Biomedical subjects

T Ramakrishna

Publications and source records attributed to T Ramakrishna.

At least 19 recordsLinked to original sources

PubMed2001

Unfolding and refolding of a quinone oxidoreductase: alpha-crystallin, a molecular chaperone, assists its reactivation.

alpha-Crystallin, a member of the small heat-shock protein family and present in vertebrate eye lens, is known to prevent the aggregation of other proteins under conditions of stress. However, its role in the reactivation of enzymes from their non-native inactive states has not been clearly demonstrated. We have studied the effect of alpha-crystallin on the refolding of zeta-crystallin, a quinone oxidoreductase, from its different urea-denatured states. Co-refolding zeta-crystallin from its denatured state in 2.5 M urea with either calf eye lens alpha-crystallin or recombinant human alpha B-crystallin could significantly enhance its reactivation yield. alpha B-crystallin was found to be more efficient than alpha A-crystallin in chaperoning the refolding of zeta-crystallin. In order to understand the nature of the denatured state(s) of zeta-crystallin that can interact with alpha-crystallin, we have investigated the unfolding pathway of zeta-crystallin. We find that it unfolds through three distinct intermediates: an altered tetramer, a partially unfolded dimer, which is competent to fold back to its active state, and a partially unfolded monomer. The partially unfolded monomer is inactive, exhibits highly exposed hydrophobic surfaces and has significant secondary structural elements with little or no tertiary structure. This intermediate does not refold into the active state without assistance. alpha-Crystallin provides the required assistance and improves the reactivation yield several-fold.

Anilino Naphthalenesulfonates↗

PubMed2001

Studies on the alpha-crystallin target protein binding sites: sequential binding with two target proteins.

PURPOSE: alpha-Crystallin belongs to a class of small heat shock proteins and is shown to prevent aggregation of several proteins. We have shown that the temperature-induced structural perturbation leads to several fold enhanced activity. The purpose of this study was to investigate the availability and specificity of the hydrophobic sites that might become available at elevated temperatures. Specifically, we address the following question: Is there an increased exposure of fixed number of hydrophobic sites as a function of temperature or does a new set of sites become available at elevated temperatures? METHODS: alpha-Crystallin target protein complexes were made at two different temperatures and this complex was investigated for its chaperone-like activity towards the same target protein and also other target proteins. DTT-induced aggregation of insulin, alpha-lactalbumin, thermal aggregation of betaL- and gamma-crystallin, and photo-aggregation of gamma-crystallin were used as model systems. Increased light scattering was used to monitor the progress of aggregation. RESULTS: alpha-Crystallin target protein complex prepared at 37 degrees C temperature was effective against thermal aggregation of betaL-crystallin as well as non-thermal aggregation at elevated temperatures. However, the complex prepared at high temperature was ineffective at lower temperatures as well as with other target proteins at both temperatures. CONCLUSIONS: More target protein binding sites become available at elevated temperatures. The sites available at low temperature are a subset of the total sites available at elevated temperatures.

Animals↗

PubMed2001

Interaction of human recombinant alphaA- and alphaB-crystallins with early and late unfolding intermediates of citrate synthase on its thermal denaturation.

We have investigated the role of recombinant human alphaA- and alphaB-crystallins in the heat-induced inactivation and aggregation of citrate synthase. Homo-multimers of both alphaA- and alphaB-crystallins confer protection against heat-induced inactivation in a concentration-dependent manner and also prevent aggregation. Interaction of crystallins with early unfolding intermediates of citrate synthase reduces their partitioning into aggregation-prone intermediates. This appears to result in enhanced population of early unfolding intermediates that can be reactivated by its substrate, oxaloacetate. Both these homo-multimers do not form a stable complex with the early unfolding intermediates. However, they can form a soluble, stable complex with aggregation-prone late unfolding intermediates. This soluble complex formation prevents aggregation. Thus, it appears that the chaperone activity of alpha-crystallin involves both transient and stable interactions depending on the nature of intermediates on the unfolding pathway; one leads to reactivation of the enzyme activity while the other prevents aggregation.

