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Biomedical subjects

G Singh

Publications and source records attributed to G Singh.

At least 289 records · Page 16Linked to original sources
PubMed1996

Bone cell matrix promotes the adhesion of human prostatic carcinoma cells via the alpha 2 beta 1 integrin.

Prostatic carcinoma cells have a propensity to metastasize to bone, and we propose that this phenomenon may be promoted by the adhesion of metastatic cells to bone matrix. Bone matrix is produced by osteoblasts, and we have developed an in vitro model of bone matrix by isolating the substratum deposited by human osteoblast-like U2OS cells. The collagenous nature of this matrix was demonstrated by the incorporation of [3H]proline and its subsequent release by purified collagenase. Both U2OS matrix and purified type I collagen stimulated the adhesion of human PC-3 prostatic carcinoma cells. Human laminin supported adhesion to a much lesser extent, and PC-3 cells did not adhere to fibronectin. Adhesion of PC-3 cells to U2OS matrix closely resembled adhesion to purified type I collagen with respect to (a) inhibition by a collagen-derived peptide and by antibodies raised against alpha 2 or beta 1 integrin collagen receptor subunits; (b) lack of inhibition by RGD (Arg-Gly-Asp) peptides; (c) stimulation by Mn2+ and Mg2+ ions but not by Ca2+ ion; and (d) stimulation by the phorbol ester PMA (phorbol 12-myristate 13-acetate). This adhesion was also stimulated (2.3-fold) by transforming growth factor beta (TGF-beta), which is a major bone-derived growth factor. We conclude that human osteoblast-like matrix is an adhesive substrate for PC-3 prostate carcinoma cells. This adhesion appears to be mediated by the interaction of alpha 2 beta 1 integrin on PC-3 cells with matrix-derived collagen. The stimulation of this adhesion by TGF-beta suggests that the co-expression of TGF-beta and type I collagen in bone may synergistically facilitate the adhesion of metastatic cells to bone matrix proteins and thereby increase their localization in the skeleton.

Amino Acid Sequence↗
PubMed1996

Transient expression of Na+/H+ exchanger isoform NHE-2 in LLC-PK1 cells: inhibition of endogenous NHE-3 and regulation by hypertonicity.

Na+/H+ exchanger isoforms NHE-2 and NHE-3 demonstrate distinct tissue expression patterns in renal epithelial cells. NHE-2 is predominantly expressed in the inner medulla whereas NHE-3 is highly expressed in the proximal tubule cells. The purpose of the current experiments was to study the characteristics of NHE-2 upon its own expression in cultured proximal tubule cells, LLC-PK1. Toward this end, LLC-PK1 cells were subjected to six cycles of proton suicide. The mutant cells, when grown to confluence and assayed for Na+/H+ exchanger by 22Na+ influx, showed significant reduction in NHE activity as compared to the parent cells (10.4 nmole/mg prot/4 min in parent cells vs. 1.8 in mutant cells, P < 0.001, n = 4). This remaining exchanger activity was mostly mediated via NHE-3 as shown by inhibition of the Na influx following PKC stimulation (65% with PMA vs. 100% without PMA. P < 0.05, n = 4). The mutant cells were transiently transfected with a pCMV/NHE-2 expression vector using calcium phosphate precipitation method. Northern blot analysis showed the expression of a 3.4 kb transcript only in the transfected cells. The expression peaked at 48 hr and diminished by 96 hr. The exchanger activity at 48 hr after transfection was mostly due to NHE-3 (as shown by inhibition in the presence of PMA) but was significantly lower than in sham transfected cells (1.2 nmoles/mg prot. in NHE-2-transfected and 2.1 in sham-transfected, P < 0.05, n = 4). At 60 hr after transfection, the cells exhibited PMA-stimulated Na influx (>28%) indicating functional expression of NHE-2. Increasing the osmolality of the media to 510 mOsm/l stimulated the Na+/H+ exchanger in NHE-2 transfected cells but inhibited the exchanger activity in sham transfected cells. In conclusion, NHE-2 appears as a 3.4 kb transcript in transfected LLC-PK1 cells and functional expression of NHE-2 is preceded by inhibition of endogenous NHE-3 activity. The NHE-2 is stimulated by hypertonicity, indicating a likely role for this isoform in cell volume regulation.

