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Biomedical subjects

C M Rao

Publications and source records attributed to C M Rao.

At least 37 records · Page 2Linked to original sources
PubMed1997

Co-refolding denatured-reduced hen egg white lysozyme with acidic and basic proteins.

Refolding of denatured-reduced lysozyme and the effect of co-refolding it with other proteins such as RNase A, bovine serum albumin, histone, myelin basic protein, alcohol dehydrogenase and DNase I on the renaturation yield and the aggregation of lysozyme have been studied. Basic proteins consistently increase the renaturation yield of the basic protein lysozyme (10-20% more than in their absence) with little or no aggregation. On the other hand, co-refolding of lysozyme with acidic proteins leads to aggregation and a significant decrease in renaturation yields. Our results show that hetero-interchain interactions (non-specific interactions) occur when the basic protein lysozyme is refolded together with acidic proteins such as bovine serum albumin, alcohol dehydrogenase or DNase I. Our results also suggest that the net charge on proteins plays a significant role in such non-specific aggregation. These results should prove useful in understanding the hetero-interchain interactions between folding polypeptide chains.

Animals↗
PubMed1997

Effect of the chaperone-like alpha-crystallin on the refolding of lysozyme and ribonuclease A.

Alpha-crystallin exhibits chaperone-like properties in preventing aggregation of proteins. We have studied the effect of alpha-crystallin on the refolding of denatured-disulfide intact and denatured-reduced lysozyme and RNase A. Alpha-crystallin does not have any effect on the refolding of both the denatured-disulfide intact enzymes. However, it inhibits the aggregation and oxidative renaturation of denatured-reduced lysozyme. Interestingly, it has no effect on the refolding of denatured-reduced RNase A. In order to probe the molecular basis of this differential behavior of alpha-crystallin towards lysozyme and RNase A, we have carried out circular dichroism and fluorescence studies on the refolding of denatured-reduced RNase A. It exhibits an extended conformation with little difference in the exposed hydrophobicity during the refolding process. We have earlier shown the presence of an aggregation-prone, refolding-competent, molten-globule-like intermediate on the refolding pathway of lysozyme. Alpha-crystallin binds to this intermediate, prevents its aggregation and inhibits its oxidative refolding. It was earlier believed that alpha-crystallin, unlike other chaperones, does not recognize intermediates on the refolding pathway but only recognizes intermediates on the unfolding pathway of proteins. Our present study clearly shows that it recognizes the refolding intermediates as well.

Chaperonins↗
PubMed1997

Regulation of cellular phosphorylation of hyaluronan binding protein and its role in the formation of second messenger.

The presence of the 34-kDa hyaluronan binding protein, a new member of the 'hyaladherins' family, was demonstrated in a wide variety of cell lines by immunoblot analysis. This protein was observed to be highly phosphorylated in transformed fibroblasts compared to normal fibroblasts. Phosphorylation was enhanced in the presence of its ligand i.e., hyaluronan, but not in the presence of other glycosaminoglycans. The phosphorylated form of this hyaluronan binding protein was shown to be present on the cell surface and could be detected in serum-free medium. The regulation of the cellular and cell surface phosphorylation of HA-binding protein by HA, PMA and calyculin-A was demonstrated in different cell lines. Hyaluronan enhanced the phosphorylation of PLC-gamma in association with increased formation of inositol 1,4,5-triphosphate, both of which were specifically blocked by pretreatment of the cells with purified anti-hyaluronan binding protein antibodies. The data presented here indicate a role for the 34-kDa hyaluronan binding protein in cellular signal transduction.

Animals↗
PubMed1997

Chaperone-like activity and temperature-induced structural changes of alpha-crystallin.

alpha-Crystallin is known to exhibit chaperone-like activity. We have studied its chaperone-like activity toward the aggregation of betaL-crystallin upon refolding of this protein from its unfolded state in guanidinium chloride. The chaperone-like activity of alpha-crystallin is less pronounced below 30 degrees C and is enhanced above this temperature. The plot of percentage protection as a function of temperature shows two transitions; one at 30 degrees C and another at around 55 degrees C. We have performed steady state fluorescence, fluorescence polarization, fluorescence quenching, circular dichroism, sedimentation analysis, and gel filtration chromatography to probe the temperature-induced structural changes of alpha-crystallin. Our results show that at above 50 degrees C, alpha-crystallin undergoes a transition to a multimeric molten globule-like state. Above 30 degrees C, a minor but detectable perturbation in its tertiary structure occurs that might lead to the observed exposure of its hydrophobic surfaces. These results support our earlier hypothesis that alpha-crystallin prevents the aggregation of other proteins by providing appropriately placed hydrophobic surfaces; a structural transition above 30 degrees C involving enhanced or reorganized hydrophobic surfaces of alpha-crystallin is important for its chaperone-like activity. It is possible that a structural alteration induced by temperature forms a part of the general mechanism of chaperone function, because they are required to function more effectively at nonpermissible temperatures.

