PubMed Health⌕ Search

Biomedical subjects

C M Rao

Publications and source records attributed to C M Rao.

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

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↗
PubMed2001

Expression of recombinant zeta-crystallin in Escherichia coli with the help of GroEL/ES and its purification.

zeta-Crystallin is a taxon-specific crystallin found in the eye lens of guinea pig and other hystricomorph rodents and camelids. It is an NADPH:quinone oxidoreductase and is also present in low amounts in other tissues where it might act as a detoxifying enzyme. A lens-specific promoter confers lens-specific expression of the gene in high amounts where it is speculated to play a structural role in maintaining the transparency of the lens ensemble. A deletion mutation leads to autosomal dominant congenital cataract and also results in the loss of NADPH binding. In order to perform structural studies with the protein with an aim to delineate the cause of cataract in these mutant guinea pigs, recombinant zeta-crystallin was cloned and expressed in Escherichia coli. The overexpression of the protein in E. coli resulted in a major fraction of it partitioning into inclusion bodies. The co-overexpression of the bacterial chaperone system GroEL/ES along with zeta-crystallin could significantly enhance the yield of soluble protein. Active zeta-crystallin could then be purified from the E. coli using Mono Q anion exchange FPLC and was found to be identical to the native zeta-crystallin isolated from the guinea pig lens with respect to size, spectral properties, and activity.

Animals↗
PubMed2001

Palpable breast cancer which is mammographically invisible.

We have evaluated tumour characteristics, local recurrence rates and prognostic markers in 40 women with symptomatic palpable breast cancer proven by cytology, but in whom routine two-view mammography failed to detect a radiological abnormality. False negative mammograms were identified by cross-referencing all negative mammograms performed at the Royal Victoria Infirmary during the period 1995-1999, with pathological records at the same institution. The average age was 48 years. The majority of the tumours were invasive ductal carcinomas, 35 with an average size of 24 mm. There were 16 Grade II and 15 Grade III tumours. Lymphovascular invasion was seen in 18 on histology and six patients had distant metastases. Of those patients treated by conservation therapy there has been only one local recurrence, with a median follow-up of 18 months. We conclude that mammographically invisible tumours are of common histological type, are frequently high grade and node positive and occur mainly in the younger age group. However, BCT remains a viable option in the treatment of these tumours.

Journal Article↗
PubMed2000

Packing-induced conformational and functional changes in the subunits of alpha -crystallin.

The heteroaggregate alpha-crystallin and homoaggregates of its subunits, alphaA- and alphaB-crystallins, function like molecular chaperones and prevent the aggregation of several proteins. Although modulation of the chaperone-like activity of alpha-crystallin by both temperature and chaotropic agents has been demonstrated in vitro, the mechanism(s) of its regulation in vivo have not been elucidated. The subunits of alpha-crystallin exchange freely, resulting in its dynamic and variable quaternary structure. Mixed aggregates of the alpha-crystallins and other mammalian small heat shock proteins (sHSPs) have also been observed in vivo. We have investigated the time-dependent structural and functional changes during the course of heteroaggregate formation by the exchange of subunits between homoaggregates of alphaA- and alphaB-crystallins. Native isoelectric focusing was used to follow the time course of subunit exchange. Circular dichroism revealed large tertiary structural alterations in the subunits upon subunit exchange and packing into heteroaggregates, indicating specific homologous and heterologous interactions between the subunits. Subunit exchange also resulted in quaternary structural changes as demonstrated by gel filtration chromatography. Interestingly, we found time-dependent changes in chaperone-like activity against the dithiothreitol-induced aggregation of insulin, which correlated with subunit exchange and the resulting tertiary and quaternary structural changes. Heteroaggregates of varying subunit composition, as observed during eye lens epithelial cell differentiation, generated by subunit exchange displayed differential chaperone-like activity. It was possible to alter chaperone-like activity of preexisting oligomeric sHSPs by alteration of subunit composition by subunit exchange. Our results demonstrate that subunit exchange and the resulting structural and functional changes observed could constitute a mechanism of regulation of chaperone-like activity of alpha-crystallin (and possibly other mammalian sHSPs) in vivo.