Animals↗
PubMed1996

Nucleotide excision repair in the human ovarian carcinoma cell line (2008) and its cisplatin-resistant variant (C13*).

Repair of cisplatin-damaged DNA was investigated in a human ovarian carcinoma cell line (2008) and its cisplatin-resistant variant (C13*) using a host-cell reactivation (HCR) assay. The HCR of cisplatin-damaged adenovirus (Ad) was not significantly different in C13* cells compared to 2008 cells. The cisplatin concentrations required to reduce the amount of viral DNA replicated to 50% were 0.12 +/- 0.02 microM and 0.10 +/- 0.01 microM after 48 h of repair in 2008 and C13* cells respectively. Similarly, the cisplatin concentration required to reduce the expression of a reporter gene inserted in the viral DNA was not significantly altered in C13* cells compared to the parental line (IC50 values were 0.28 +/- 0.04 microM in 2008 cells and 0.17 +/- 0.06 microM in C13* cells after 48 h of repair). Pretreatment of the cells with cisplatin, immediately prior to Ad infection, did not significantly alter the HCR of cisplatin-damaged Ad in either cell type. In addition, a cisplatin-sensitive variant derived from the C13* cells, namely the RH4 cells, did not differ significantly from either the 2008 or C13* cells in their ability to reactivate cisplatin-damaged Ad. Furthermore, a component of the nucleotide excision repair (NER) pathway, DNA polymerase alpha, was investigated using the competitive inhibitor aphidicolin. The combination of cisplatin and aphidicolin resulted in similar synergistic growth inhibition in both the 2008 and C13* cells providing additional support to the HCR results which suggest that enhanced NER is not responsible for the cisplatin resistance in C13* cells.

Adenoviridae↗
PubMed1996

Reduced anesthetic requirements in aged rats: association with altered brain synaptic plasma membrane Ca(2+)-ATPase pump and phospholipid methyltransferase I activities.

Aging is associated with a decrease in anesthetic requirements. Animal models of aging manifest alteration of brain Ca2+ homeostasis and increased methyltransferase I (PLMTI) activity. In this study we evaluated concurrently anesthetic requirements and brain plasma membrane Ca(2+)-ATPase (PMCA) and PLMTI activities in young and aged rats. Halothane, desflurane, isoflurane and xenon MEDs (lowest partial pressures that suppress a pain response) were measured in 2 and 25 month old, male Fisher-344 rats. Halothane MED was also measured in 2 and 30 month old F344/BNF1 rats, a strain that undergoes aging with less debilitation. PMCA pumping and PLMTI activities were measured in synaptic plasma membranes (SPM) prepared from the cortex and diencephalon-mesencephalon (DM). For aged Fisher-344 rats, MEDs for halothane, desflurane, isoflurane and xenon were reduced to 81%, 82%, 67% and 86%, respectively, of young controls; PMCA activity was diminished to 91% in cortical SPM and 82% in DM SPM; and cortical and DM PLMTI activities were increased to 131% and 114% of young control. For F344/BNF1 rats, MED for halothane was reduced to 87%, PMCA activity was diminished to 90% in cortical SPM and 72% DM SPM, and PLMTI activity was increased to 133% in cortical SPM and 112% in DM SPM. The strong association between age and reduced anesthetic requirements for inhalational agents on the one hand and altered PMCA and PLMTI activity on the other lends support to the underlying hypothesis that PMCA and PLMTI may be involved in the production of the anesthetic state.

Aging↗
PubMed1996

Increased anesthetic requirements for isoflurane, halothane, enflurane and desflurane in obese Zucker rats are associated with insulin-induced stimulation of plasma membrane Ca(2+)-ATPase.

