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Biomedical subjects

U Kumar

Publications and source records attributed to U Kumar.

At least 37 records · Page 2Linked to original sources
PubMed2000

Sequence, expression and reconstitution of an HCV genome from a British isolate derived from a single blood donation.

Morphological analysis of hepatitis C virus and development of antiviral drugs to eradicate this agent have been seriously hampered by the low viraemias observed during natural infection and the unavailability of a cell culture system for virus propagation. Recently a low-grade hepatitis has been reported in chimpanzees after intrahepatic transfection of full-length synthetic HCV RNA and successful infections shown to be critically dependent on the integrity and genetic homogeneity of the reconstituted clone. In this study we describe and characterize a full HCV RNA sequence derived from a case of chronic sporadic hepatitis. The genotype was shown to be 1a with a low level of intraclonal sequence heterogeneity, and processing of both structural and nonstructural proteins has been documented. The assembly of the full genome has also been achieved. The low level of intraclonal variation observed may reflect infection with a single isolate and the fact that cloning was performed on virus obtained from a single blood donation makes this clone a good candidate for future in vivo and in vitro transfection studies.

Animals↗
PubMed2000

Simple nephrectomy: transperitoneal approach.

Transperitoneal laparoscopic nephrectomy is performed much as originally described by Clayman and associates, although preoperative embolization of the kidney is no longer routine. Some surgeons use a hand-assisted technique, especially in difficult cases or when the organ is being removed intact, as in live-donor nephrectomy.

Adult↗
PubMed1999

Agonist-dependent up-regulation of human somatostatin receptor type 1 requires molecular signals in the cytoplasmic C-tail.

We have previously reported that the human somatostatin receptor type 1 (hSSTR1) stably expressed in Chinese hamster ovary-K1 cells does not internalize but instead up-regulates at the membrane during continued agonist treatment (1 microM somatostatin (SST)-14 x 22 h). Here we have investigated the molecular basis of hSSTR1 up-regulation. hSSTR1 was up-regulated by SST in a time-, temperature-, and dose-dependent manner to saturable levels, in intact cells but not in membrane preparations. Although hSSTR1 was acutely desensitized to adenylyl cyclase coupling after 1 h SST-14 treatment, continued agonist exposure (22 h) restored functional effector coupling. Up-regulation was unaffected by cycloheximide but blocked by okadaic acid. Confocal fluorescence immunocytochemistry of intact and permeabilized cells showed progressive, time-dependent increase in surface hSSTR1 labeling, associated with depletion of intracellular SSTR1 immunofluorescent vesicles. To investigate the structural domains of hSSTR1 responsible for up-regulation, we constructed C-tail deletion (Delta) mutants and chimeric hSSTR1-hSSTR5 receptors. Human SSTR5 was chosen because it internalizes readily, displays potent C-tail internalization signals, and does not up-regulate. Like wild type hSSTR1, Delta C-tail hSSTR1 did not internalize and additionally lost the ability to up-regulate. Swapping the C-tail of hSSTR1 with that of hSSTR5 induced internalization (27%) but not up-regulation. Substitution of hSSTR5 C-tail with that of hSSTR1 converted the chimeric receptor to one resembling wild type hSSTR1 (poor internalization, 71% up-regulation). These results show that ligand-induced up-regulation of hSSTR1 occurs by a temperature-dependent active process of receptor recruitment from a pre-existing cytoplasmic pool to the plasma membrane. It does not require new protein synthesis or signal transduction, is sensitive to dephosphorylation events, and critically dependent on molecular signals in the receptor C-tail.

Adenylate Cyclase Toxin↗
PubMed1999

Localization of Niemann-Pick C1 protein in astrocytes: implications for neuronal degeneration in Niemann- Pick type C disease.

Niemann-Pick type C disease (NP-C) is an inherited neurovisceral lipid storage disorder characterized by progressive neurodegeneration. Most cases of NP-C result from inactivating mutations of NPC1, a recently identified member of a family of genes encoding membrane-bound proteins containing putative sterol sensing domains. By using a specific antipeptide antibody to human NPC1, we have here investigated the cellular and subcellular localization and regulation of NPC1. By light and electron microscopic immunocytochemistry of monkey brain, NPC1 was expressed predominantly in perisynaptic astrocytic glial processes. At a subcellular level, NPC1 localized to vesicles with the morphological characteristics of lysosomes and to sites near the plasma membrane. Analysis of the temporal and spatial pattern of neurodegeneration in the NP-C mouse, a spontaneous mutant model of human NP-C, by amino-cupric-silver staining, showed that the terminal fields of axons and dendrites are the earliest sites of degeneration that occur well before the appearance of a neurological phenotype. Western blots of cultured human fibroblasts and monkey brain homogenates revealed NPC1 as a 165-kDa protein. NPC1 levels in cultured fibroblasts were unchanged by incubation with low density lipoproteins or oxysterols but were increased 2- to 3-fold by the drugs progesterone and U-18666A, which block cholesterol transport out of lysosomes, and by the lysosomotropic agent NH4Cl. These studies show that NPC1 in brain is predominantly a glial protein present in astrocytic processes closely associated with nerve terminals, the earliest site of degeneration in NP-C. Given the vesicular localization of NPC1 and its proposed role in mediating retroendocytic trafficking of cholesterol and other lysosomal cargo, these results suggest that disruption of NPC1-mediated vesicular trafficking in astrocytes may be linked to neuronal degeneration in NP-C.

