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Biomedical subjects

A K Verma

Publications and source records attributed to A K Verma.

At least 55 records · Page 3Linked to original sources
PubMed1997

Replacement of Val674 by Pro increases the sensitivity of the plasma membrane Ca2+ pump to inhibition by Mg2+.

A cDNA encoding a plasma membrane Ca2+ pump mutant V674P(ct120) was constructed and expressed in COS-1 cells. Immunoblots of transfected COS-1 membranes showed that the V674P(ct120) and the wild-type hPMCA4b(ct120) proteins were expressed at similar levels. The change of Val674 to Pro reduced the activity of the hPMCA4b(ct120) to an extent similar to that observed previously in the full-length Ca2+ pump (Adamo et al. (1995) J. Biol. Chem. 270, 30111-30114). Despite its lower activity, the apparent affinity for Ca2+ of the V674P(ct120) enzyme was at least as high as that of hPMCA4b(ct120), indicating that substitution of Val674 by Pro did not impair the interaction of the enzyme with Ca2+. The sensitivity of the V674P(ct120) enzyme to inhibition by vanadate was not significantly different from that of the hPMCA4b(ct120), supporting the idea that the mutation did not alter the equilibrium between E2-E1. The study of the Mg2+ dependency of the Ca2+ transport showed that the V674P(ct120) mutant reached maximum activation at 100 microM Mg2+ in contrast with 500 microM in the hPMCA4b(ct120). Furthermore, while at 2 mM Mg2+ the hPMCA4b(ct120) showed no sign of inhibition, the activity of the mutant decreased to less than 50% of the maximum activity observed at 100 microM Mg2+. These results indicate that the decrease in the activity observed upon substitution of Val674 by Pro was due to a higher sensitivity to Mg2+ as inhibitor.

Animals↗
PubMed1997

Improvement of limb circulation in peripheral vascular disease using epidural spinal cord stimulation: a prospective study.

Spinal cord stimulation was used in 46 patients for pain associated with lower extremity ischemic vascular disease that was considered to be nonreconstructable. Thirty-nine patients who had a follow-up examination between 2 and 36 months after the procedure form the basis of this report. Thirty (77%) of 39 cases were considered successful. Clinical endpoints indicating failure included amputation, vascular reconstruction, poor pain relief, or hardware malfunction. The transcutaneous partial pressure of oxygen (TcPO2) increased in both target and control feet. In patients with good outcome with a preimplantation TcPO2 of less than 30 mm Hg, TcPO2 increased significantly (p < 0.05). Pulse volume recording improved significantly (p < 0.05) at the thigh, metatarsal, and great toe levels in successfully treated patients. Peak blood flow velocity also showed a significant increase in patients with good outcome (p < 0.05). Patients with a TcPO2 of less than 10 mm Hg following stimulation tended to undergo amputation within the first 3 months. Improvement in pain control, combined with an increase in TcPO2 values that was greater than 10 mm Hg, were significant early predictors of long-term success. An initial increase in peak blood flow velocities (measured in Doppler studies) of greater than 10 mm also signified a good long-term outcome. Spinal cord stimulation appears to be a useful therapeutic modality for controlling pain and improving perfusion in a select group of patients with end-stage ischemic vascular disease considered nonreconstructable. The best results were seen in patients with severe claudication and rest pain without trophic changes in the foot. The mechanism of this beneficial effect is not yet completely understood.

Aged↗
PubMed1997

Comparative evaluation of breast lesions with the help of impression smears, histopathology and mammography.

A total of 148 surgically removed benign and malignant breast lesions were studied to correlate cytomorphologial features in impression smears and histopathological sections. Mammograms were taken prior to surgery. Role of mammography in detection of non-palpable breast lesions was enlightened. Impression smears helped in quick diagnosis (intraoperative) and overall accuracy obtained was 97.4%. Cluster predominant and Grade II nuclear grading on smear pattern was seen in 58.8% and 57.4% cases respectively amongst the 68 malignant cases. 45% cases revealed non-palpable breast lesions on mammography which was later confirmed on histopathology.

Breast Diseases↗
PubMed1996

Protein kinase C activates the plasma membrane Ca2+ pump isoform 4b by phosphorylation of an inhibitory region downstream of the calmodulin-binding domain.