A wide spectrum of structurally disparate inhalational anesthetics reduce brain synaptic plasma membrane Ca(2+)-ATPase (PMCA) activity, whereas phospholipid methyltransferase I (PLMTI) is enhanced by anesthetics. Several rat models with incidental or disease-induced reduction of PMCA and enhancement of PLMTI activities manifest increased sensitivity to inhalational anesthetics. Because insulin is known to stimulate PMCA, anesthetic requirements in hyperinsulinemic obese Zucker rats (fa/fa) and in normoinsulinemic lean Zucker heterozygotes (fa/+) were examined, and brain synaptic PMCA and PLMTI activities were determined in both genotypes. Significantly higher partial pressures of halothane, enflurane, isoflurane, and desflurane were required to inhibit the pain response in obese rats compared to lean Zucker rats. Dose dependent stimulation of PMCA pumping was observed in synaptic membranes from both types, but insulin concentrations in extracts of diencephalon-mesencephalon, cerebellum, and medulla (but not cortex) were higher in obese than in lean Zucker rats. Microdialysis of three subcortical regions showed marked increases in insulin levels with halothane exposure in obese rats, compared to lean controls. These observations in an anesthetic resistant rat model lend further support to the hypothesis that the calcium pump plays a functional role in production of the anesthetic state.

Anesthetics, Inhalation↗
PubMed1996

The use of organic compounds of phosphorus in clinical dentistry.

Organic compounds of phosphorus have been developed for a range of applications in clinical dentistry. These include dentine bonding agents, restorative materials and therapeutic agents, such as active ingredients in anticaries mouthwashes. A characteristic feature of all of these applications is the good bonding of the phosphorus compound to the tooth. This review highlights the progress that has been made to date in preparing functional and durable organophosphorus bonding agents and cements, and includes coverage of recent synthetic work aimed at preparing improved organophosphorus molecules for this application. The review concludes that this remains a promising field of chemistry to explore in the search for improved, clinically useful dental materials.

Dental Bonding↗
PubMed1996

Inhibition of glycosylation decreases Na+/H+ exchange activity, blocks NHE-3 transport to the membrane, and increases NHE-3 mRNA expression in LLC-PK1 cells.

Recent studies have shown that NHE-3 is the luminal Na+/H+ exchanger isoform in cultured renal proximal tubule cells LLC-PK1 and OK (J Biol Chem 1994; 269:15613-8). The purpose of the current experiments was to study the role of NHE-3 glycosylation on antiporter activity in LLC-PK1 cells. Treatment of LLC-PK, cells with 1.5 microgram/ml tunicamycin for 24 hours, which blocks glycosylation in the endoplasmic reticulum, significantly decreased antiporter activity as asses sed by acid-stimulated sodium 22 uptake (9.52 +/- 1.0 nmol/mg protein in control cells vs 5.85 +/- 0.7 nmol/mg protein in tunicamycin-treated cells, p < 0.01, n = 4) and sodium-dependent pHi recovery from an acid load (0.46 +/- 0.05 pH/min in control cells vs 0.35 +/- 0.04 pH/min in tunicamycin-treated cells, p < 0.02, n = 6). Lactate dehydrogenase (LDH) concentration in the medium was the same in both groups (p > 0.05), indicating that the inhibitory effect of tunicamycin was not caused by cell toxicity. Northern hybridization of poly(A)+ RNA from LLC-PK1 cells illustrated that in tunicamycin-treated cells, NHE-3 mRNA expression increased threefold over control cells. Immunoblots of luminal membranes from control LLC-PK, cells with specific NHE-3 antiserum showed a doublet at 94 to 95 kd and a band at 90 kd. Luminal membranes from tunicamycin-treated cells showed only one strong band at 95 kd. NHE-3 immunoblots of whole cell extract from tunicamycin-treated cells showed that in addition to the 95 kd protein, an 87 kd band was also detected. These results are consistent with the possibility that the two bands in the 94 and 90 kd areas became deglycosylated and did not reach the membrane in the presence of tunicamycin. We conclude that glycosylation of the Na+/H+ exchanger isoform NHE-3 is essential for antiporter activity in LLC-PK, cells. The results further suggest that glycosylation of NHE-3 mediates the translocation and insertion of this exchanger in the plasma membrane.