Chromatography, Gel↗

PubMed2001

Redox-regulated chaperone function and conformational changes of Escherichia coli Hsp33.

We have studied the chaperone activity and conformation of Escherichia coli heat shock protein (Hsp)33, whose activity is known to be switched on by oxidative conditions. While oxidized Hsp33 completely prevents the heat-induced aggregation of zeta-crystallin at 42 degrees C at a ratio of 1:1 (w/w), the reduced form exhibits only a marginal effect on the aggregation. Far UV-circular dichroism (CD) spectra show that reduced Hsp33 contains a significant alpha-helical component. Oxidation results in significant changes in the far UV-CD spectrum. Near UV-CD spectra show changes in tertiary structural packing upon oxidation. Polarity-sensitive fluorescent probes report enhanced hydrophobic surfaces in the oxidized Hsp33. Our studies show that the oxidative activation of the chaperone function of Hsp33 involves observable conformational changes accompanying increased exposure of hydrophobic pockets.

Amino Acid Sequence↗

PubMed2001

Enzymatic, clinical and histologic evaluation of corneal tissues in experimental fungal keratitis in rabbits.

Mycotic keratitis, being frequently refractive to most of the currently available antifungal therapy, continues to pose a therapeutic challenge to the clinician. In keratitis of infectious etiology stromal dissolution may be brought about by a combination of agent and host factors. An understanding of the source and nature of corneal tissue damage is essential for evolving more effective therapeutic modalities in the treatment of fungal keratitis. In the present study, we have characterized the extracellular proteases produced in vitro by corneal fungal pathogens namely the Aspergillus flavus and Fusarium solani when collagen was provided as the sole nitrogen source. In addition, fungal infected rabbit corneas were investigated for proteolytic activities and nature of inflammatory reaction. Gelatin zymography detected protease bands with molecular mass ranging from 100 to 200 kDa in the culture extracts of A. flavus, and a single major band of molecular mass approximately 200 kDa in the culture extracts of F. solani. A basal proteolytic activity of mass 65 kDa was visualized in all uninfected and infected rabbit corneal extracts. Infected corneas in addition revealed the presence of additional proteolytic species of mass 92 and 200 kDa. The enzyme inhibitory profile suggested that fungal cultures in vitro contained predominantly serine protease activity and to a lesser extent metalloprotease activity. However, fungal infected corneal homogenates showed the presence of metalloproteinase activity alone, the enzymatic activities entirely being sensitive to ethylene diamine tetra acetate (EDTA), a metalloprotease inhibitor. Interestingly, the serine proteolytic activity detected in fungal cultures in vitro was not present in the fungal infected corneas in vivo. However, the possible role of fungal serine proteases in the activation of corneal matrix metalloproteinases (MMPs) cannot be ruled out. Based on the criteria of molecular mass, proteolytic activity in the presence of calcium at neutral pH, and sensitivity to inhibition by a metalloprotease inhibitor, the 65 and 92 kDa gelatinases were identified as MMP 2 and MMP 9, respectively. The expression of 92 and 200 kDa gelatinases correlated positively with the amount of polymorphonuclear cells present in the infected tissues. Activated resident corneal cells or inflammatory cells may largely contribute to the increased proteolytic activities in fungal infected corneas resulting in tissue matrix degradation in fungal keratitis.

Animals↗

PubMed1999

Structural and functional consequences of the mutation of a conserved arginine residue in alphaA and alphaB crystallins.