Amino Acid Sequence↗
PubMed1999

Apolipoprotein D gene expression in the rat brain and light and electron microscopic immunocytochemistry of apolipoprotein D expression in the cerebellum of neonatal, immature and adult rats.

Apolipoprotein D gene and protein expression were investigated in the rat brain and cerebellum, respectively, during development. Apolipoprotein D gene expression was first observed in embryonic day 12 rat brain, with a moderate increase in apolipoprotein D messenger RNA levels towards the later part (embryonic days 15-17) of gestation. In the postnatal rat brain, a marked induction of apolipoprotein D messenger RNA occurred at postnatal day 10, with progressively higher levels of apolipoprotein D messenger RNA observed up to postnatal day 20. Somewhat lower, but none the less high, levels of apolipoprotein D messenger RNA continued to be present in brains of adult animals. In the immature cerebellum (day 3 up to one- to two-week-old rats), there were many densely labeled apolipoprotein D-immunoreactive cells that had features of oligodendrocyte precursors. Purkinje neurons showed apolipoprotein D immunoreactivity in one- to two-week-old animals, after which there appeared to be some decrease in staining. Oligodendrocytes in the cerebella of two-week-old animals were strongly apolipoprotein D positive, with immunoreactivity declining in older animals. These results reveal a maturation-associated induction of apolipoprotein D gene expression in the rat brain, and expression of apolipoprotein D in glial (immature oligodendrocyte) cells in the immature cerebellum, followed by specific expression of apolipoprotein D in Purkinje neurons.

Aging↗
PubMed1999

Unusual cases of congenital cholesteatoma of the ear.

Congenital cholesteatoma may originate at various sites in the temporal bone. For example, in the petrous apex, the cerebellopontine angle, the middle ear cavity, the mastoid process or the external auditory canal. The least common site being the mastoid process. We present two cases of congenital cholesteatoma of the mastoid process, each presenting with different symptoms and at different ages. Both patients underwent surgical treatment, which confirmed the diagnosis and radiological findings.

Adolescent↗
PubMed1999

Differential regulation of somatostatin receptor types 1-5 in rat aorta after angioplasty.

Treatment of restenosis after angioplasty with octapeptide somatostatin (SST) analogs has met with variable success. These analogs bind with high affinity to only two SST receptor (SSTR) subtypes (2 and 5), display moderate affinity for SSTR3, and low affinity for SSTR1 and 4. To optimize the vasculoprotective effect of SST, we have investigated the pattern of expression of all five SSTRs in rat thoracic aorta in the resting state and at 15 min, 3, 7, and 14 days after balloon endothelial denudation. SSTR1-5 were analyzed as mRNA by semiquantitative reverse transcriptase-polymerase chain reaction and as protein by immunocytochemistry. All five SSTRs were expressed in rat aorta both as mRNA and protein and displayed a time-dependent, subtype-selective response to endothelial denudation. mRNA for SSTR1 and 2 increased acutely (SSTR1 > SSTR2) on days 3 and 7, coincident with smooth muscle cell (SMC) proliferation, and declined to basal levels by day 14. SSTR3 and 4 displayed a different pattern with a delayed, more gradual increase in mRNA beginning at days 3-7 and continued to increase thereafter. SSTR5 mRNA was constitutively expressed at a low level and showed no change during the 2 wk postinjury period. By immunohistochemistry, SSTR1-5 antigens were localized predominantly in SMC that were present in the media or had migrated into the intima; antigen expression correlated with receptor mRNA expression. Notably, only SSTR1,3,4 were expressed in the intima: SSTR1 and 4 during the proliferative burst and SSTR3 and 4 after proliferation, when SMC migration into the intima continues. These results demonstrate dynamic changes in SSTR1-5 expression after vascular trauma localized to areas of vascular SMC migration and replication. In view of their early and prominent induction, SSTR1 may be the optimal subtype to target for inhibition of myointimal proliferation, and SSTR3 and 4 for migration and remodeling.

