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Biomedical subjects

C M Rao

Publications and source records attributed to C M Rao.

At least 55 records · Page 3Linked to original sources
PubMed1994

Red edge excitation shifts of crystallins and intact lenses. A study of segmental mobility and inter-protein interactions.

The shift that occurs in the fluorescence emission wavelength upon changing the excitation wavelength towards the red edge of the absorption band is termed red edge excitation shift (REES). We have monitored the REES of intrinsic protein fluorescence of freshly isolated intact lenses, of individual crystallins in their native, denatured and photodamaged states and also of crystallin mixtures. The observed REES values for the lenses from different species are different suggesting that the mobilities and packing of the crystallins may vary with the species. Lens photodamage in all the cases resulted in an increase of REES. Denaturation of crystallins in solution reduces REES and renaturation restores it. Mixtures of alpha- and beta-crystallins prepared either by directly mixing equimolar solutions or mixing them in 4 M urea followed by dialysis (reconstituting) gave similar REES values indicating the absence of any specific interactions in dilute solutions. Possible existence of induced alterations facilitating inter-crystallin interactions at high protein concentration is suggested.

Animals↗
PubMed1993

Specificity in the binding of inhibitors to the active site of human/primate aspartic proteinases: analysis of P2-P1-P1'-P2' variation.

To understand the differences in the binding specificities within the aspartic proteinase family of enzymes, we have carried out studies to determine the inhibition constants of a set of related compounds with various members of the human enzyme family. The inhibition constants (Ki values) were determined by competitive inhibition of the hydrolysis of chromogenic octapeptide substrates in the pH range of 3-5. For comparison, inhibition of monkey renin was studied by RIA at pH 6.0. All inhibitors were based on the general structure 4-(morpholinylsulfonyl)-L-Phe-P2-(cyclohexyl)Ala psi[isostere]-P1'-P2'. The isosteric replacements of the scissile peptide bond included difluorohydroxyethylene, 1,2-diols, 1,3-diols, and difluoroketones. Side chain substituents in P2 include hydrogen, allyl, ethylthio, (methoxycarbonyl)methyl, N-methylthiouridobutyl, imidazolylmethyl, and 4-amino-2-thiazolylmethyl. Our measurements have identified potent and selective inhibitors which are useful in evaluating the differences in the specificities among selected enzymes of this family.

Amino Acid Sequence↗
PubMed1992

Levels of reduced pyridine nucleotides and lens photodamage.

Since most of the known factors that are associated with cataract formation are oxidative in nature, one would expect that a highly reductive environment might arrest or retard the progress of cataract formation. Reduced nucleotides, both NADH and NADPH, are potent reductants with a large negative redox potential of -320 mV. Lenses of certain species contain high levels of these nucleotides, presumably due to the presence of taxon specific crystallins. We have utilized this situation to investigate whether the levels of reduced pyridine nucleotides modulate photo-oxidative damage to the lens. We have monitored the time dependent loss of tryptophan fluorescence upon photodamage for lenses from guinea pig, rabbit and frog (Rana) that contain high levels of pyridine nucleotides and compared with the lenses from rat, Xenopus and a mutant strain of guinea pig that contain significantly lower amounts of these nucleotides. About 75% and 90% of the initial fluorescence intensity is lost in the case of rat and Xenopus lenses, respectively, after a total of 35 min exposure. Rabbit, guinea pig and frog lenses, under identical conditions, show only about 35-40% loss of the initial fluorescence. It appears that the lenses that contain high levels of reduced nucleotides are less susceptible to photodamage. The observed anti-oxidative role of reduced nucleotides in the lenses indicates the possibility of testing reductants (NADPH, NADH and their functional analogues) as potential candidates to therapeutically intervene in the process of cataractogenesis.

Animals↗
PubMed1991

Synchronous scan fluorescence spectroscopy of proteins and human eye lenses.

Scanning both the excitation and emission monochromators synchronously while recording the fluorescence spectrum results in a considerable decrease in the apparent band width and shift in the peak position. We demonstrate the potential of this approach in the studies on proteins and their interactions as well as fluorophores in condensed media. We have chosen crystallins, eye lens proteins and human lenses. Synchronous scan spectra of alpha-, beta- and gamma-crystallins are clearly distinguishable and appear to provide specific signatures. The spectrum of the mixed solution could be simulated by the linear combination of components indicating that these proteins might not have any specific interaction in the dilute solutions. Synchronous spectra of the human lenses, both normal and cataractous, show several distinguishable features.

Animals↗
PubMed1991

Identification of mutations that occurred on the genome of Japanese encephalitis virus during the attenuation process.

