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Biomedical subjects

D Joshi

Publications and source records attributed to D Joshi.

At least 19 recordsLinked to original sources
PubMed1996

Tamoxifen (estrogen antagonist) inhibits voltage-gated calcium current and contractility in vascular smooth muscle from rats.

Tamoxifen (Tx) has been used in breast cancer treatment and prophylaxis because of its antiestrogenic activity; however, Tx may also have beneficial cardiovascular effects and other actions mediated by mechanisms other than estrogen receptors. Previous studies showing interactions of Tx with Ca+(+)-channel blockers suggested that Tx may affect Ca++ channels, an hypothesis that was investigated using whole cell patch clamp techniques in vascular smooth muscle cells (cell line A7r5 and freshly dissociated cells) and by determining effects on contractions of isolated blood vessels. Tx reduced current through L-type Ca+2 channels, with an ID50 of 2 x 10(-6) M when applied by cumulative addition to A7r5 cells. With acute application, 10(-6) M Tx significantly reduced L-type current in A7r5 cells within 2 min to 88% of control (vehicle, 0.1% ethanol) in A7r5 cells, 67% of control in aortic vascular smooth muscle cells, and 60% of control in tail artery vascular smooth muscle cells. Tx also decreased the rate of inactivation of L-type current. Inhibition of T-type current by Tx was less than for L-type current but was significant at 10(-5) M Tx. Treatment of tail artery rings with Tx (10(-5) M, 15 min; 10(-6) M, 4 hr) reduced K+-elicited contractions. Since therapeutic concentrations of Tx during treatment may exceed 10(-6) M, these effects of Tx on vascular smooth muscle Ca++ channels and vessel contractions may have a role in the efficacy and side-effects of Tx treatment.

Animals↗
PubMed1995

Age-related alterations in the expression of prohormone convertase messenger ribonucleic acid (mRNA) levels in hypothalamic proopiomelanocortin mRNA neurons in the female C57BL/6J mouse.

POMC processing is mediated by the prohormone convertases (PC1 and PC2). The cleavage of beta-endorphin-(1-31) is mediated by PC2. PC2 can also further cleave beta-endorphin-(1-31) to beta-endorphin-(1-27). We previously reported a significant increase in the proportion of beta-endorphin-(1-27) and -(1-27) forms in the arcuate nucleus (ARC) of the hypothalamus in middle-aged females with irregular estrous cycles (5-7 days) compared to young female C57BL/6J mice with regular cycles (4-5 days). Changes in processing enzymes may be a mechanism underlying this change. We compared ARC messenger RNA (mRNA) levels of PC1, PC2, and furin by Northern blot and in situ hybridization analyses in young, middle-aged, and old mice. Antisense complementary RNA probes to mouse PC1, PC2, and furin were radiolabeled and used in single label studies, alone or in combination with a mouse POMC digoxigenin-labeled complementary RNA probe for double label studies. For Northern blot analysis, young (4- to 5-month-old) normally cycling (4-5 days) mice at diestrus were compared to middle-aged (12- to 13-month-old) irregularly cycling (5-7 days) mice at diestrus. By Northern blot analysis, a significant increase (P < 0.05) in ARC PC2 mRNA levels was detected in middle-aged compared to young mice, but ARC PC1 and furin mRNA levels were unaltered. Single label in situ hybridization analysis confirmed these findings in the general neuron population. We also observed a significant reduction in ARC furin mRNA levels in old mice compared to either young or middle-aged mice. Double labeling in situ hybridization histochemistry demonstrated that PC2 mRNA levels were significantly increased (at least 2-fold) in POMC mRNA-containing neurons of middle-aged compared to young mice. Selective changes in PC2 mRNA levels in ARC POMC neurons are correlated with changes in beta-endorphin-(1-31) processing to beta-endorphin-(1-27)/(1-26) in middle-aged animals. Our data suggest that the natural age-related shift in beta-endorphin peptide processing is mediated by PC2.