Ammonium Chloride↗
PubMed1996

Artificial urinary sphincter in patients with neurogenic bladder dysfunction.

OBJECTIVE: To assess the role of the artificial urinary sphincter (AUS) in the management of sphincter weakness incontinence in neuropathic patients. PATIENTS AND METHODS: Ninety patients (75 male and 15 female, mean age 26 years, range 13-62) with neurogenic bladder dysfunction (71 with congenital and 19 with acquired cord lesions) who underwent implantation of an AUS were reviewed. All patients were followed up for a minimum of one year after the initial implantation of the sphincter (mean 4 years, range 1-10). Pre-operative video-urodynamics demonstrated sphincter weakness incontinence in all patients, with 24 patients having an acontractile-type bladder. Of 66 patients with intermediate-type bladders, 52 underwent cystoplasty. RESULTS: Eighty-three patients (92%) were continent both night and day, three were occasionally damp and controlled by pharmacotherapy, and four had a persistent poor result. Of the 66 patients with detrusor hyper-reflexia, 52 (79%) required a cystoplasty to achieve continence and 14 (21%) were controlled with anti-cholinergic therapy. The re-operation rate was 28% (25/90) and complications included six infections, seven erosions, eight system failures, two pump failures, one sheered tube and one rectal and one bladder perforation. Seventy (78%) of the patients currently perform intermittent catheterization to treat high post-voiding residual urine volumes. CONCLUSIONS: A rate of continence > 90% was achieved in these neurogenic patients after implantation of an AUS and we recommend a simultaneous cystoplasty in patients with detrusor overactivity.

Adolescent↗
PubMed1996

Artificial urinary sphincter for post-prostatectomy incontinence.

OBJECTIVE: To assess the role of the artificial urinary sphincter in the management of patients with post-prostatectomy incontinence. PATIENTS AND METHODS: The study comprised 28 patients (mean age 71 years, range 45-81) with post-prostatectomy incontinence caused by sphincter deficiency who underwent implantation of an artificial sphincter and were followed for a mean of 41 months (range 6-79). RESULTS: Twenty-one (75%) patients had a bulbar cuff and seven (25%) had a membranous urethral cuff implanted. Problems with the bulbar urethral cuff included persistent stress leakage in six, system failure in two and infection in one patient. Nine patients with the bulbar urethral cuff and one patient with the membranous urethral cuff required revision. Of the nine re-operations in those fitted with a bulbar urethral cuff, five required a higher pressure balloon, one a smaller cuff, two had system failures replaced and one had a membranous urethral cuff implanted because of infection. Three of these nine patients needed a third operation and in all of these a membranous urethral cuff was inserted. A total of 33 operations were performed in 21 patients with bulbar urethral cuffs (a re-operation rate of 57%) and eight operations were carried out in seven patients with membranous urethral cuffs (a re-operation rate of 14%). Three patients needed anticholinergic therapy and one needed a clam cystoplasty for intractable instability. Twenty-four patients (86%) are dry and fully continent, three (11%) have occasional stress leakage and one patient had a cerebro-vascular accident 9 months after surgery and now has an indwelling catheter. CONCLUSIONS: Patients who had artificial sphincters implanted to treat post-prostatectomy incontinence achieved a social continence rate of 96%. With bulbar urethral sphincters there was a high incidence of post-implantation incontinence and a high re-operation rate to achieve this success.

Aged↗
PubMed1996

Role of endothelin in the cardiovascular effects of diaspirin crosslinked and stroma reduced hemoglobin.

