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Biomedical subjects

K K Sharma

Publications and source records attributed to K K Sharma.

At least 37 records · Page 2Linked to original sources
PubMed2002

Evaluation of the analgesic effect of neurosteroids and their possible mechanism of action.

The present study investigates the effect of progesterone (P), a pregnane precursor of neurosteroids and 4-chlordiazepam (4-CD), a high affinity ligand for mitochondrial diazepam binding inhibitor receptor (MDR) that stimulates neurosteroid synthesis, in both acute, (tail flick latency test, TFL) and chronic, (formalin-induced pain response, FT), models. Both P and 4-CD showed an analgesic response in these models. The effect of P and 4-CD was antagonized by bicuculline on TEL but not in FT. However, naloxone attenuated the antinociceptive response of P and 4-CD in TFL as well as FT. Further, P and 4-CD pretreatment potentiated the analgesic effect of morphine and nimodipine in both the models of pain sensitivity. Thus, neurosteroids produce an antinociceptive effect which may be mediated by modulation of GABAergic and/or opiodergic mechanisms as well as voltage gated calcium channels.

Analgesics↗
PubMed2002

Differential effects of benzodiazepines on immune responses in non-stressed and stressed animals.

Benzodiazepines (BZDs) used extensively as antianxiety agents are known for their low toxicity. However, a long lasting depression of mitogen stimulated secretion of macrophage-derived cytokines has been shown in offsprings of rats that were exposed to diazepam during pregnancy. The Present study investigates the effects of long term administration of diazepam and alprazolam on humoral and cell-mediated immune responses in adult male Wistar rats and Swiss albino mice. Administration of diazepam (5 mg/kg/day x 7-14 d) and alprazolam (1 mg/kg/day x 7-14 d) produced a significant reduction of anti-SRBC antibody titre, a measure of humoral immune response, and foot pad thickness and % leucocyte migration inhibition (% LMI), measures of cell-mediated immune responses. Administration of diazepam (5 mg/kg, i.p.) or alprazolam (1 mg/kg, i.p.) before subjecting the animals to restraint stress (RS) reversed the immunosuppressive effects of RS. Both per se immunosuppressive effects and attenuation of RS-induced immunosuppression of BZDs was antagonized by flumazenil (10 mg/kg, i.p.), a central BZD receptor antagonist. Thus, BZDs appear to modulate the immune system in non-stressed and stressed adult animals in a differential manner and these effects are mediated via central benzodiazepine receptors.

Animals↗
PubMed2002

Effect of minocycline and tetracycline on immunological responses in experimental animals.

In recent years, there has been an increase in the use of antibiotics, primarily tetracycline anlogues, like minocy cline to treat rheumatoid arthritis. However, the mechanism of action of these analogues is not clearly defined. The present study investigates the effects of minocycline and tetracycline on some immunological parameters in Wistar rats and Swiss albino mice. Haemagglutination (HA) titre was employed as parameter of humoral immune response and % leukocyte migration inhibition (% LMI) and footpad thickness tests were used as measures of cell mediated immune response. Both minocycline and tetracycline significantly improved humoral immune response in rats as indicated by an increase in anti-SRBC antibody titre. In the LMI test, depending on the time period of drug administration, there was an increase or a decrease in the % LMI. When drugs were administered on days 1-7 after sensitization, both the compounds caused a significant increase in % LMI. However, the % LMI was significantly decreased when the drugs were administered on days 7-13 of sensitization, indicating variable effects of these agents on the Immune mechanism depending on the time of administration in relation to the development of immune responsiveness. Both minocycline as well as tetracycline produced a significant decrease in the paw volume in the footpad-thickness test which indicates a decrease in lymphokine production/release. The present study thus shows that minocycline and tetracycline exhibit immunomodulatory properties, which may contribute significantly to their beneficial effects in rheumatoid arthritis.

Animals↗
PubMed2001

Phe71 is essential for chaperone-like function in alpha A-crystallin.