The total nucleotide sequences of the genomes of two Japanese encephalitis virus (JEV) strains, the attenuated vaccine strain SA14-14-2 and its parental virulent strain SA14, were determined by using the molecular cloning technique. The sequence analysis revealed that both virion RNA molecules were 10,976 nucleotides long with 95 and 585 flanking bases at the 5' and 3' untranslated sequences, respectively. A single, long open reading frame spanning 10,296 nucleotides was observed to encode a polyprotein of 3432 amino acid residues. When these sequences were compared with each other, 57 nucleotide substitutions were found to be scattered all over the genome. Of these, 24 resulted in amino acid changes within viral proteins. Structural proteins C and E contain one and eight amino acid changes, respectively. Of the nonstructural proteins, NS1 contains three, NS2a two, NS2b two, NS3 four, NS4a one, NS4b one, and NS5 two amino acid substitutions. The 5'- and 3'-terminal untranslated regions contain one- and two- point mutations, respectively. These data and comparative studies with other JEV strain genomes provide a molecular basis for investigating attenuation mechanisms of JEV.

Amino Acid Sequence↗
PubMed1991

Effect of histamine on wound healing.

Using incision, excision and dead space wound models in rats, a study was conducted on the effect of histamine on wound healing. Exogenous histamine given either ip or locally was without any effect. Semicarbazide as (histamine synthesis inhibitor) suppressed healing process (breaking strength of skin incision wound), decreased breaking strength and hydroxyproline content of granulation tissue and delay in period of epithelization. On the other hand compound 48/80 (a promoter of histamine forming capacity) was found to promote wound healing. Exogenous histamine (topical) reversed the anti-healing effect of semicarbazide on incision and excision wounds.

Animals↗
PubMed1991

A simple method to quantify maturation of wound collagen.

While investigating the effect of NSAIDs and Zn(II) (NSAIDs)2 complexes on healing of dead space wounds we found that both NSAIDs and Zn(II) (NSAIDs)2 reduced collagen content to a comparable extent yet only NSAIDs reduced breaking strength (BS) of wounds. Since collagen content and maturation are the two important determinants of BS it was obvious that only NSAIDs reduced maturation. A simple method to assess the extent of maturation in available collagen is being reported in the present study. While all NSAIDs significantly reduced the maturation of collagen, Zn(II) (NSAIDs)2 complexes per se did not affect the maturation of collagen of 10-day old. Though, chronologically, granulation tissues obtained from both the treated groups were 10-day old, maturation-wise the age of NSAIDs and Zn(II) (NSAIDs)2 treated granulation was 6 and 12 days respectively.

Animals↗
PubMed1991

Effects of antihistamines on wound healing.

Role of antihistamines (H1 and H2 blockers) in wound healing by utilizing incision and dead space wound models in albino rats was investigated. H1 blockers (mepyramine and promethazine) were found to decrease breaking strength of 10 day old dermal incision wounds and collagen content (as hydroxyproline) and breaking strength of granulation tissue harvested over tubular implant. On the other hand H2 blockers (Cimetidine and ranitidine) did not alter the above parameters. The findings that H1 blockers suppress healing implicate H1 receptors in alleged prohealing effect of histamine, and suggest clinical evaluation of these agents for suppression of overhealing states like keloid, adhesions and strictures.

Animals↗
PubMed1990

The conformational status of a protein influences the aerobic photolysis of its tryptophan residues: melittin, beta-lactoglobulin and the crystallins.

We have studied the aerobic photolysis of the tryptophan residues of the proteins melittin and beta-lactoglobulin when the proteins are in ordered conformations and when they are in randomly coiled states. The results suggest that the conformational status of the protein is a factor that influences the photolysis of the constituent tryptophan residues. This point appears to be of relevance to the photo-oxidation of the tryptophan residues of the eye lens proteins crystallins.

Aerobiosis↗
PubMed1990

Time profile of hemin aggregation: an analysis.

The status of hemin, whether monomeric or aggregated, appears to be a key factor in deciding its biological activity. The molecular basis for the tendency of hemin to aggregate and the factors that regulate it are not yet fully understood. We have investigated the time profile of aggregation of hemin in aqueous solutions and the effect of temperature on the process of aggregation. Aggregation increases with increase in temperature. The time profile data, as monitored by change in absorbance at 398 nm, fits a nonlinear equation with three time constants, suggesting a possibility of three processes. Interestingly, the variation of these three time constants with temperature are different.

Hemin↗
PubMed1990

Effects of procoagulants on wound healing.

As blood coagulation is a prelude for wound healing, a systemic haemocoagulant (Botropase) and local procoagulants (thrombin and fibrin) were evaluated on physical (wound breaking strength, wound half-closure time and period of epithelization), biochemical (granuloma-hydroxyproline and hexosamine) and histological attributes of healing wounds in albino rats. Botropase prompted all phases of tissue repair. Thrombin delayed wound contraction whereas fibrin had no discernable action. The findings that procoagulants modify healing process has bearing on their surgical use.

Animals↗
PubMed1990

Comparative evaluation of tolmetin & tolmetin-zinc on wound-repair & inflammation.