The carboxyl-terminal region of the plasma membrane Ca2+ pump isoform 4b contains two autoinhibitory regions which keep the pump inactive in the absence of activators such as calmodulin. One of these regions is approximately coterminous with the calmodulin-binding domain, while the second region is downstream (Verma, A. K., Enyedi, A., Filoteo, A. G., and Penniston, J. T. (1994) J. Biol. Chem. 269, 1687-1691). The carboxyl-terminal region has also been identified as the site for phosphorylation of this isoform by protein kinase C (Wang, K. K. W., Wright, L. C., Machan, C. L., Allen, B. G., Conigrave, A. D., and Roufogalis, B. D. (1991) J. Biol. Chem. 266, 9078-9085). Using constructs lacking various numbers of residues at the carboxyl terminus, we studied the degree of phosphorylation by protein kinase C and the resultant activation of Ca2+ transport. The results showed that the most specific and easy phosphorylation occurred in a region of about 20 residues which is downstream of the calmodulin-binding domain, and that the downstream inhibitory domain had also about the same size and location. Phosphorylation partially activated the pump by removing only the inhibition due to this region. Binding of calmodulin to the calmodulin-binding domain activated the pump more fully by removing the inhibition due to both regions, regardless of the state of phosphorylation at the downstream inhibitory region.

Amino Acid Sequence↗
PubMed1996

Detection of isoform 4 of the plasma membrane calcium pump in human tissues by using isoform-specific monoclonal antibodies.

The epitope location and specificity of monoclonal antibodies JA9, 5F10 and JA3, raised against the human plasma membrane Ca2+ pump (hPMCA), were analysed by using synthetic peptides of the corresponding epitopes as well as the complete isoforms, hPMCA4b, hPMCA4a and hPMCA1b, expressed in COS-1 cells. The experiments with the peptides showed that JA9 reacted specifically with a region containing residues 51-75 of hPMCA4 (a or b), but not with the same region of isoforms 1, 2 or 3. JA3 reacted with residues 1156-1180, a region unique to hPMCA4b. 5F10 reacted in the region of residues 719-738, which is highly conserved in all PMCA isoforms. Indeed, 5F10 recognized all three isoforms expressed in COS-1 cells. JA9, in contrast, reacted with both variants a and b of hPMCA4 but not with hPMCA1, and JA3 recognized exclusively hPMCA4b. We used these antibodies to discern the distribution of hPMCA4a and hPMCA4b in human brain, heart, kidney and lung. In Western blots of human brain samples, we could identify both hPMCA4a and hPMCA4b. Heart tissue also showed isoform 4b, and probably 4a. In contrast, kidney and lung showed primarily hPMCA4b. In brain, overlapping bands that did not correspond to either variant of hPMCA4 were detected, and in kidney a band migrating in the same position as hPMCA1b was observed. The distribution of the a and b forms of hPMCA4 at the protein level, as analysed by these antibodies, is consistent with the available data about the abundance of mRNAs for the hPMCA isoforms. The presence of hPMCA4b in all the samples supports the proposed role of this isoenzyme as a constitutive form of the pump.

Alternative Splicing↗
PubMed1996

Functional expression and characterization of the mouse epitope tag-protein kinase C isoforms, alpha, beta I, beta II, gamma, delta and epsilon.

To simplify the detection of the exogenously produced protein kinase C (PKC) isoforms, we constructed the T7-Tag PKC alpha, betaI, betaII, gamma, delta and epsilon expression plasmids. T7-Tag sequences (AlaSerMetThrGlyGlyGlnGlnMetGlyArg) were inserted at the 5'-end of the translational initiation site. Transient transfection of T7-Tag PKC expression plasmids into CV-1 cells increased the levels of PKC protein, and PKC activity. T7-Tag-PKC epsilon, like native PKC epsilon, transactivated the transcription of a NF-kappa B reporter construct. These results indicate that the plasmids encoding T7-Tag PKC are functional and may be useful to define the role PKC isoforms play in cell proliferation, differentiation and tumor promotion.

Amino Acid Sequence↗
PubMed1996

Superinduction of mouse epidermal ornithine decarboxylase activity by repeated 12-o-tetradecanoylphorbol-13-acetate treatments.