Experiments with mini-alphaA-crystallin (KFVIFLDVKHFSPEDLTVK) showed that Phe(71) in alphaA-crystallin could be essential for the chaperone-like action of the protein (Sharma, K. K., Kumar, R. S., Kumar, G. S., and Quinn, P. T. (2000) J. Biol. Chem. 275, 3767-3771). In the present study we replaced Phe(71) in rat alphaA-crystallin with Gly by site-directed mutagenesis and then compared the structural and functional properties of the mutant protein with the wild-type protein. There were no differences in molecular size or intrinsic tryptophan fluorescence between the proteins. However, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid interaction indicated a higher hydrophobicity for the mutant protein. Both wild-type and mutant proteins displayed similar secondary structure during far UV CD experiments. Near UV CD signal showed a slight difference in the tertiary structure around the 285-295 region for the two proteins. The mutant protein was totally inactive in suppressing the aggregation of reduced insulin, heat-denatured citrate synthase, and alcohol dehydrogenase. However, a marginal suppression of beta(L)-crystallin aggregation was observed when mutant alphaA-crystallin was included. These results suggest that Phe(71) contributes to the chaperone-like action of alphaA-crystallin. Therefore we conclude that the 70-88-region in alphaA-crystallin, identified by us earlier, is the functional chaperone site in alphaA-crystallin.

Amino Acid Sequence↗
PubMed2001

Analysis of alpha-crystallin chaperone function using restriction enzymes and citrate synthase.

PURPOSE: To compare the abilities of [alpha]A-crystallin, [alpha]B-crystallin, and mini-[alpha]A-crystallin (a synthetic peptide chaperone representing the functional unit of [alpha]A-crystallin) to protect against heat-induced inactivation of citrate synthase (CS) and restriction enzymes, SmaI and NdeI. METHODS: Restriction enzymes, SmaI and NdeI were heated at different temperatures in the presence of various amounts of molecular chaperones and tested for their ability to cleave plasmid DNA. The aggregation of CS was measured at 43 degrees C while the loss in activity was monitored at 37 degrees C in the presence of various crystallins. RESULTS: Restriction enzyme activities were protected by the crystallin subunits up to 37 degrees C for SmaI and 43 degrees C for NdeI. However, the mini-[alpha]A-crystallin was unable to protect endonuclease activity. The crystallin subunits and the peptide chaperone were able to suppress thermal aggregation of CS at 43 degrees C, but failed to stabilize its activity at 37 degrees C. CONCLUSIONS: The ability of [alpha]-crystallin subunits to stabilize denaturing proteins varies from enzyme to enzyme as evidenced by the inactivation of CS and protection of SmaI and NdeI activity in the presence of [alpha]-crystallin subunits. Additionally, our results show that there could be more than one site in [alpha]A-crystallin responsible for its chaperone-like action. By addition of crystallin subunits to restriction enzymes prior to or during storage, transport, or assay would maintain or improve their activity thereby decreasing their cost.

Animals↗
PubMed2001

Studies on trypsin-modified bovine and human lens acylpeptide hydrolase.

Acylpeptide hydrolase removes the N -acetylated amino acids from the peptide substrates but not from intact proteins. Cleavage between amino acid residues 203--204 of the native acylpeptide hydrolase results in the formation of a 55 kDa truncated active enzyme in the bovine lens, in vivo. In this study we explored the hydrolytic properties of the truncated enzyme using lens beta- and gamma-crystallins as substrates. SDS--PAGE analysis indicated that the beta B2-crystallin was cleaved by truncated acylpeptide hydrolase into several protein fragments (10--26 kDa). No cleavage of the gamma-crystallins was observed under similar conditions. Both the acylpeptide hydrolase activity and the protease activity of the 55 kDa enzyme were completely inhibited by diisopropylfluorophosphate, p -chloromercuribenzoate and ebelactone, and moderately inhibited by N -tosyl phenylalanine chloromethyl ketone. SDS--PAGE analysis followed by fluorography of ((3)H) diisopropylfluorophosphate labeled human lens acylpeptide hydrolase preparation showed the presence of the 55 kDa truncated form of the enzyme, as observed in the bovine lens. The peptide (d)-AIKGDQFL-NH(2)--the amino acid sequence 200--207 of the native bovine acylpeptide hydrolase with an in vivo cleavage site of native protein--was hydrolysed by the lens protease(s) suggesting that the in vivo generation of the 55 kDa acylpeptide hydrolase may be mediated through a proteolytic processing. The protease(s) responsible for the cleavage of this peptide was inhibited by diisopropylfluorophosphate and p -chloromercuribenzoate.