Aging↗
PubMed1995

Hypothalamic processing of beta-endorphin in female C57BL/6J mice is altered at middle age.

beta-Endorphin (beta-endo) (1-31) is the active opioid peptide product of pro-opiomelanocortin processing. Further post-translational modifications of beta-endo(1-31) yield beta-endo(1-27), (1-26) and their acetylated forms which are considered to be opiate receptor antagonists. Mechanistically, alteration in opiatergic properties is likely to result in the loss of a number of physiological functions including reproductive capacity. The purpose of this study was to determine whether there are changes in the way beta-endo neurones process the peptide with age in female C57BL/6J mice. Pooled extracts of arcuate nucleus (ARC) and preoptic area (POA) of 3- to 4-month-old normally cycling (4-5 days at dioestrus), 12- to 13-month-old irregularly cycling (5-7 days at dioestrus), 23- to 24-month-old acyclic (in persistent dioestrus) animals were subjected to reversed-phase HPLC (n = 4 experiments). Column fractions were assayed for beta-endo-like-immunoreactivity by sequence-specific RIAs. The opiate receptor active as well as opiate receptor antagonist forms of beta-endo were present in both ARC and POA at all three age groups although their ratios varied. beta-Endo(1-31), the active opiate, was the predominant form in young animals. At middle age there was a threefold (P < 0.05, ANOVA) increase in the antagonist forms of beta-endo and this was associated with a significant (P < 0.05, ANOVA) increase in the ratio of antagonist to active forms. This was accompanied by a trend toward an increase in acetylated forms of beta-endo in middle-aged mice. HPLC profiles from hypothalami of old animals more closely resembled those of young females. The increase in the antagonist forms of beta-endo at middle age may contribute to a decline of opiatergic influences in the female C57BL/6J mouse and suggest a mechanism whereby alterations in opiate influence over gonadotrophin control may occur.

Aging↗
PubMed1995

Luteinizing hormone response to N-methyl-D, L-aspartic acid in the presence of physiological estradiol concentrations: influence of age and the ovary.

We have previously reported that the pituitary of intra-atrially cannulated old female C57BL/6J mice is as capable of responding to a GnRH challenge as is that of young females (10). We have observed elevated luteinizing hormone (LH) levels in ovariectomized (OVX) intra-atrially cannulated mice. Sustained physiologic levels of estradiol (E2) for 6 days suppressed circulating LH to intact levels. However, in that model, a bolus of E2 following E2 priming was unable to elicit an LH surge (Joshi et al., unpublished findings). The present studies were designed to examine: first, whether GnRH neurons are competent to release GnRH in the presence of tonic physiologic levels of E2 and, second, whether either age or the ovary can influence GnRH neuronal responsiveness. The N-methyl-D, L-aspartic acid (NMA)-evoked GnRH response was assessed indirectly by measuring LH in two groups of OVX C57BL/6J mice: short-term OVX (S-OVX) (1 week) mice were either prepubertal (5 weeks), postpubertal (10 weeks), young (5 months), middle aged (12 months), or old (24 months). Long-term OVX (L-OVX) mice were either young (5 months), or old (24 months) and OVX at puberty; middle-aged L-OVX mice were OVX at 8 months and examined at 12 months of age. Animals were administered physiologic levels of E2 by subcutaneous silastic capsule for 1 week before testing. LH secretion was inhibited by E2 in S-OVX mice of all ages. In no case did NMA overcome this inhibition in E2 primed S-OVX females. E2 also inhibited LH secretion in L-OVX mice of all ages, but NMA was able to overcome the E2 inhibition of LH secretion in L-OVX mice (young: 0.5 +/- 0.1, 0.84 +/- 0.19 ng/ml, first and second challenge, respectively; middle-aged: 0.46 +/- 0.1, 1.08 +/- 0.16 ng/ml; and old: 1.44 +/- 0.19, 0.99 +/- 0.27 ng/ml). This last effect was independent of animal maturity at the time of OVX or animal age at the time of experiment. These findings suggest that although the ovaries in the 24-month-old S-OVX mice had not produced enough E2 to alter the vaginal cytology for 2 +/- 0.5 months before the experiment, the ovarian modulation of the inhibitory effect of E2 on NMA-induced LH secretion was still present. The nature of the ovarian factor(s) modulating this effect is unknown.(ABSTRACT TRUNCATED AT 400 WORDS)

Age Factors↗
PubMed1993

Bedside measurement of creatine kinase to guide thrombolysis on the coronary care unit.

Thrombolysis is effective in treating patients with acute myocardial infarction when started within 12 h of onset, and thus is in widespread use. Early diagnosis is essential but some patients admitted to the coronary care unit with myocardial infarction do not meet the diagnostic criteria on arrival, and thus do not undergo thrombolysis. We studied 117 patients admitted consecutively to our coronary care unit with suspected but unproven myocardial infarction; normally none would have received thrombolytic agents. In each patient creatine kinase levels were measured on admission and after intervals by nurses using capillary blood samples and a dry chemistry system. Infarction was subsequently confirmed in 29 patients. Of these, 17 (59%) were correctly diagnosed and underwent thrombolysis within 12 h on the basis of a raised creatine kinase measured at the bedside. Our findings suggest that use of a bedside assay by nurses allows additional patients with myocardial infarction to receive the benefit of thrombolytic therapy within the first 12 h.