A correlation of the levels of epidermal protein kinase C (PKC) isozymes, steady state levels of ornithine decarboxylase (ODC) mRNA, and ODC antizyme with the induction of ornithine decarboxylase (ODC) activity by a second repeat 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment to mouse skin was determined. A single application of TPA to female CD-1 mouse skin leads to a dramatic induction of ODC activity (approximately 3 nmol CO2/60 min/mg protein) which peaks at about 5 h after treatment. However, a superinduction of ODC activity (approximately 13 CO2/60 min/mg protein) is observed upon the second TPA application at 48 or 72 h after the first TPA treatment. Prior application of a tumor initiating dose of 7,12-dimethylbenz[a]anthracine to mouse skin did not influence the degree of induction of ODC by a repeat TPA treatment. Western Blot analyses using antibodies specific to PKC alpha, beta, gamma, delta and epsilon indicate detectable levels of PKC alpha, beta, delta and epsilon in mouse epidermal extracts. A time course of the effects of a single topical application of 20 nmol of TPA to the mouse skin indicate that none of PKC isozymes (alpha, beta, gamma, delta and epsilon) were completely downregulated at times (72 h) when ODC was overinduced by TPA. TPA-induced steady state levels of ODC mRNA did not correlate with the degree of superinduction of ODC activity by TPA. The second TPA treatment, 72 h after the first TPA treatment, which leads to superinduction of ODC activity did not decrease the levels of the ODC-antizyme. The results indicate that superinduction of mouse epidermal ODC activity is regulated in part post-transcriptionally and may not be the result of either a loss of PKC isoform(s) or a decrease in the levels of ODC antizyme.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

Plasma membrane calcium pump isoform 4a has a longer calmodulin-binding domain than 4b.

Alternate splicing of human plasma membrane calcium pump isoform 4 (hPMCA4) transcripts causes the expression of two variants, hPMCA4a and hPMCA4b, which have different downstream regulatory regions. Of the two, hPMCA4a has a lower affinity for calmodulin and a lower effective affinity for Ca2+ (Enyedi, A., Verma, A. K., Heim, R., Adamo, H. P., Filoteo, A. G., Strehler, E. E., and Penniston, J. T. (1994) J. Biol. Chem. 269, 41-43). Additional consequences of the alternate splice were studied by analyzing the characteristics of constructs (expressed in COS-1 cells) containing different portions of the carboxyl terminus of hPMCA4a. Our results show striking differences in the structure of the calmodulin-binding and autoinhibitory domains of the two variants. The calmodulin-binding region of hPMCA4b is a region of about 28 residues, whereas that of hPMCA4a is about 49 residues long and is probably interrupted by a region not involved in the binding. The autoinhibitory region of hPMCA4b (a part of the downstream region that keeps the molecule inactive in the absence of Ca2+-calmodulin) is divided between the 28-residue calmodulin-binding region and a downstream region, whereas in hPMCA4a, all of it is contained within the 49-residue calmodulin-binding region.

Adenosine Triphosphate↗
PubMed1996

Localization of the 12-O-tetradecanoylphorbol-13-acetate response of the human ornithine decarboxylase promoter to the TATA box.

In a previous study, we narrowed the region of the human ornithine decarboxylase (ODC) promoter responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA) to nt -42 to +54 around the transcription initiation site (Kim YJ, Pan H, Verma AK, Mol Carcinog 10:169-179, 1994). Here we report defining the role of the TATA box in TPA-induced transcription from the -42/+54 ODC promoter fragment. A transversion mutation at the third position of the TATA box (TATAAGT-->TAAAAGT) reduced TPA responsiveness of the reporter construct -42/+54 ODC-Luc by 49%. Electrophoretic mobility shift assays (EMSAs) using HeLa cell nuclear protein extracts revealed no differences in the binding pattern between the natural -42/+54 ODC promoter element and the -42/+54 ODC promoter element containing the T-->A mutation. However, antibodies to the general transcription factor TFIIB disrupted the DNA-protein complexes normally formed with the -42/+54 ODC promoter element in EMSAs. A consensus TATA box oligonucleotide formed two bands, with the faster mobility band displaying enhanced binding with nuclear protein extracts from TPA treated cells. Furthermore, incubation of HeLa cell nuclear extracts with an oligonucleotide containing the ODC TATA box also caused formation of two specific bands in EMSA. Both bands exhibited augmented binding to nuclear proteins from TPA-treated cells. Introduction of the T-->A transversion mutation in the ODC TATA oligonucleotide eliminated binding of the faster migrating band formed with the natural ODC TATA oligonucleotide. These results indicate that TPA modulation of the general transcription machinery may play a role in the TPA-activated transcription of the human ODC promoter.

Cell Nucleus↗
PubMed1996

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity by genistein, a tyrosine kinase inhibitor.