In view of the reported healing-suppressant activity of some NSAIDs and absence of this adverse effect in their zinc-complexes, tolmetin (Tol), a recently introduced NSAID and its zinc-complex (Tol-Zn) were compared for their wound healing and antiinflammatory profiles in male albino rats. Tolmetin-zinc (Tol-Zn) significantly reversed (P less than 0.01) the suppressant effect of Tol on gain in breaking strength of skin incisions and dead space wounds (breaking strength, g: for control, Tol and Tol-Zn were: 313 +/- 7, 250 +/- 11, 294 +/- 16 in skin wounds; 244 +/- 7, 137 +/- 18, 195 +/- 16 in dead space wounds). Tol-Zn shared the significant (P less than 0.001) suppressant effect of Tol on granuloma formation (granuloma weight, mg: control - 69 +/- 3, Tol - 36 +/- 3.03, Tol-Zn - 37 +/- 2.75) and its collagen content (total hydroxyproline per tissue, microgram: control - 1955 +/- 55, Tol - 1400 +/- 200, Tol-Zn - 1410 +/- 150). Rat paw edema induced by carrageenin was significantly (P less than 0.001) reduced by Tol as well as Tol-Zn. In the chronic model both the agents suppressed significantly (P less than 0.001) granuloma formation by 50 per cent. Zinc sulphate by itself reduced the rat paw edema by 39 per cent (P less than 0.02) and did not affect the other parameters. Zinc-complex appears to be an improved version of tolmetin as an antiinflammatory agent with no adverse effect on the healing process. Tolmetin-zinc promotes gain in breaking strength, not by increasing the collagen content, but by favouring better maturation of available collagen at the wound site.

Animals↗
PubMed1989

Binding site conformation dictates the color of the dye stains-all. A study of the binding of this dye to the eye lens proteins crystallins.

The interaction of the cationic carbocyanine dye Stains-all (1-ethyl-2-[3-(1-ethyl-naphthol[1,2-d]thiazolin-2-ylidene)-2- methylpropenyl]naphthol[1,2-d]thiazolium bromide) with the eye lens proteins crystallins has been studied. alpha- and gamma-crystallins do not bind the dye, while beta- and delta-crystallins do, consistent with the fact that the latter two proteins bind the calcium ion. beta-Crystallin resembles parvalbumin in that it induces only the J-band of the bound dye. delta-crystallin, on the other hand, induces only the gamma-band. Analysis of the metachromasia induced in the dye by these and other proteins suggests that Stains-all is responsive to the conformational status of the region to which it binds in a protein. The J-band of the dye is activated when it binds to a globular domain, and the gamma-band is activated when it binds to a helical stretch of the protein.

Animals↗
PubMed1989

Calcium ion binding to delta- and to beta-crystallins. The presence of the "EF-hand" motif in delta-crystallin that aids in calcium ion binding.

Abnormal levels of endogenous calcium ions are known to induce eye lens opacity, and a variety of causative factors has been proposed, including calcium-mediated aggregation and precipitation of the lens proteins crystallins. We have specifically looked in some detail at the interaction of Ca2+ with various crystallins and its consequences. Lenses incubated in solutions containing 10 mM Ca2+ or 5 mM Tb3+ opacified. Fluorescence titration of crystallins with TbCl3 revealed that this ion binds to delta- and beta-crystallins in solution. Equilibrium dialysis showed that four Ca2+ ions bind to one delta-crystallin tetramer with an affinity of 4.3 x 10(3) M-1. Analysis of the amino acid sequence of delta-crystallin reveals the presence of a calmodulin-type "helix-loop-helix" or "EF-hand" calcium ion binding conformational motif in the region comprising residues 300-350. This is a novel feature of the molecule not reported so far. No other crystallins appear to have this motif. beta-Crystallin also binds four Ca2+ ions/aggregate unit of mass 160 kDa, with an affinity of 2.6 x 10(3) M-1, presumably in the midregion of the molecule that is rich in anionic and polar residues. Circular dichroism spectroscopy shows that the binding of calcium ion leads to subtle conformational changes in the molecules, notably in the tertiary structure.

Amino Acid Sequence↗
PubMed1989

Red edge excitation effect in intact eye lens.

Shift in the wavelength of emission upon shift in the excitation wavelength towards the red edge of the absorption band is termed Red Edge Excitation Shift (REES). This effect is observed only in situation where the fluorophore mobility with respect to the surrounding matrix is considerably reduced. We have observed such red edge excitation effect in the intact eye lens. The REES observed for a normal lens is different from that seen in a photodamaged lens and hence appears to be a potential tool to monitor the changes in the state of the lens. Photodamage experiments with tryptophan in polyethylene glycol (PEG) and intact eye lens indicate that the red edge photon can also cause photodamage.

Animals↗