Clinical Enzyme Tests↗
PubMed1993

Clinical application of motorsensory differentiated nerve repair.

Since 1979 acetylcholinesterase has been used in clinical practice for motor sensory differentiation. It was first used for median and ulnar nerve injuries at the wrist. Recently the application was extended to secondary nerve repair, including plexus reconstruction. The aim of this study was to present, for the first time, clinical results of motor sensory differentiated median and ulnar nerve repair and to show the advantage of this method in secondary repair. We compared a group of nine patients with motor sensory differentiated median and ulnar nerve repair with a group of 13 patients without motor sensory differentiation. Sensibility testing, strength measurements, and anatomical examinations were performed. The hand function was expressed in percentage values. Compound muscle action potentials and sensory antidromic conduction velocities were measured electroneurographically. Sensibility recovery was significantly better after motor sensory differentiated median nerve repair (P < 0.05). In secondary nerve repair acetylcholinesterase was used additionally for evaluating the level of resection of the proximal stump. The time required for perioperative histochemical differentiation has now been reduced to 2 hr.

Acetylcholinesterase↗
PubMed1993

Modulation of hypothalamic mu-opioid receptor density by estrogen: a quantitative autoradiographic study of the female C57BL/6J mouse.

The labelling of hypothalamic binding sites by [125I]-FK, a specific mu-opioid receptor ligand, was studied in female C57BL/6J mice to test whether removal of ovarian steroids affected the density of distribution of receptor binding. Labelling densities in the forebrain of normally cycling (intact) females (N = 12), were compared to those in mice that had been ovariectomized (OVX) for 6 weeks (n = 8) and in mice that had been OVX and implanted with an estradiol (E2) capsule (OVX+E2) for 6 weeks (n = 11). Frozen sections from the rostral forebrain were incubated with 1 nM [125I]-FK and processed for light microscopic autoradiography. The diagonal band of Broca (DBB), organum vasculosum lamina terminalis (OVLT), preoptic area (POA), septum, parietal cortex, and striatum were analyzed using computerized image analysis. The distribution of labelling was similar in all three experimental groups in all the regions; however, labelling was significantly reduced in the ventrolateral POA of OVX animals compared to intact females. Labelling densities in the OVX animals replaced with the gonadal steroid estradiol were not significantly different from those in normally cycling mice. This study demonstrates a region-specific loss of mu-opiate receptor labelling following long-term deprivation of gonadal steroids, and supports the hypothesis that estrogen directly or indirectly influences the density of mu-opioid receptors in the rostral forebrain of female mice.

Animals↗
PubMed1993

Peripheral nerve repair in the hand with and without motor sensory differentiation.

To investigate the value of motor sensory differentiated nerve repair, we examined a group of 9 patients with motor sensory differentiated nerve repair and a group of 13 patients without motor sensory differentiated nerve repair. The clinical and electroneurographic findings were compared. For the clinical examination, Millesi's scoring system was used. The hand function after motor sensory differentiated median nerve repair was 72% +/- 16% compared with 57% +/- 14% without motor sensory differentiation. The hand function after motor sensory differentiated median and ulnar nerve repair was 53% +/- 12% compared with 43% +/- 24% without motor sensory differentiation. After ulnar nerve repair the achieved values for hand function were high even without motor sensory differentiation. Our results indicate that intraoperative motor sensory differentiation of injured nerves is helpful to reestablish particularly the sensory function in median nerve injuries.

Action Potentials↗
PubMed1993

A rapid method for microbial sample preparation for the scanning electron microscope.

A synthetic aromatic polymer has been used for preparing replicas of different microorganisms. This method of preparing highly concentrated (9.6 k) microbiological samples for scanning electron microscopy was compared with a standard method. The micrographs of the replicated samples are satisfactory. This method is rapid, cost effective and produces good results, especially in the case of spore-forming mycelial microorganisms.

Aspergillus fumigatus↗
PubMed1993

Gonadotropin hormone-releasing hormone induced luteinizing hormone responses in young and old female C57BL/6J mice.