The effects of the protein tyrosine kinase inhibitor genistein on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity in monkey kidney epithelial CV-1 cells were determined. CV-1 cells were pretreated with genistein for 2 hr before treatment with 100 nM TPA. ODC activity was determined 9 hr after TPA treatment. Genistein inhibited TPA-induced ODC activity at 0.1, 1, 10, 25, 50, 100, 200, and 400 microM by 0%, 0%, 42%, 59%, 62%, 81%, 91%, and 100%, respectively (IC50 = 20 microM). Genistein inhibited TPA-induced mitogen-activated protein kinase (MAPK) tyrosine phosphorylation and the accumulation of steady state levels of ODC mRNA at 400 microM but not at 25 microM. Genistein, at 25 microM, did not alter the TPA-induced phosphorylation of p90 ribosomal S6 kinase but caused a approximately 50% decrease of the TPA-induced phosphorylation of p70 S6 kinase (p70S6K), a protein kinase involved in the control of translational efficiency. Taken together, these data indicate that genistein may inhibit TPA-induced ODC activity at the transcriptional and translational levels through the inhibition of MAPK and p70S6K activation, respectively. The regulation of MAPK and p70S6K may be mediated through different protein tyrosine kinases that have differential sensitivity to genistein.

Animals↗
PubMed1995

Two residues that may ligate Ca2+ in transmembrane domain six of the plasma membrane Ca(2+)-ATPase.

In order to identify Ca2+ ligands in the putative transmembrane domain 6 of the plasma membrane Ca2+ pump, amino acids Asn879, Met882, Asp883, and Ser887 were singly altered. Asn879, Met882, and Asp883 were chosen because the corresponding amino acids have been proposed as Ca2+ ligands in the sarcoplasmic reticulum Ca2+ pump (Clarke, D. M., Loo, T. W., and MacLennan, D. H. (1990) J. Biol. Chem. 265, 6262-6267). For the alterations, a fully active truncated version of the pump was used, because the interaction of Ca2+ with the pump could be studied without interference from calmodulin binding. The mutants at Asn and Asp did not carry out ATP-supported Ca2+ uptake and formed no acylphosphate from [gamma-32P]ATP, suggesting that, like the corresponding amino acids in the sarcoplasmic reticulum Ca2+ pump, these two are Ca2+ ligands. However, all the mutants at the position of Met882 showed some activity. Indeed, the Met882--> Ile mutant was fully active at a saturating Ca2+ concentration and only the K1/2 for Ca2+ activation was shifted slightly upward. Converting the Met to Thr (which is the corresponding residue in the sarcoplasmic reticulum Ca2+ pump) reduced the activity to 20% of the wild type, further emphasizing the differences between the two Ca2+ pumps. The mutant Ser887--> Ala was expressed in greater amounts than, and had a specific activity about 50% higher than, the wild type, indicating that this serine also could not be a Ca2+ ligand and could not replace the missing Thr at position Met882.

Amino Acid Sequence↗
PubMed1995

Lack of 12-O-tetradecanoylphorbol-13-acetate responsiveness of ornithine decarboxylase introns which have AP-1 consensus sequences.

The AP-1 consensus sequences (TGAGTCA) are the major 12-O-tetradecanoylphorbol113-acetate (TPA) responsive elements shared by several TPA inducible genes, such as c-sis, c-fos, c-myc, collagenase, stromelysin, hMTIIA and SV40. However, the role of AP-1 binding sites, which are present in the introns 3, 5, and 11 of ODC gene, in the regulation of TPA-induced ornithine decarboxylase (ODC) gene transcription are unknown. We determined the TPA responsiveness of the AP-1 sequences in the introns of ODC gene in CV-1 cells which induce ODC activity and mRNA in response to TPA treatment. ODC introns containing AP-1 sequences were inserted into the chloramphenicol acetyltransferase (CAT) reporter gene. Transient transfection of CV-1 cells with the intron-CAT constructs followed by TPA treatment did not induce CAT activity. However, when flanking regions of the AP-1 site in intron 3 were narrowed down to 74 bp, TPA induced CAT activity by 5- to 7-fold. The TPA-inducibility could be eliminated by mutation of the AP-1 site (TGAGTCA-->TGATGCCA or TGATGA) in 74 bp of intron 3. These results indicate that the AP-1 sequences in the intact ODC introns may not be responsive to TPA. The flanking sequences of the AP-1 site may be crucial to determine whether the AP-1 site is accessible to the TPA-induced transcriptional factor(s).

Animals↗
PubMed1995

Community perception of mosquitos and mosquito control in some areas of urban Delhi and rural Alwar, Rajasthan.