Chaperonins↗
PubMed1997

Depth profiling of mammalian cells by photoacoustic spectroscopy: localization of ligands.

Phase-resolved monitoring of photoacoustic signals can provide information about the depth profile of a sample. We describe an application of this principle to determine the depth profiles of ligands and antitumor agents in mammalian cells. Measurements of the in-phase and quadrature components of the photoacoustic spectra (which yield information from the surface and the interior, respectively) of a tumor cell line, AK-5, treated with the antitumor agent coralyne chloride have been made. They clearly show that the drug accumulates in the cell interior and is not seen on the cell surface, providing in situ evidence for the localization of this drug. Histochemical dyes which stain cells uniformly give identical in-phase and quadrature spectra; spectra of cells incubated with nuclear stains demonstrate a differential staining of the nucleus and the cytoplasm. These results demonstrate the usefulness of phase-resolved photoacoustic spectroscopy in monitoring differential interactions of drugs and other ligands with cells.

Animals↗
PubMed1997

Effects of 2.5 s mouse nerve growth factor on regeneration of injured sciatic nerves in mice and rats.

AIM: To study the promoting activity of 2.5 s murine nerve growth factor (NGF) for the regeneration of injuried sciatic nerve. METHODS: The sciatic nerves of mice and rats were crushed. The axon counts and differential axon counts were made. Evoked potential latency (NMEPL) was recorded at sites of proximal (Pro) and distal (Dis) segment of soleus muscles. RESULTS: In mice, NGF im 0.5-1 kBU.kg-1 increased the axon counts of regenerated nerves 20 d after crush; 2-4 kBU.kg-1 simultaneously alleviated the denervated atrophy of soleus muscles. In rats, NGF i.m. increased the axon counts at dose of 1 kBU.kg-1 for 40 d, 2 kBU.kg-1 for 30 d and 40 d, and 4 kBU.kg-1 for 20 d, 30 d and 40 d after crush; the number of large axons of regenerated nerves were also increased by NGF i.m. 2 kBU.kg-1 for 40 d and 4 kBU.kg-1 for 30 d and 40 d; Pro NMEPL was shortened by NGF 1, 2, and 4 kBU.kg-1, Dis-NMEPL was shortened by NGF 2 and 4 kBU.kg-1 for 40 d. CONCLUSION: NGF im evidently promotes the regeneration of injuried sciatic nerve, and attenuates the skeletal muscular atrophy caused by nerve injury.

Animals↗
PubMed1997

An experimental model to produce partial thickness burn wound.

Simple burn wound models are always warranted. In view of this a partial thickness reproducible 2 degrees C burn wound has been produced using hot molten wax. This model is simple, convenient, and could be used to monitor wound contraction and epithelization in burn wounds.

Animals↗
PubMed1996

Molten-globule state of carbonic anhydrase binds to the chaperone-like alpha-crystallin.

alpha-Crystallin, a multimeric protein, exhibits chaperone-like activity in preventing aggregation of several proteins. We have studied the chaperone-like activity of alpha-crystallin toward heat-induced aggregation of bovine and human carbonic anhydrase. Human carbonic anhydrase aggregates at 60 degrees C, while bovine carbonic anhydrase does not aggregate significantly at this temperature. Removal of the enzyme-bound metal ion, Zn2+, by EDTA modulates the aggregation behavior of bovine carbonic anhydrase. Fluorescence and circular dichroism studies show that removal of the metal ion from the bovine carbonic anhydrase by a chelator such as EDTA enhances the propensity of the enzyme to adopt the molten-globule state. alpha-Crystallin binds to this state of the enzyme and prevents aggregation. Fluorescence and circular dichroism studies on the alpha-crystallin-enzyme complexes show that the enzymes in the complex are in the molten-globule state. These results are of relevance to the interaction of chaperones with the partially unfolded states of target proteins.

Animals↗
PubMed1996

In vivo characteristics and localisation of carotenoid pigments in psychrotrophic and mesophilic Micrococcus roseus using photoacoustic spectroscopy.

Photoacoustic spectra of cells of M. roseus were recorded to study the in vivo characteristics and localisation of the carotenoid pigments in these cells. The PA spectra indicated that both the psychrotrophic and mesophilic strains had similar chromophores. The cis carotenoids were prominent in the psychrotrophic M.roseus whereas shorter polyenes were more prominent in mesophilic M.roseus. Further, depth profiling photoacoustic studies revealed that in both the strains of M.roseus the bulk of the chromophore was associated with the cell membrane.

Acoustics↗
PubMed1996

Refolding of denatured and denatured/reduced lysozyme at high concentrations.