Angioplasty, Balloon↗
PubMed1999

Subtype-selective expression of the five somatostatin receptors (hSSTR1-5) in human pancreatic islet cells: a quantitative double-label immunohistochemical analysis.

We have developed a panel of rabbit polyclonal antipeptide antibodies against the five human somatostatin receptor subtypes (hSSTR1-5) and used them to analyze the pattern of expression of hSSTR1-5 in normal human islet cells by quantitative double-label confocal fluorescence immunocytochemistry. All five hSSTR subtypes were variably expressed in islets. The number of SSTR immunopositive cells showed a rank order of SSTR1 > SSTR5 > SSTR2 > SSTR3 > SSTR4. SSTR1 was strongly colocalized with insulin in all beta-cells. SSTR5 was also an abundant isotype, being colocalized in 87% of beta-cells. SSTR2 was found in 46% of beta-cells, whereas SSTR3 and SSTR4 were relatively poorly expressed. SSTR2 was strongly colocalized with glucagon in 89% of alpha-cells, whereas SSTR5 and SSTR1 colocalized with glucagon in 35 and 26% of alpha-cells, respectively. SSTR3 was detected in occasional alpha-cells, and SSTR4 was absent. SSTR5 was preferentially expressed in 75% of SST-positive cells and was the principal delta-cell SSTR subtype, whereas SSTR1-3 were colocalized in only a few delta-cells, and SSTR4 was absent. These studies reveal predominant expression of SSTR1, SSTR2, and SSTR5 in human islets. Beta-cells, alpha-cells, and delta-cells each express multiple SSTR isoforms, beta-cells being rich in SSTR1 and SSTR5, alpha-cells in SSTR2, and delta-cells in SSTR5. Although there is no absolute specificity of any SSTR for an islet cell type, SSTR1 is beta-cell selective, and SSTR2 is alpha-cell selective. SSTR5 is well expressed in beta-cells and delta-cells and moderately well expressed in alpha-cells, and thereby it lacks the islet cell selectivity displayed by SSTR1 and SSTR2. Subtype-selective SSTR expression in islet cells could be the basis for preferential insulin suppression by SSTR1-specific ligands and of glucagon inhibition by SSTR2-selective compounds.

Animals↗
PubMed1999

Absorbable mesh envelope facilitates handling of pediatric en bloc kidneys during transplantation.

There is increasing evidence to favor the use of pediatric en bloc kidneys in renal transplantation. However, from a technical standpoint, pediatric en bloc renal transplantation is more complex because of difficulty in optimal intraoperative handling and positioning, as well as a higher potential for vascular complications. We describe our experience with a technique that has helped us to minimize the technical difficulties associated with the surgical procedure. Use of a tailored absorbable mesh envelope improves handling of the en bloc pediatric kidneys during performance of the vascular anastomoses and reduces the risk of torsion of renal pedicles.

Absorbable Implants↗
PubMed1999

Evaluation of thyroid hormones in chronic renal failure.

When chronic renal failure becomes advanced, the serum levels of most hormones are altered because of several interplaying mechanisms. This study was carried out to evaluate the level of total thyroxine (T4), triiodothyronine (T3) and thyrotropin (TSH) in 96 clinically euthyriod patient with chronic renal failure and 25 healthy individual as control. The patients were grouped into two groups, 62 patient on conservative management and 34 patients on chronic haemodialysis. The patient of both groups showed significant decrease in total T3 and T4 level as compared to normal control. Serum TSH level were similar in both groups as well as in control and were with in normal limit except 3 patients on conservative management which showed TSH level above normal chronic haemodialysis did not have positive effect in alteration of serum T3, T4 and TSH level.

Adult↗
PubMed1998

The cytoplasmic tail of the human somatostatin receptor type 5 is crucial for interaction with adenylyl cyclase and in mediating desensitization and internalization.