OBJECTIVES: Diaspirin crosslinked hemoglobin is a resuscitative solution with excellent oxygen-carrying capacity. Diaspirin crosslinked hemoglobin produces an immediate increase in blood pressure and marked regional circulatory changes in rats and pigs. Our objective was to determine the role of endothelin in the cardiovascular actions of diaspirin crosslinked hemoglobin (modified) and (unmodified) stroma reduced hemoglobin solutions. DESIGN: Prospective, randomized comparison of cardiovascular effects of diaspirin crosslinked and stroma reduced hemoglobin in control rats and in rats pretreated with cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123), an endothelin-A receptor antagonist. SETTING: Research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Modified, highly purified, and heat pasteurized (diaspirin crosslinked) and unmodified (stroma reduced) hemoglobin in control (untreated) and BQ-123 (5 mg/kg/hr iv)-treated rats. MEASUREMENTS AND MAIN RESULTS: Infusion of stroma reduced hemoglobin (400 mg/kg iv) in control rats produced an increase in blood pressure (43%) and total peripheral resistance (65%) without any change in heart rate, cardiac output, and stroke volume. Stroma reduced hemoglobin decreased blood flow to the kidneys and liver, increased blood flow to the heart, and had no effect on blood flow to the brain, gastrointestinal tract, spleen, musculoskeletal system, skin, and mesentery and pancreas. Infusion of stroma reduced hemoglobin in rats treated with BQ-123 (5 mg/kg/hr iv) increased the blood pressure to a similar degree when compared with control rats, but the increase in total peripheral resistance was significantly attenuated. The stroma reduced hemoglobin-induced decrease in blood flow to the kidneys and liver was significantly attenuated in BQ-123-treated rats as compared with control rats. However, the stroma reduced hemoglobin-induced increase in blood flow to the heart of BQ-123-treated rats was similar to the increase in control rats. Infusion of diaspirin crosslinked hemoglobin (400 mg/kg iv) produced increases in blood pressure (81%), cardiac output (36%), stroke volume (30%), and total peripheral vascular resistance (45%), along with increases in blood flow to the heart, spleen, gastrointestinal tract, and skin of control rats. The blood flows to the brain, kidneys, liver, musculoskeletal system, and mesentery and pancreas were not altered by diaspirin crosslinked hemoglobin in control rats. The increases in blood pressure, cardiac output, stroke volume, and total peripheral vascular resistance by diaspirin crosslinked hemoglobin were significantly blocked in BQ-123-treated rats as compared with control rats. The increases in blood flow to the heart, spleen, and skin by diaspirin crosslinked hemoglobin were significantly blocked in BQ-123-treated rats as compared with control rats. Diaspirin crosslinked hemoglobin produced an increase in the blood flow to the brain and a decrease in blood flow to the kidney and musculoskeletal system of BQ-123-treated rats as compared with control rats. Blood plasma endothelin-1-like immunoreactivity was found to be significantly increased after treatment with diaspirin crosslinked hemoglobin or stroma reduced hemoglobin. CONCLUSIONS: The endothelin-A receptor antagonist, BQ-123, could attenuate the systemic hemodynamic and regional circulatory effects of diaspirin crosslinked hemoglobin and stroma reduced hemoglobin. However, the increase in blood flow to the heart induced by stroma reduced hemoglobin could not be attenuated by BQ-123.

Animals↗
PubMed1996

Segmental enteritis: "enteritis necroticans". A clinicopathologic study.

Acute segmental enteritis, also called "enteritis necroticans" is characterized by nonocclusive intestinal ischemia in the absence of any precipitating cause. We studied 30 such patients over a 5.5-year period. All patients had acute abdominal symptoms requiring emergency laparotomy, and in only 30% was a preoperative diagnosis of segmental enteritis proposed. The jejunum alone (63.3%), or with the ileum (26.7%), was the most common site of necrotic patches (86.7%), frank gangrene (50.0%), free perforation (36.7%), and circumferential dusky lesions (30.0%). Twenty-eight patients required resection of the affected bowel. Specimen angiography in two cases showed normal mesenteric vasculature. In 12 patients, a detailed microbiological profile from peritoneal fluid, intestinal contents, and intestinal wall was performed, but Clostridium welchii was isolated from none. The resected intestinal specimens showed mucosal ulceration, submucosal edema, mixed inflammatory infiltrate, and patchy necrosis of the muscularis propria. The intramural vessels were patent in all cases. Wound-related complications occurred in 14, pulmonary complications in 14, renal failure in eight, and fecal fistulas in five; 23.3% died.

Abdomen, Acute↗