Circular Dichroism↗
PubMed2000

Inability of chaperones to fold mutant zeta crystallin, an aggregation-prone eye lens protein.

PURPOSE: zeta-crystallin is a quinone oxido-reductase, recruited in the eye lens of hystricomorphic rodents and camels. A deletion mutation constituting the NADPH-binding domain causes congenital cataract in a strain of guinea pigs. The presence of large quantities of a-crystallin, a molecular chaperone, does not provide any protection against this. In order to investigate whether the underlying reason for the lack of protection is the formation of a folding-incompetent protein, we have expressed the mutant protein in a heterologous system along with other known chaperones. METHODS: We expressed the mutant zeta-crystallin in E. coli along with other chaperones such as GroEL/ES and DnaK/DnaJ/GrpE and then analyzed whether these chaperones could increase the amount of protein partitioning into the soluble fraction of E. coli cells. RESULTS: These chaperones were unable to rescue the mutant protein from partitioning into inclusion bodies, although they could increase the yield of soluble wild-type zeta-crystallin. CONCLUSIONS: The deletion of 34 amino acids, constituting the NADPH-binding domain of zeta-crystallin, makes the protein incompetent to fold correctly and thus form insoluble aggregates. It perhaps suggests why the mutant strain of guinea pigs have cataract at birth even though their lenses contain high amounts of alpha-crystallin. This study also shows that certain mutations can render proteins incompetent to fold into soluble molecules despite abundant assistance.

Amino Acid Sequence↗
PubMed2000

Domain swapping in human alpha A and alpha B crystallins affects oligomerization and enhances chaperone-like activity.

alphaA and alphaB crystallins, members of the small heat shock protein family, prevent aggregation of proteins by their chaperone-like activity. These two proteins, although very homologous, particularly in the C-terminal region, which contains the highly conserved "alpha-crystallin domain," show differences in their protective ability toward aggregation-prone target proteins. In order to investigate the differences between alphaA and alphaB crystallins, we engineered two chimeric proteins, alphaANBC and alphaBNAC, by swapping the N-terminal domains of alphaA and alphaB crystallins. The chimeras were cloned and expressed in Escherichia coli. The purified recombinant wild-type and chimeric proteins were characterized by fluorescence and circular dichroism spectroscopy and gel permeation chromatography to study the changes in secondary, tertiary, and quaternary structure. Circular dichroism studies show structural changes in the chimeric proteins. alphaBNAC binds more 8-anilinonaphthalene-1-sulfonic acid than the alphaANBC and the wild-type proteins, indicating increased accessible hydrophobic regions. The oligomeric state of alphaANBC is comparable to wild-type alphaB homoaggregate. However, there is a large increase in the oligomer size of the alphaBNAC chimera. Interestingly, swapping domains results in complete loss of chaperone-like activity of alphaANBC, whereas alphaBNAC shows severalfold increase in its protective ability. Our findings show the importance of the N- and C-terminal domains of alphaA and alphaB crystallins in subunit oligomerization and chaperone-like activity. Domain swapping results in an engineered protein with significantly enhanced chaperone-like activity.

Base Sequence↗
PubMed1999

Differential temperature-dependent chaperone-like activity of alphaA- and alphaB-crystallin homoaggregates.