Adolescent↗
PubMed2001

Interaction of alpha-lactalbumin with mini-alphaA-crystallin.

alphaA-Crystallin can function like a molecular chaperone. We have recently shown that residues 71-88 in alphaA-crystallin represent the "chaperone active site" of the protein. A peptide containing the sequence of alphaA-crystallin sequence DFVIFLDVKHFSPEDLTVK (mini alphaA-crystallin) by itself displays the antiaggregation property of alphaA-crystallin. We have prepared a complex of reduced alpha-lactalbumin and mini-alphaA-crystallin and investigated the nature, conformation, and properties of the complex by dynamic light scattering, HPLC analysis, CD spectroscopy, and fluorescence studies. Although mini-alphaA was able to prevent the precipitation of reduced alpha-lactalbumin, large aggregates (50-500 nm) of the complex were formed during the assay. Amino acid composition estimation revealed that alpha-lactalbumin and mini-alphaA-crystallin were present in 1:2 ratio in the aggregates. During our study significant red shift in the Trp fluorescence emission maximum and an increase in Bis-ANS binding to the mini alphaA-crystallin-bound alpha-lacatalbumin were observed. The CD spectra of the complex showed a significant loss of alpha-helical content but the beta-sheet content appeared to be less affected, indicating the molten-globule state of the reduced lactalbumin in the complex. These data show that the active site of alphaA-crystallin by itself can maintain a significantly denatured and unfolded protein in soluble form.

Anilino Naphthalenesulfonates↗
PubMed2001

Conformational specificity of mini-alphaA-crystallin as a molecular chaperone.

The chaperone activity and biophysical properties of the 19 amino acid peptide DFVIFLDVKHFSPEDLTVK, identified as the functional element in alphaA-crystallin and here referred to as mini-alphaA-crystallin, were studied using light scattering and spectroscopic methods after altering its sequence and enantiomerism. The all-D and all-L conformers of the peptide do not show marked differences in their chaperone-like activity against heat-induced aggregation of alcohol dehydrogenase at 48 degrees C and dithiothreitol-induced aggregation of insulin. The retro peptide does not show any secondary structure and is also unable to act like a chaperone. Both all-L and all-D peptides lose their beta-sheet conformations, hydrophobicity and chaperone-like activity at temperatures > 50 degrees C. However, upon cooling, a significant portion of those properties was regained, suggesting temperature-dependent, reversible structural alterations in the peptides under investigation. We propose that both the hydrophobicity and beta-sheet conformation of the functional element of alphaA-crystallin are essential for chaperone-like activity.

Amino Acid Sequence↗
PubMed2001

Development of differential tolerance to the sedative and anti-stress effects of benzodiazepines.

Differential degree of tolerance has been reported to develop for anticonvulsant, sedative and skeletal muscle relaxant effects of benzodiazepines (BZDs). Acute treatment with BZDs reportedly reduces the formation of gastric stress ulcers and attenuates stress-induced immunosuppression. The present study investigates whether tolerance develops to these antistress effects of BZDs by using diazepam and chlordiazepoxide as representative drugs. A single dose of diazepam (5 mg/kg, i.p.) or chlordiazepoxide (20 mg/kg, i.p.) produced a significant reduction in locomotor activity, a measure of sedative effect and antagonized the effect of restraint stress (RS) on gastric mucosal lesions and anti-sheep red blood cell (SRBC) antibody titre. With chronic treatment (X 7 d), there was a marked tolerance to the sedative effect of both the studied BZD drugs, while much less tolerance developed to their ulcer protective action. However, no tolerance was observed to the attenuating effect of diazepam and chlordiazepoxide on RS-induced immunosuppression. Thus, the results of the present study indicate that different mechanisms may be involved in the development of tolerance to the sedative, antiulcer and immunomodulatory effects of BZDs.

Animals↗
PubMed2001

Cybersex with minors: forensic implications.

This paper is designed to assist forensic psychiatrists/psychologists who evaluate adults who commit sexual crimes against children on the Internet. The typical offender is an adult male who logs onto the Internet and enters a chat room in which children congregate. Unbeknownst to the offender, undercover police officers are posing as minors in the chat rooms. The undercover officer (pretend kid) and offender engage in increasingly explicit, sexual conversation; the offender may transmit erotic photographs to the undercover officer and/or arrange to meet at a motel in order to have sexual intercourse. The authors will discuss the relevant legal, clinical, and ethical aspects of examining these offenders, and describe specific cases that the author (2) evaluated.

Adolescent↗
PubMed2001

Possible mechanism of anticonvulsant effect of ketamine in mice.