There is little information regarding community perception of mosquitos and and their control in India. A qualitative study based on In depth discussions with the people in Urban Delhi, and rural Alwar was carried out in August-September, 1995. In the Ghazipur and Munirka areas of Delhi, 63 respondent groups with 171 head counts, and 59 respondent groups and 305 persons in Alwar were met. Community responses to uniform set of questions on mosquito types, seasonality, biting, breeding places, ongoing control activities, and methods of personal protection were recorded. Most people in the areas surveyed viewed mosquitos primarily as a biting nuisance, and secondarily as agents of disease and debilitators of health. In general the knowledge regarding mosquitos was deficient. An overwhelming tendency to attribute mosquitogenic conditions to the unsavoury features of one's physical environment was observed in both areas. The methods of personal protection and attitudes to organised control are discussed.

Animals↗
PubMed1994

Combining polyamine depletion with radiation therapy for rapidly dividing head and neck tumors: strategies for improved locoregional control.

PURPOSE: Locoregional control is adversely affected as clonogens from rapidly proliferating tumors repopulate during a course of radiation therapy. The cytostatic agent alpha-difluoromethylornithine (DFMO) was investigated for its capacity to slow proliferation kinetics in human squamous cell carcinomas (SCC) of the head and neck (H&N), with the ultimate objective of improving locoregional control in rapidly dividing tumors treated with radiation therapy. METHODS AND MATERIALS: Three human SCC cell lines established from primary H&N tumors were evaluated in vitro (cell culture) and in vivo (SCC tumor xenografts in athymic mice) for the capacity of DFMO to induce growth inhibition. Flow cytometry analysis of SCC tumor growth kinetics and quantitative assessment of polyamine biosynthesis inhibition was performed to verify DFMO activity. DFMO effects on in vitro SCC radiosensitivity using clonogenic survival were also studied. RESULTS: A noncytotoxic exposure to DFMO (5mM x 72 hours) induced pronounced growth inhibition in all three SCC cell lines (70-90% at 7 days), and induced a 2-3 fold delay in volume doubling time for SCC tumor xenografts when administered orally in the drinking water (1.5%) to athymic mice. Kinetic analysis via flow cytometry confirmed that DFMO produced a lengthening of SCC cell cycle times, but did not alter in vitro radiosensitivity. Inhibition of ornithine decarboxylase (ODC) activity and depletion of endogenous polyamines (putrescine and spermidine), were confirmed in normal tissue (mouse skin) and in human SCC tumor xenografts of athymic mice receiving continuous oral DFMO. CONCLUSION: These data indicate that antiproliferative agents, such as DFMO, are capable of altering human SCC growth kinetics without altering intrinsic radiosensitivity. Such kinetic modulation may therefore provide a strategy to reduce the adverse impact of tumor cell proliferation during a radiotherapy treatment course for rapidly dividing tumors such as those in the H&N.

Animals↗
PubMed1994

Regulatory region of plasma membrane Ca2+ pump. 28 residues suffice to bind calmodulin but more are needed for full auto-inhibition of the activity.

The Ca(2+)-pumping activity of constructs containing various portions of the putative 28-residue calmodulin-binding domain (C domain) of hPMCA4b were compared. As the length of the C domain in the pump increased, the pump's activity decreased and the ability of calmodulin to stimulate the activity increased. Study of the calmodulin dependence of activity showed that the construct containing all 28 residues of the C domain had a K1/2 for calmodulin equal to that of the complete molecule; the constructs containing less of the C domain interacted less strongly with calmodulin. On the other hand, incorporation of all 28 residues of the C domain did not decrease the activity of the pump (in the absence of calmodulin) as low as the activity of the complete molecule. This indicates that other segments of the molecule, further toward the COOH terminus, are also required for the degree of inhibition seen in the complete molecule.

Amino Acid Sequence↗
PubMed1994

The Ca2+ affinity of the plasma membrane Ca2+ pump is controlled by alternative splicing.

The plasma membrane Ca2+ pump is a calmodulin-regulated P-type ATPase that is an essential element in controlling intracellular Ca2+ concentration. Studies on the gene structure of this pump have revealed an alternate splice option that changes the structure of the calmodulin-binding domain. This change in the structure of the enzyme results in a reduced calmodulin affinity. Tests of the enzyme's activity in the presence of a high calmodulin concentration, approximating that found inside living cells, show that this reduced calmodulin affinity causes a reduced apparent affinity of the enzyme for Ca2+. This shift in the Ca2+ activation occurs in a Ca2+ concentration range crucial to cellular function and is probably the physiologically important consequence of the alternate splice.

Alternative Splicing↗