The estrogen-induced luteinizing hormone (LH) surge is markedly attenuated in old (24 months) female mice compared with young (6 months) females. To test whether or not this attenuated LH response is due to a diminished capacity of the pituitary to respond to hypothalamic gonadotropin hormone-releasing hormone (GnRH), the pituitary response to exogenous GnRH was measured in young (5-6 months), normally cycling (n = 6) and old (24 months), acyclic constant diestrus (n = 6) C57BL/6J female mice. Mice were ovariectomized and estrogen-treated for 7 days. After intrajugular catheterization on Day 6, serial blood samples were taken at 15-min intervals for 165 min on Day 7. Serum LH was measured in samples obtained before and after infusion of either saline or GnRH (5 micrograms/5 microliters saline/kg body wt) and 15 micrograms/15 microliters saline/kg body wt 1 hr apart). Saline-treated animals demonstrated no LH response in either young (0.09 +/- 0.02 ng/ml baseline) or old (0.11 +/- 0.01 ng/ml baseline) females. However, a significant release of LH was obtained in response to each challenge of GnRH in both young (0.3 +/- 0.04 ng/ml first challenge, 0.69 +/- 0.1 ng/ml second challenge) and old (0.78 +/- 0.1 ng/ml first challenge, 1.76 +/- 0.2 ng/ml second challenge) mice. The LH response in the aged group was significantly greater (P < 0.05, analysis of variance) than in the young group. These results show that pituitaries of old female mice were at least as capable of responding to exogenous stimulation by GnRH as those of young. We conclude that alteration in the capacity of the pituitary to respond to GnRH is not likely to be a factor contributing to altered LH secretion with age in C57BL/6J mice.

Aging↗
PubMed1992

Reversed-phase liquid chromatographic purification and isolation of a radio-iodinated selective probe for mu opioid receptors in the brain.

A Guard-PAK precolumn system was used for the reversed-phase liquid chromatography purification of a small, synthetic radiolabeled opioid peptide, FK 33-824 (D-Ala2, methyl-phe4, Met (O)ol5 enkephalin) (FK). This procedure involves trace enrichment of iodinated peptide onto the precolumn while iodination reagents are not retained. Radioactive contamination of high-performance liquid chromatography columns and injectors is thus avoided. Precolumn chromatography has sufficient resolving power to separate not only labeled from unlabeled peptide but also mono- from di-iodinated peptide. Purified 125I-labeled FK (estimated specific activity 85.9-153.7 Ci/mmol) showed high specific binding to mouse corpus striatum, neocortex, cingulate cortex, nucleus accumbens septi, diagonal band of Broca, nucleus medialis septi, area preopticus magnocellularis, and the nucleus of the caudate/putamen. Radioligand binding was inhibited by both antagonists (naloxone and naltrexone); and agonists D-Ala2, N-methyl-phe4, gly-ol5-enkephalin [DAGO]; FK; and beta-endorphin at all concentrations tested (1 x 10(-8) to 1 x 10(-4) M). Adrenocorticotropin hormone (ACTH) did not block ligand binding at any concentration tested. Distribution of mu opioid receptors was analyzed by light microscopic autoradiography. Sections incubated with 125I-labeled FK in the presence of agonists and antagonists demonstrated decreasing ligand binding with increasing doses of competitor. ACTH did not block ligand binding at any concentration tested. HPLC analyses of ligand which had been iodinated 1.5 half lives before the date of the experiment demonstrated a single peak similar to that of freshly iodinated ligand. Similar binding kinetics and autoradiographic labeling patterns were observed as compared to those obtained with freshly iodinated peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Light flashes of different durations (0.063-3.33 msec) phase shift the circadian flight activity of a bat.

The phase-response curve (PRC) for the circadian rhythm in the flight activity of a cave-dwelling bat, Hipposideros speoris, constructed with 0.063-msec light flashes, reported here is the first of its kind for any circadian system and is unlike any other phase-response curves constructed for other nocturnal animals. The phase responding with maximal advances (90 degrees) and the phase responding with maximal delays (0 degree) of this PRC were exposed to light flashes of systematically varying durations from 0.083 to 3.33-msec. For 0 degree phase, the flashes of 0.063-3.33 msec effected delay phase shifts of comparable magnitude. For 90 degrees phase, the flashes of 0.063-1.0 msec effected advance phase shifts, whereas 3.33-msec flashes effected unmistakable delay phase shifts with advancing transients. Phase shifts evoked with such light flashes are further compared with phase shifts evoked with pulses of longer durations (15 min to 2.8 hr) for H. speoris.

Animals↗