The study was designed to investigate the effect of ketamine on convulsive behaviour using maximal electroshock (MES) test. An attempt was also made to study the possible receptor mechanisms involved. MES seizures were induced in mice via transauricular electrodes (60 mA, 0.2sec). Seizure severity was assessed by the duration of tonic hindlimb extensor phase and mortality due to convulsions. Intraperitoneal administration of ketamine produced a dose-dependent (5-50 mg/kg) protection against hindlimb extensor phase. The anticonvulsant effect of ketamine was antagonized neither by naloxone (low as well as high doses) nor sulpiride, but was attenuated by haloperidol, a dopamine (D2)/sigma receptor antagonist. Co-administration of gamma-aminobutyric acid (GABA)-ergic drugs (GABA, muscimol, diazepam and baclofen) and N-methyl-D-aspartate (NMDA) receptor antagonist, dizocilpine (MK801) with ketamine facilitated the anticonvulsant action of the latter drug. In contrast, flumazenil, a benzodiazepine (BZD)-GABAA receptor antagonist, reversed the facilitatory effect of diazepam on the anti-MES effect of ketamine. Similarly, delta-aminovaleric acid (DAVA), antagonized the facilitatory effect of baclofen on anti-MES action of ketamine. These BZD-GABAergic antagonists, flumazenil or DAVA per se also attenuated the anti-MES effect of ketamine given alone. The results suggest that besides its known antagonistic effect on NMDA channel, other neurotransmitter systems i.e. sigma, GABAA-BZD-chloride channel complex and GABAB receptors may also be involved in the anti-MES action of ketamine.

Animals↗
PubMed2001

Antinociceptive activity of a neurosteroid tetrahydrodeoxycorticosterone (5alpha-pregnan-3alpha-21-diol-20-one) and its possible mechanism(s) of action.

The present study investigates the effects of a neurosteroid tetrahydrodeoxycorticosterone (5alpha-pregnan-3alpha-21-diol-20-one) in two experimental models of pain sensitivity in mice. Tetrahydrodeoxycorticosterone (2.5, 5 mg/kg, i.p.) dose dependently decreased the licking response in formalin test and increased the tail flick latency (TFL) in tail flick test. Bicuculline (2 mg/kg, i.p.), a GABA(A) receptor antagonist blocked the antinociceptive effect of tetrahydrodeoxycorticosterone in TFL test but failed to modulate licking response in formalin test. Naloxone (1 mg/kg, i.p.), an opioid antagonist effectively attenuated the analgesic effect of tetrahydrodeoxycorticosterone in both the models. Tetrahydrodeoxycorticosterone pretreatment potentiated the antinociceptive response of morphine, an opioid compound and nimodipine, a calcium channel blocker in formalin as well as TFL test. Thus, tetrahydrodeoxycorticosterone exerts an analgesic effect, which may be mediated by modulating GABA-ergic and/or opioid-ergic mechanisms and voltage-gated calcium channels.

Analgesics↗
PubMed2001

Status of lifestyle modifications in hypertension.

Hypertension is essentially the elevation of arterial blood pressure beyond an arbitrary cut off point, though the dividing line between normal and elevated BP is lacking. Hypertension can be classified into primary, essential or idiopathic hypertension on one hand, and secondary one due to some disease itself. In treating hypertension, antihypertensives have their role, but attention may be directed towards some lifestyle modifications. As regarding dietary interventions, calorie restriction may influence the minimisation of BP. Body weight reduction, less alcohol consumption, salt restriction, potassium and calcium supplementation can enhance the process of lowering BP. The role of magnesium in hypertension is debatable. Serum cholesterol level is commonly elevated in hypertensive patients and its reduction reduces the risk of non-fatal coronary events. Diet rich in plant fibres either alone or with a low fat, low sodium could lower the BP by about 5 mm Hg in hypertensives. The omega-3-polyunsaturated fatty acids found in highest concentrations in cold water fishes have a modest antihypertensive effect. Caffeine contained in two cups of coffee may raise the BP by 5 mm Hg in infrequent users but in habitual users, caffeine has no role. Deficiency of vitamin C might lead to hypertension. As regarding behavioural changes, stopping smoking, regular physical exercise, relaxation therapies like yoga, etc, have definite beneficial effect on hypertensives. The antihypertensive effect of lifestyle modifications may obviate drug therapy. For this one or more of the lifestyle modifications should be tried initially in all hypertensive patients.

Alcohol Drinking↗
PubMed2001

Potentiation of analgesia and reversal of tolerance to morphine by calcium channel blockers.