Refolding of proteins at high concentrations often results in aggregation. To gain insight into the molecular aspects of refolding and to improve the yield of active protein, we have studied the refolding of lysozyme either from its denatured state or from its denatured/reduced state. Refolding of denatured lysozyme, even at 1 mg/ml, yields fully active enzyme without aggregation. However, refolding of denatured/reduced lysozyme into buffer that lacks thiol/disulfide reagents leads to aggregation. Thiol/disulfide redox reagents such as cysteine/cystine and reduced/oxidized glutathione facilitate the renaturation, with the yield depending on their absolute concentrations. We have obtained an approximately 70% renaturation yield upon refolding of lysozyme at 150 microgram/ml. The cysteine/cystine redox system is more efficient compared with the glutathione redox system. When lysozyme is refolded in the absence of redox reagents, a transient intermediate that has regained a significant amount of secondary structure is formed. The tryptophans in this intermediate are as exposed to water as in the fully unfolded protein. It shows increased exposure of hydrophobic surfaces compared with the native or completely unfolded enzyme. This aggregation-prone intermediate folds to active enzyme upon addition of oxidized glutathione before the aggregation process starts. These properties of the intermediate in the refolding pathway of lysozyme are similar to those proposed for the molten globule.

Animals↗
PubMed1996

Hyaluronic acid induced hyaluronic acid binding protein phosphorylation and inositol triphosphate formation in lymphocytes.

In this report, the role of 34 kDa HA-binding protein in hyaluronic acid-induced cellular signalling in lymphocytes has been examined. The binding of 125I-HA to lymphocytes in vivo was found to be inhibited by pre-incubation of the cells with anti-34 kDa HA-binding protein antibodies, thus confirming 34 kDa HA-binding protein as the specific HA-receptor in lymphocytes. This observation was substantiated by anti-34 kDa HA-binding protein antibodies immunoblotting and 125I-HA ligand blotting of lymphocytes cell lysate. The HA-induced cell aggregation, tyrosine phosphorylation and cytoskeletal protein phosphorylation demonstrate the HA-induced early cellular signalling events in lymphocytes. Further, to study the involvement of 34 kDa HA-binding protein in mitogen induced lymphocyte signalling, we studied in vivo phosphorylation and secondary messenger formation. The enhanced 34 kDa HA-binding protein phosphorylation by HA and the inhibition of cellular aggregation and IP3 formation by anti-HA-binding protein antibodies revealed that 34 kDa HA-binding protein is one of the potential mediators in HA-induced signal transduction.

Animals↗
PubMed1995

Rapid refolding studies on the chaperone-like alpha-crystallin. Effect of alpha-crystallin on refolding of beta- and gamma-crystallins.

alpha-Crystallin, a multimeric protein present in the eye lens, is shown to have chaperone-like activity in preventing thermally induced aggregation of enzymes and other crystallins. We have studied the rapid refolding of alpha-crystallin, and compared it with other calf eye lens proteins, namely beta- and gamma-crystallins. alpha-Crystallin forms a clear solution upon rapid refolding from 8 M urea. The refolded alpha-crystallin has native-like secondary, tertiary, and quaternary structures as revealed by circular dichroism and fluorescence characteristics as well as gel filtration and sedimentation velocity measurements. On rapid refolding, beta- and gamma-crystallins aggregate and form turbid solutions. The presence of alpha-crystallin in the refolding buffer marginally increases the recovery of beta- and gamma-crystallins in the soluble form. However, unfolding of these crystallins together with alpha-crystallin using 8 M urea and subsequent refolding significantly increases the recovery of these proteins in the soluble form. These results indicate that an intermediate of alpha-crystallin formed during refolding is more effective in preventing the aggregation of beta- and gamma-crystallins. This supports our earlier hypothesis (Raman, B., and Rao, C. M. (1994) J. Biol. Chem. 269, 27264-27268) that the chaperone-like activity of alpha-crystallin is more pronounced in its structurally perturbed state.

Animals↗
PubMed1995

Temperature dependent chaperone-like activity of alpha-crystallin.