We have investigated the role of the cytoplasmic tail (C-tail) of the human somatostatin receptor type 5 (hSSTR5) in regulating receptor coupling to adenylyl cyclase (AC) and in mediating agonist-dependent desensitization and internalization responses. Mutant receptors with progressive C-tail truncation (Delta347, Delta338, Delta328, Delta318), Cys320 --> Ala substitution (to block palmitoylation), or Tyr304 --> Ala substitution of a putative NPXXY internalization motif were stably expressed in Chinese hamster ovary K1 cells. Except for the Tyr304 --> Ala mutant, which showed no binding, all other mutant receptors exhibited binding characteristics (Kd and Bmax) and G protein coupling comparable with wild type (wt) hSSTR5. The C-tail truncation mutants displayed progressive reduction in coupling to AC, with the Delta318 mutant showing complete loss of effector coupling. Agonist pretreatment of wt hSSTR5 led to uncoupling of AC inhibition, whereas the desensitization response of the C-tail deletion mutants was variably impaired. Compared with internalization (66% at 60 min) of wt hSSTR5, truncation of the C-tail to 318, 328, and 338 residues reduced receptor internalization to 46, 46, and 23%, respectively, whereas truncation to 347 residues slightly improved internalization (72%). Mutation of Cys320 --> Ala induced a reduction in AC coupling, desensitization, and internalization. These studies show that the C-tail of hSSTR5 serves a multifunctional role in mediating effector coupling, desensitization, and internalization. Whereas coupling to AC is dependent on the length of the C-tail, desensitization and internalization require specific structural domains. Furthermore, internalization is regulated through both positive and negative molecular signals in the C-tail and can be dissociated from the signaling and acute desensitization responses of the receptor.

Adenylyl Cyclases↗
PubMed1998

Expression of somatostatin receptor subtypes in human brain tumors.

Expression of mRNA for the 5 somatostatin receptors (sst1-5) was characterized by Northern blot and RT-PCR analysis in 20 meningioma and 9 glioma samples. sst1 mRNA was detectable by Northern blots of poly-A+ RNA in meningiomas but not gliomas. In contrast, sst2 mRNA was readily detected by Northern blots of total RNA as a major 2.3 kb transcript and 2 minor 4.3 kb and 8 kb transcripts in all meningiomas and 6 out of 9 gliomas. Quantitation of the 2.3 kb sst2 mRNA showed that 15 out of 20 tumors expressed 1.3- to 33-fold higher levels than control normal human brain. Mean sst2 mRNA for the 20 meningioma samples was 978% that of normal brain. Three gliomas showed 7- to 14-fold higher sst2 mRNA than normal brain whereas the remaining samples displayed very low or undetectable levels. Immunocytochemistry of meningioma and glioma samples, with a sst2-specific antibody revealed immunoreactivity in tumor cells and peritumoral tissue, with prominent expression in blood vessels. mRNA for sst3,4,5 could not be detected by Northern blots in any of the tumors. RT-PCR analysis of meningiomas and gliomas revealed the following percent of tumors positive for a given sst mRNA: sst1 (86%), sst2 (100%), sst3 (60%), sst4 (58%), and sst5 (67%); 85% of tumors expressed 3 of the 5 subtypes. No correlation was found between the pattern of expression of sst mRNA and tumor type, location, and histology for either the meningiomas or gliomas. Our results show that meningiomas and gliomas are all positive for at least one sst subtype, the majority expressing multiple subtypes. sst2 is the most abundant isoform with a rich expression in both tumor and peritumoral tissue especially blood vessels.

Adult↗
PubMed1998

Comparison of the rate of sequence variation in the hypervariable region of E2/NS1 region of hepatitis C virus in normal and hypogammaglobulinemic patients.

The hypervariable region (HVR) of the E2/NS1 region of hepatitis C virus (HCV) varies greatly between viral isolates with high rates of genomic change reported during the course of chronic infection. The HVR is thought to encode a structurally unconstrained envelope protein containing several linear B cell epitopes recognized by neutralizing antibody. It has been postulated that amino acid changes in the HVR could result from humoral immune pressure leading to the selection of escape mutants. The aim of this study was to compare the rates of nucleotide and amino acid variation in the HVR of control patients to patients with common variable immunodeficiency (CVID) where the effect of the humoral immune system is reduced. Five controls and four patients with CVID were studied. Serum samples were taken over periods of between 1 and 6 years. HCV was detected by polymerase chain reaction (PCR) with primers derived from conserved flanking regions of the HVR. PCR products were cloned into a plasmid vector and recombinant clones identified by restriction enzyme digestion. Purified DNA from at least three individual clones from each time point was sequenced by the dideoxynucleotide chain-termination method. Consensus sequences were extracted from the three clones, and the DNA and deduced protein sequences were compared. Control patients had a mean rate of nucleotide change of 6.954 nucleotide substitutions per year, compared with patients with CVID with a rate of 0.415 nucleotide substitutions per year (P < .02). The corresponding rates for amino acid variation were 3.868 amino acid substitutions per year for the control patients compared with 0.185 amino acid substitutions per year for the patients with CVID. These findings suggest that in the absence of humoral immune selective pressure, the frequency of occurrence of genetic variation in the major viral species is reduced. The mutations occur, but in the absence of immune selection remain as minor species. The evolution of viral mutants capable of evading the host's immune system may contribute to the ability of HCV to establish chronic infection.

Adult↗