alpha-Crystallin, a heteromultimeric protein made up of alphaA- and alphaB-crystallins, functions as a molecular chaperone in preventing the aggregation of proteins. We have shown earlier that structural perturbation of alpha-crystallin can enhance its chaperone-like activity severalfold. The two subunits of alpha-crystallin have extensive sequence homology and individually display chaperone-like activity. We have investigated the chaperone-like activity of alphaA- and alphaB-crystallin homoaggregates against thermal and nonthermal modes of aggregation. We find that, against a nonthermal mode of aggregation, alphaB-crystallin shows significant protective ability even at subphysiological temperatures, at which alphaA-crystallin or heteromultimeric alpha-crystallin exhibit very little chaperone-like activity. Interestingly, differences in the protective ability of these homoaggregates against the thermal aggregation of beta(L)-crystallin is negligible. To investigate this differential behavior, we have monitored the temperature-dependent structural changes in both the proteins using fluorescence and circular dichroism spectroscopy. Intrinsic tryptophan fluorescence quench-ing by acrylamide shows that the tryptophans in alphaB-crystallin are more accessible than the lone tryptophan in alphaA-crystallin even at 25 degrees C. Protein-bound 8-anilinonaphthalene-1-sulfonate fluorescence demonstrates the higher solvent accessibility of hydrophobic surfaces on alphaB-crystallin. Circular dichroism studies show some tertiary structural changes in alphaA-crystallin above 50 degrees C. alphaB-crystallin, on the other hand, shows significant alteration of tertiary structure by 45 degrees C. Our study demonstrates that despite a high degree of sequence homology and their generally accepted structural similarity, alphaB-crystallin is much more sensitive to temperature-dependent structural perturbation than alphaA- or alpha-crystallin and shows differences in its chaperone-like properties. These differences appear to be relevant to temperature-dependent enhancement of chaperone-like activity of alpha-crystallin and indicate different roles for the two proteins both in alpha-crystallin heteroaggregate and as separate proteins under stress conditions.

Acrylamide↗
PubMed1999

An atypical form of alphaB-crystallin is present in high concentration in some human cataractous lenses. Identification and characterization of aberrant N- and C-terminal processing.

Two unique polypeptides, 22.4 and 16.4 kDa, were prominent in some human cataracts. Both proteins were identified as modified forms of the small heat shock protein, alphaB-crystallin. The concentration of total alphaB-crystallin in most of these cataracts was significantly increased. The 22.4-kDa protein was subsequently designated as alphaB(g). Mass spectrometric analyses of tryptic and Asp-N digests showed alphaB(g) is alphaB-crystallin minus the C-terminal lysine. alphaB(g) constituted 10-90% of the total alphaB-crystallin in these cataracts and was preferentially phosphorylated over the typical form of alphaB-crystallin. Human alphaB(g) and alphaB-crystallin were cloned and expressed in Escherichia coli. The differences in electrophoretic mobility and the large difference in native pI values suggest some structural differences exist. The chaperone-like activity of recombinant human alphaB(g) was comparable to that of recombinant human alphaB-crystallin in preventing the aggregation of lactalbumin induced by dithiothreitol. The mechanism involved in generating alphaB(g) is not known, but a premature termination of the alphaB-crystallin gene was ruled out by sequencing the polymerase chain reaction products of the last exon for the alphaB-crystallin gene from lenses containing alphaB(g). The 16.4-kDa protein was an N-terminally truncated fragment of alphaB(g). The high concentration of alphaB-crystallin in these cataracts is the first observation of this kind in human lenses.

Amino Acid Sequence↗
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

Possible role of the 34-kilodalton hyaluronic acid-binding protein in visceral Leishmaniasis.

Leishmania donovani, the causative organism of human visceral leishmaniasis, invades host macrophages through its interaction with the cell surface molecules of target cells. The presence of a cell surface protein (Mr 34 kDa) having specific affinity toward hyaluronan (HA), a major extracellular matrix component, has been previously reported in macrophage cell lines. In order to identify the possible role of this HA-binding protein (HABP) in leishmaniasis, initially we demonstrated its overexpression in spleen, liver, macrophages, and serum of hamsters infected with L. donovani. We further observed higher levels of HABP in the macrophage cell line J774.G8 upon infection with L. donovani. Finally, we observed a significant increase in the level of HABP in the serum of patients with kala-azar. In order to understand its functional role in leishmaniasis, we report here a significant inhibition of cellular phosphorylation of HABP in hamster macrophages infected with L. donovani. Interestingly, the 34-kDa HABP was shown to bind with 2 proteins of promastigotes as well as amastigotes of L. donovani (with molecular masses of 55 kDa and 30 kDa respectively), suggesting a possible role for HABP in adhesion during the interaction of promastigotes and macrophages.