Alpha-crystallin, a multimeric protein present in the eye lens, is known to have chaperone-like activity in preventing the aggregation of enzymes and other crystallins. We have studied the chaperone-like activity of this protein towards the aggregation of insulin B chain, induced by reducing the interchain disulphide bond with dithiothreitol. At room temperature, there is no detectable protection (at a 1:1 (w/w) ratio of insulin: alpha-crystallin) against the aggregation of insulin B chain by alpha-crystallin, whereas it completely prevents this aggregation at 40 degrees C. We have monitored the temperature dependence of the protection of aggregation by alpha-crystallin; the protection increases sharply above 30 degrees C and reaches almost 100% by 41 degrees C. Probing the hydrophobic surfaces of alpha-crystallin with the hydrophobic fluorphore 8-anilino-1 naphthalene sulfonate suggests that the hydrophobic surfaces of alpha-crystallin are exposed to a greater extent above 30 degrees C. A complete prevention of the aggregation is achieved at 27.6 degrees C by increasing the concentration of alpha-crystallin by more than 8 fold. Similar temperature dependent chaperone-like activity of alpha-crystallin is observed towards the aggregation of zeta-crystallin, an enzyme crystallin from guinea pig. We have earlier shown that alpha-crystallin exposes hydrophobic surface(s) at temperatures above 30 degrees C. These results support our earlier hypothesis [Raman, B. and Rao, Ch.M. (1994) J. Biol. Chem. 269, 27264-27268] that the chaperone-like activity of alpha-crystallin is more pronounced in its structurally perturbed state.

Animals↗
PubMed1995

Fluorescent staining for proteins on polyacrylamide gels with 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride).

A simple and sensitive post electrophoresis fluorescent staining technique for proteins on polyacrylamide gels using 5-dimethylamino-1-naphthalene sulfonyl chloride (dansyl chloride) has been developed. Dansyl chloride staining increases the sensitivity, 0.125 micrograms protein per band can be visualised by this technique. The staining method appears to be applicable to all types of proteins including proteoglycans.

Dansyl Compounds↗
PubMed1995

Effect of smoke condensate on the physiological integrity and morphology of organ cultured rat lenses.

Smoke, either from cigarette smoking or from burning of organic fuels, has been proposed to be a major environmental risk factor for a variety of human diseases. Recently, smoke was implicated in cataract, an eye lens opacification which is a major cause of blindness. We have undertaken a study to investigate the effect of wood smoke condensate on the physiological integrity and morphology of organ cultured lenses. Lenses in organ culture are metabolically active and have functional defense systems, thus they provide an appropriate model for studying effects of smoke condensate. Our present study indicates that metabolites of wood smoke condensate accumulate in the lens. The ability of the lenses to accumulate rubidium-86 (mimic of potassium) and choline from the medium is compromised by exposure to smoke condensate. Rubidium efflux studies suggest that the damage is primarily at the uptake level and does not involve an overall increase in membrane permeability. Protein leakage experiments corroborate this suggestion. Histological data show distinct morphological changes such as hyperplasia, hypertrophy and multilayering of epithelial cells.

Animals↗
PubMed1995

Wound healing profiles of ketorolac, metronidazole and tinidazole administered post-surgically.

On dead space wounds, drugs (ketorolac, metronidazole and tinidazole) caused a significant (P < 0.01) decrease in breaking strength, granulation tissue weight and hydroxyproline content in male rats. Both the parameters of excision wound were significantly (P < 0.01) hastened by metronidazole and tinidazole only. Post operative management of wounds with ketorolac (a potent analgesic), metronidazole and tinidazole (for anaerobic infections) may be dealt with the risk of a delay in healing. Both metronidazole and tinidazole promote the epithelization process.

Animals↗
PubMed1994

Chaperone-like activity and quaternary structure of alpha-crystallin.

alpha-Crystallin has been shown to function as a molecular chaperone in preventing thermal aggregation of crystallins and other proteins. The molecular mechanism of this protection is not yet clear. gamma-Crystallin aggregates upon exposure to UV light. We have investigated the effect of the presence of alpha-crystallin in the photoaggregation process and find that alpha-crystallin does not prevent photoaggregation at low temperatures. The protection starts around 30 degrees C and steeply increases with temperature. The plot of protection ability versus temperature is sigmoidal, indicating a structural transition. Perturbation of the quaternary structure of alpha by non-thermal mode, such as 3 M urea, also results in enhanced protection. Pyrene, a hydrophobic fluorophore, is sparingly soluble in water. alpha-Crystallin enhances the solubility of pyrene by severalfold. Temperature dependence of this solubilization shows a transition around 30 degrees C (another at about 50 degrees C). Fluorescence intensity ratio of third and first peaks of pyrene emission (I3/I1,), indicative of hydrophobicity of the reporting area, also shows similar transitions suggesting enhanced hydrophobicity. Gel filtration experiments of irradiated samples indicate the complex formation between gamma- and alpha-crystallins. alpha-Crystallin does not prevent cold precipitation of gamma-crystallin. On the basis of these results, we hypothesize that alpha-crystallin prevents aggregation of non-native structures by providing appropriately placed hydrophobic surfaces. A structural transition above 30 degrees C enhances the protective ability, perhaps by increasing or reorganizing the hydrophobic surfaces. A similar temperature dependence has been reported for GroEL. Whether a structural switch, either activated by temperature, solvent conditions, or small molecule binding, forms a part of the general mechanism of chaperone activity needs to be investigated.

Animals↗