealed by in situ hybridization at the electron microscopic level, the mRNA is located in proximal and distal dendritic segments and is exclusively confined to regions containing rough endoplasmic reticulum. These results suggest that dendrites of hypothalamic neurons may be capable of local precursor synthesis independent of that occurring in the cell somata. A heterologous system has been employed to define cis-acting elements within the vasopressin mRNA which may be involved in dendritic compartmentalization. Expression vector constructs consisting of the cytomegalovirus promoter coupled to the rat vasopressin cDNA have been injected into the cell nuclei of cultured neurons derived from embryonic rat superior cervical ganglia. Vector-encoded vasopressin transcripts were also sorted to dendrites of these neurons indicating that the molecular determinants of dendritic mRNA transport are not cell specific. Mapping of the targeting elements revealed two segments within the vasopressin mRNA that are able to confer dendritic compartmentalization to alpha-tubulin mRNA which is normally confined to the cell body.
Decreased vascular endothelial growth factor expression associated with tumor regression induced by (E)-2'-deoxy-2'-(fluoromethylene)cytidine (MDL 101,731).
The effect of the antitumor drug MDL 101,731 [(E)-2'-deoxy-2'-(fluoromethylene)cytidine] on tumor growth and on steady-state vascular endothelial growth factor (VEGF) mRNA levels in MDA-MB-231, PC-3, MCF-7, and HT-29 human tumor xenografts grown in nude mice was examined, using quantitative in situ hybridization. MDL 101,731 caused regression of MDA-MB-231 and PC-3 tumor xenografts, but only inhibition of growth (without regression) of MCF-7 xenografts. The drug caused inhibition of growth of HT-29 xenografts at low doses, and regression at high doses. When treatment with MDL 101,731 led to tumor regression, VEGF mRNA levels were decreased. When treatment led only to inhibition of growth, there was no significant change in VEGF mRNA. Further examination of the tumor xenografts revealed that elevated VEGF mRNA was associated with hypoxic zones surrounding areas of necrosis in the tumors, and that the drop in VEGF mRNA observed in tumors from mice treated with MDL 101,731 correlated with a loss of zones of necrosis. In contrast, treatment with cisplatin led to either an increase (PC-3) or no change (MDA-MB-231) in VEGF mRNA levels, and no loss of necrotic zones. Quantitative analysis of changes in VEGF mRNA levels was supported by immunohistochemical analysis of VEGF protein in the same tumor specimens. In vitro, MDL 101,731 was a potent inhibitor of VEGF secretion in cells exposed to hypoxia, whereas there was no effect of cisplatin on VEGF secretion by three of the four cell lines tested. These findings suggest that inhibition of VEGF expression by MDL 101,731 may distinguish this compound from other classes of cytotoxic agents, such as cisplatin.
RAPID and opposite effects of BDNF and NGF on the functional organization of the adult cortex in vivo.
The adult cortex is thought to undergo plastic changes that are closely dependent on neuronal activity (reviewed in ref. 1), although it is not yet known what molecules are involved. Neurotrophins and their receptors have been implicated in several aspects of developmental plasticity, and their expression in the adult cortex suggests additional roles in adult plasticity. To examine these potential roles in vivo, we used intrinsic-signal optical imaging to quantify the effects of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) on the functional representation of a stimulated whisker in the 'barrel' subdivision of the rat somatosensory cortex. Topical application of BDNF resulted in a rapid and long-lasting decrease in the size of a whisker representation, and a decrease in the amplitude of the activity-dependent intrinsic signal. In contrast, NGF application resulted in a rapid but transient increase in the size of a representation, and an increase in the amplitude of the activity-dependent intrinsic signal. These results demonstrate that neurotrophins can rapidly modulate stimulus-dependent activity in adult cortex, and suggest a role for neurotrophins in regulating adult cortical plasticity.
Pharmacokinetics of radiolabelled buffalo follicle-stimulating hormone (125I-buFSH) in female buffalo calves (Bubalus bubalis).
Explore the source record for details and available documents.
Identification of potential redox-sensitive cysteines in cytosolic forms of fructosebisphosphatase and glyceraldehyde-3-phosphate dehydrogenase.
Tertiary-structure modeling suggests the occurrence of disulfide bonds in the cytosolic form of fructose-bisphosphatase (EC 3.1.3.11) in spinach (Spinacia oleracea L.), sugarbeet (Beta vulgaris L.) and potato (Solanum tuberosum L.). Redox modulation could then control the AMP sensitivity of fructosebisphosphatase in the cytosol, as suggested by the experiments of E. Khayat et al. (1993, Plant Physiol. 101, 57-64). Modeling also reveals two cysteine residues correctly positioned to form a disulfide bond and hence potentially redox-sensitive in the cytosolic glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from the facultative crassulacean metabolism plant Mesembryanthemum crystallinum L.
Enzyme-enzyme interaction in the chloroplast: glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase and aldolase.
Apparent physical interaction between pea chloroplast (Pisum sativum L.) glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase (EC 4.1.2.13) is seen in phase-partitioning, fluorescent-anisotropy and isoelectric-focusing experiments. Similarly, results obtained in phase-partitioning and isoelectric-focusing experiments indicate physical interaction between aldolase and triose-phosphate isomerase (EC 5.3.1.1). Kinetic experiments suggest that both aldolase-bound glyceraldehyde-3-phosphate can act as substrate for glyceraldehyde-3-phosphate dehydrogenase. These results are consistent with the notion that there is interaction between these three enzymes both during photosynthetic CO2 fixation and during glycolysis in the chloroplast.
Association of symptoms with Helicobacter pylori infection in children.
The aim of this study was to determine prospectively whether Helicobacter pylori-associated gastritis is associated with specific symptoms by evaluating whether these symptoms are relieved by treatment of the infection. Symptoms resolved after the eradication of H. pylori in only three of eight children with H. pylori-associated gastritis alone, in comparison with all six children with duodenal ulcer disease.
Gestational choriocarcinoma of the fallopian tube--a case report.
Gestational Choriocarcinoma occurring in a female aged 20 years is being reported following ectopic tubal pregnancy. The case responded well to the conventional Chemotherapy regimen.
Leishmania donovani: hemolytic activity of promastigotes.
A hemolytically active component was found to be present in Leishmania donovani promastigotes for the first time. It lysed human and rabbit erythrocytes to varying degrees. The optimal pH for the activity was found to be 5.8. The rate of hemolysis was dependent on both erythrocyte and parasite concentrations. Parasites in the log phase were more effective in lysing erythrocytes than those in the stationary phase. Centrifugation at 10,000g showed major activity in the pellet fraction of the autolysate. Boiling in a water bath for 5 min reduced 98% of the hemolytic activity, which was also found to be inhibited by trypsin. The presence of such a hemolytic factor appears to be important in the nutrition of this parasite.