A point mutation of a highly conserved arginine residue in alphaA and alphaB crystallins was shown to cause autosomal dominant congenital cataract and desmin-related myopathy, respectively, in humans. To study the structural and functional consequences of this mutation, human alphaA and alphaB crystallin genes were cloned and the conserved arginine residue (Arg-116 in alphaA crystallin and Arg-120 in alphaB crystallin) mutated to Cys and Gly, respectively, by site-directed mutagenesis. The recombinant wild-type and mutant proteins were expressed in Escherichia coli and purified. The mutant and wild-type proteins were characterized by SDS-polyacrylamide gel electrophoresis, Western immunoblotting, gel permeation chromatography, fluorescence, and circular dichroism spectroscopy. Biophysical studies reveal significant differences between the wild-type and mutant proteins. The chaperone-like activity was studied by analyzing the ability of the recombinant proteins to prevent dithiothreitol-induced aggregation of insulin. The mutations R116C in alphaA crystallin and R120G in alphaB crystallin reduce the chaperone-like activity of these proteins significantly. Near UV circular dichroism and intrinsic fluorescence spectra indicate a change in tertiary structure of the mutants. Far UV circular dichroism spectra suggest altered packing of the secondary structural elements. Gel permeation chromatography reveals polydispersity for both of the mutant proteins. An appreciable increase in the molecular mass of the mutant alphaA crystallin is also observed. However, the change in oligomer size of the alphaB mutant is less significant. These results suggest that the conserved arginine of the alpha-crystallin domain of the small heat shock proteins is essential for their structural integrity and subsequent in vivo function.

Arginine↗

PubMed1999

Detection and assay of proteases using calf lens beta-crystallin aggregate as substrate.

The eye lens protein, betaL-crystallin, aggregates and yields a turbid solution upon refolding from its denatured state. We have observed that the addition of trace amounts of protease results in clearing of this turbidity. Based on this observation, we have developed a simple and rapid method for the detection and assay of proteases. This assay can be performed in the pH range of 6.0-9.0. We could assay the activity of trypsin at a concentration as low as 5 microg/ml.

Animals↗

PubMed1999

Vitamins and brain development.

Effects of deficiency of vitamins on early development of brain have been reviewed. Unusual developmental problems in neurogenesis specific for the brain and impairment of its functional capacities due to vitamin deficiency have been discussed. The species-specific "critical periods" in development of various systems have been mentioned. Indices such as reflex activity, locomotion, special senses, cognition and adaptive behavior were used for assessing brain maturation in experimental models and humans. Significant examples include brain anomalies in humans and other mammals caused by retinoid excess or deficit; increase in calbindin D28K, a vitamin D dependent calcium-binding protein during postnatal period in rat; hydrocephalus and exencephaly in prenatal rats and subarachnoidal or intracerebral hemorrhage in infants caused by vitamin E deficiency. Peripheral neuropathic lesions leading to infantile beriberi is caused by thiamine deficiency. Impaired growth in retinal layers leading to delay in maturation of electroretinogram and depth-perception in postnatal rats occur due to pyridoxine deficiency. Infants of severely vitamin B12 deficient mothers show abnormalities in behavior involving basal ganglia and pyramidal tract. Folic acid deficiency results in delayed maturation of the basic electroencepalographic patterns. In addition, vitamin-interactions leading to developmental errors have been pointed out. Vitamin B6 deficiency impairs vitamin B12 absorption and biotin deficiency may be aggravated by pantothenic acid deficiency. Vitamin C deficiency resulting in impaired metabolism may produce symptoms of deficiency of folic acid. Another characteristic examples is that iron absorption from dietary sources is dependent on ascorbic acid.

Animals↗

PubMed1998

The chaperone-like alpha-crystallin forms a complex only with the aggregation-prone molten globule state of alpha-lactalbumin.