Animals↗
PubMed1999

Effect of recombinant human basic fibroblast growth factor on stomach ulcers in rats and mice.

AIM: To study the curative effects of recombinant human basic fibroblast growth factor (rh-bFGF) on gastric ulcer healing. METHODS: Pylorus ligated, water immersion stress-induced, reserpine-induced, and acetic acid-induced gastric ulcers in rats or mice were prepared. Morphometric analyses on ulcer were performed using microscope and true color image analysis system. The DNA and RNA contents were measured by diphenylamine method and orcinol method, respectively. RESULTS: In acetic acid-induced gastric ulcers in rats, rh-bFGF 2.5-40 kU.kg-1 i.g. accelerated the chronic ulcer healing with a bell-shaped dose-effect curve and the best dosage was 10 kU.kg-1. The regenerated gland epithelium width, density of capillaries in granulation tissue, and collagen content in scar tissues obviously increased in rh-bFGF-treated groups. Simultaneously, rh-bFGF promoted the differentiation and maturation of regenerated glands around ulcers. rh-bFGF 2-4 kU.kg-1 s.c. also increased the synthesis of RNA in ulcer tissues. In acute gastric ulcers, rh-bFGF i.g. was only effective on pylorus ligated ulcers, but showed no effect on total acid output and pepsin activity in gastric juice of rats. CONCLUSION: rh-bFGF promoted the gastric ulcer healing and improved the quality of gastric ulcer healing.

Acetic Acid↗
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↗
PubMed1998

Clinical observations of juvenile myoclonic epilepsy in 131 patients: a study in South India.

We studied clinical features of 131 patients with juvenile myoclonic epilepsy (JME). The prevalence was 7.7% among the epileptic patients registered. The mean age at onset was 13.37+/-4.93 years and the diagnosis was established at a mean age of 19.53+/-7.85 years. Absence seizures were reported by 27 (20.6%) patients, myoclonic jerks by 131 (100%) and generalized tonic-clonic seizures (GTCS) by 111 (84.7%). The triad of absence seizures, myoclonic jerks and GTCS was noted in 23 (17.5%) patients, 88 (67.2%) had myoclonic jerks and GTCS, 4 (3%) had absence seizures and myoclonic jerks and 16 (12.2%) had only myoclonic jerks. Early onset absences were seen in 21 (16%) patients and the onset was late in 6 (4.6%). Absences antedated other types of seizures in all the patients. Myoclonic jerks were predominantly unilateral or had unilateral onset in 22 (16.8%). In 17 (13%) patients GTCS antedated myoclonic jerks. Myoclonic jerks had characteristic circadian distribution in 112 (85.5%) patients. On awakening GTCS occurred in 87 (78.4%) patients and in 4 (3.6%) patients they were purely nocturnal. Sleep deprivation was the most important precipitating factor (54.2%). Initial electroencephalogram (EEG) showed classical generalized spike or multiple-spike slow-wave paroxysms in 81% of records. Focal EEG abnormalities were noted in 20.6% of records. The most common focal abnormality was voltage asymmetry. A family history of epilepsy was noted in 31 (23.6%) probands. Diagnosis of JME was made in all the cases in the clinic. The factors responsible for delay in diagnosis of the 36 patients seen by neurologists included failure to ask or interpret the history which was otherwise suggestive of myoclonic jerks in all 36 (100%) cases, the type of seizure for which the patients sought medical attention, and misinterpretation of EEGs in 28 patients. Diagnosis of partial epilepsy was made in seven patients. The factors responsible for such diagnoses were, unilateral jerks in one patient, unilateral jerks and absence seizures in three patients and focal EEG abnormalities in three patients.

Adolescent↗