Effect of four calcium channel blockers (CCBs) belonging to different chemical classes, alone and in combination with morphine was investigated on two models of pain sensitivity, i.e. formalin and tail flick tests in mice. All the studied CCBs, i.e. diltiazem, flunarizine, nimodipine and verapamil inhibited formalin-induced pain responses; however, with verapamil, though there was a trend towards a reduction of paw-licking response to formalin, it was not found to be statistically significant. In contrast, none of the CCBs affected the tail flick latency at any of the doses studied. Morphine, a mu-receptor agonist exerted a significant analgesic effect in formalin as well in tail flick tests. Pretreatment with all CCBs significantly enhanced the analgesic effect of morphine in both tests of nociception. Further, concomitant administration of one of the CCBs, diltiazem with morphine prevented the development of tolerance to the latter. However, combination of diltiazem with morphine, like morphine alone was found to be ineffective in morphine tolerant animals. Results, thus, show that CCBs produced an analgesic effect of their own in formalin-induced tonic pain and potentiated the analgesic activity of morphine. They also modulated opioid-induced tolerance.

Analgesia↗
PubMed2000

Synthesis and characterization of a peptide identified as a functional element in alphaA-crystallin.

Eye lens alpha-crystallin is a member of the small heat shock protein (sHSP) family and forms large multimeric structures. Earlier studies have shown that it can act like a molecular chaperone and form a stable complex with partially unfolded proteins. We have observed that prior binding of the hydrophobic protein melittin to alpha-crystallin diminishes its chaperone-like activity toward denaturing alcohol dehydrogenase, suggesting the presence of mutually exclusive sites for these proteins in alpha-crystallin. To investigate the mechanism of the interaction between alpha-crystallin and substrate proteins, we determined the melittin-binding sites in alpha-crystallin by cross-linking studies. Localization of melittin-binding sites in alpha-crystallin resulted in the identification of RTLGPFYPSR and FVIFLDVKHFSPEDLTVK of alphaA-crystallin and FSVNLDVK of alphaB-crystallin as the chaperone sites. Of these sites, FVIFLDVKHFSPEDLTVK and FSVNLDVK were identified earlier as 1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid (bis-ANS)-binding hydrophobic sites. Here we also report the synthesis and characterization of the peptide, KFVIFLDVKHFSPEDLTVK, having the melittin as well as bis-ANS-binding sequence of alphaA-crystallin. We show that this peptide has characteristics similar to that of alphaA-crystallin by in vitro thermal aggregation assay, gel filtration study, CD spectroscopy, and bis-ANS interaction studies. The peptide sequence corresponds to the beta3 and beta4 region present in the alpha-crystallin domain of sHSP 16.5. We hypothesize that the alpha-crystallin domain in other sHSPs may have a similar function and would likely possess the anti-aggregation property even when separated from the native protein.

Alcohol Dehydrogenase↗
PubMed2000

Off-pump redo coronary artery bypass grafting.

BACKGROUND: Conventional redo coronary artery bypass grafting is associated with significant morbidity. The danger of reoperation is mainly in reopening the sternum and in the manipulation of the heart and the old grafts. Therefore, off-pump redo coronary artery bypass grafting with a patient-specific approach in selected cases seems an ideal technique. METHODS: Between October 1995 to September 1999, 50 patients with mean age of 61.8+/-8 years underwent reoperative coronary artery bypass grafting without cardiopulmonary bypass. Isolated left internal mammary artery (LIMA) to left anterior descending artery (LAD) anastomosis was carried out in 25 cases through left anterior minithoracotomy. In 1 patient LIMA was grafted on a previous vein graft to LAD, which was critically stenosed proximally but distal anastomosis was patent. In another case LIMA was grafted to Ramus intermedius branch. Midsternotomy approach was used to carry out LAD and right coronary artery grafting in 21 cases. In 2 patients a posterolateral thoracotomy approach was used to bypass obtuse marginal branches without cardiopulmonary bypass; in these cases proximal anastomosis was performed on the descending aorta. RESULTS: Mortality rate was 4% (2 deaths). Two patients sustained perioperative myocardial infarction. No patient was reexplored for hemorrhage and 38 patients did not require homologous blood transfusion. Sixteen patients underwent check angiogram and all of them were found to have patent redo grafts. Cardiac recovery room stay was 22+/-7 hours and hospital stay 5+/-2 days. CONCLUSIONS: In selected patients, reoperative coronary artery bypass grafting can be performed without cardiopulmonary bypass with a low perioperative morbidity and mortality and satisfactory graft patency.

Coronary Artery Bypass↗