The chaperone-like alpha-crystallin prevents aggregation of several proteins by interacting with their non-native states. Alpha-Lactalbumin adopts different non-native states under different experimental conditions. We have investigated the interaction of alpha-crystallin with three non-identical non-native states, using fluorescence, circular dichroism, and gel filtration chromatography. The compact molten globule state of apo-alpha-lactalbumin in tris buffer does not interact with alpha-crystallin. The expanded, flexible molten globule-like state of reduced apo-alpha-lactalbumin (formed at pH 7.2) also does not interact with alpha-crystallin. Only the aggregation-prone non-native state of reduced apo-alpha-lactalbumin formed at pH 6.0 interacts with alpha-crystallin to form a stable complex. The alpha-crystallin bound reduced apo-alpha-lactalbumin exhibits properties similar to those of a molten globule. Our results show that alpha-crystallin interacts only with the aggregation prone molten globule state of reduced apo-alpha-lactalbumin but not with the other non-aggregating molten globule states of the protein.

Animals↗

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

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↗

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

Preservation of native conformation during aluminium-induced aggregation of tau protein.

Aluminium exposure has been shown to result in aggregation of microtubule-associated protein tau in vitro. In the light of recent observations that the native random structure of tau protein is maintained in its monomeric and dimeric states as well as in the paired helical filaments characteristic of Alzheimer's disease, it is likely that factors playing a causative role in neurofibrillary pathology would not drastically alter the native conformation of tau protein. We have studied the interaction of tau protein with aluminium using circular dichroism (CD) and 27Al NMR spectroscopy. The CD studies revealed a five-fold increase in the observed elipticity of the tau-aluminium assembly. The increase in elipticity was not associated with a change in the general conformation of the protein and was most likely due to an aggregation of the tau protein induced by aluminium. 27Al NMR spectroscopy confirmed the binding of aluminium to tau protein. Hyperphosphorylation of tau in Alzheimer's disease is known to be associated with defective microtubule assembly in this condition. Abnormally phosphorylated tau exists in a polymerized form in the paired helical filaments (PHF) which constitute the neurofibrillary tangles found in Alzheimer's disease. While it is hypothesized that its altered biophysical characteristics render abnormally phosphorylated tau resistant to proteolysis, causing the formation of stable deposits, the sequence of events resulting in the polymerization of tau are little understood, as are the additional factors or modifications required for this process. Based on the results of our spectroscopic studies, a model for the sequence of events occurring in neurofibrillary pathology is proposed.

Aluminum↗

PubMed1996

Heart rate in female rat in response to snout contact by normal, gonadectomised and testosterone--pretreated gonadectomised male.

The question whether or not there will be an enhanced autonomic response in terms of heart rate in female rat, during sexual encounter with male rat was investigated. The heart rate recorded from partially restrained estrous female rat before and after the snout contact by the normal male rat showed a significant rise. There was no such rise in response to the snout contact by neonatally gonadectomised male rat of the same age as that of normal male. Treatment of testosterone propionate given to the gonadectomised male twelve hours prior to the experiment did not bring about significant rise in the heart rate of the estrous female. It is apparent that the perturbation in the structure of sexually dimorphic medial preoptic area (MPOA) in the male rat as a result of neonatal gonadectomy obliterates certain essential cues such as pheromone(s), visual and ultrasound communications which normally elicit a rise in the heart rate of the estrous female during sexual encounter.

Animals↗

PubMed1996

The role of sexually dimorphic medical preoptic area of the hypothalamus in the sexual behaviour of male and female rats.

The neonatally gonadectomized male rats failed to show typical male clasping of the flanks of the female rat in oestrous stage and also could not achieve intromission and ejaculation when studied at 90 days of age. The replacement of testosterone propionate did not abolish the deficit in sexual behaviour observed. The multiunit electrical activity (MUA) in the medial preoptic area (MPOA) of intact males as well as of oestrous females increased significantly in amplitude and frequency from the low basal levels during mounting, but reverted almost to the basal levels after mounting. The basal MUA in the gonadectomized male rats was of relatively high frequency and amplitude when compared to the intact males and oestrous females, and did not show significant changes during and after mounting. It is suggested that the dimorphic organization of MPOA, developing under the influence of neonatal testosterone, plays a significant role in the male sexual behaviour in adulthood.

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↗