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Biomedical subjects

G Krishnamurthy

Publications and source records attributed to G Krishnamurthy.

At least 19 recordsLinked to original sources
PubMed2001

RFI-641 inhibits entry of respiratory syncytial virus via interactions with fusion protein.

BACKGROUND: RFI-641, a small dendrimer-like compound, is a potent and selective inhibitor of respiratory syncytial virus (RSV), which is currently a clinical candidate for the treatment of upper and lower respiratory tract infections caused by RSV. RFI-641 inhibits RSV growth with an IC(50) value of 50 nM and prevents syncytia formation in tissue culture. RSV contains of three surface glycoproteins, a small hydrophobic (SH) protein of unknown function, and attachment (G) and fusion (F) proteins that enable binding and fusion of virus, respectively, with target cells. Because of their role in attachment and fusion, the G and F surface proteins are prominent targets for therapeutic intervention. RFI-641 was previously shown to bind purified preparations of RSV fusion protein. Based on this observation, in conjunction with the biological results, it was speculated that the fusion event might be the target of these inhibitors. RESULTS: A fusion assay based upon the relief of self-quenching of octadecyl rhodamine R18 was used to determine effects of the inhibitors on binding and fusion of RSV. The results show that RFI-641 inhibits both RSV-cell binding and fusion events. The inhibition of RSV is mediated via binding to the fusion protein on the viral surface. A closely related analog, WAY-158830, which is much less active in the virus-infectivity assay does not inhibit binding and fusion of RSV with Vero cells. CONCLUSIONS: RFI-641, an in vivo active RSV inhibitor, is shown to inhibit both binding and fusion of RSV with cells, events that are early committed steps in RSV entry and pathogenicity. The results described here demonstrate that a non-peptidic, small molecule can inhibit binding and fusion of enveloped virus specifically via interaction with the viral fusion protein.

Animals↗
PubMed2000

Quantitation of pharmaceutically important phenothiazines by oxidimetry.

A new spectrophotometric method for the assay of phenothiazines in pure form as well as in pharmaceutical formulations with the chromium(VI)-metol-sulfanilic acid system has been developed. The method is based on the oxidation of the drugs by a known excess of chromium(VI) and subsequent determination of the unreacted oxidant by interacting with metol and sulfanilic acid. The reacted oxidant corresponds to the drug content. The coloured species exhibits maximum absorbance at 530 nm. Beer's law is obeyed over the concentration range 5-60 micrograms ml-1 and the relative standard deviation is found to be less than 2%. The apparent molar absorptivities are in the range 3.77 x 10(3)-3.98 x 10(3) l mol-1 cm-1, the detection limits being in the range 0.6133-1.1349 micrograms ml-1. The method was successfully applied to the determination of the studied drugs in their formulations and the mean percentage recoveries were found to be 97.32-102.80%.

Chemistry, Pharmaceutical↗
PubMed1997

Pex/PEX tissue distribution and evidence for a deletion in the 3' region of the Pex gene in X-linked hypophosphatemic mice.

PEX, a phosphate-regulating gene with homology to endopeptidases on the X chromosome, was recently identified as the candidate gene for X-linked hypophosphatemia. In the present study, we cloned mouse and human Pex/PEX cDNAs encoding part of the 5' untranslated region, the protein coding region, and the entire 3' untranslated region, determined the tissue distribution of Pex/PEX mRNA, and characterized the Pex mutation in the murine Hyp homologue of the human disease. Using the reverse transcriptase/polymerase chain reaction (RT/PCR) and ribonuclease protection assays, we found that Pex/PEX mRNA is expressed predominantly in human fetal and adult mouse calvaria and long bone. With RNA from Hyp mouse bone, an RT/PCR product was generated with 5' but not 3' Pex primer pairs and a protected Pex mRNA fragment was detected with 5' but not 3' Pex riboprobes by ribonuclease protection assay. Analysis of the RT/PCR product derived from Hyp bone RNA revealed an aberrant Pex transcript with retention of intron sequence downstream from nucleotide 1302 of the Pex cDNA. Pex mRNA was not detected on Northern blots of poly (A)+ RNA from Hyp bone, while a low-abundance Pex transcript of approximately 7 kb was apparent in normal bone. Southern analysis of genomic DNA from Hyp mice revealed the absence of hybridizing bands with cDNA probes from the 3' region of the Pex cDNA. We conclude that Pex/PEX is a low-abundance transcript that is expressed predominantly in bone of mice and humans and that a large deletion in the 3' region of the Pex gene is present in the murine Hyp homologue of X-linked hypophosphatemia.

Amino Acid Sequence↗
PubMed1996

Flavins inhibit human cytomegalovirus UL80 protease via disulfide bond formation.

Among the most potent inhibitors of human cytomegalovirus protease identified by random screening of a chemical library was 1,4-dihydro-7,8-dimethyl 6H-pyrimido[1,2-b]-1,2,4,5-tetrazin-6-one (1) (PTH2). The oxidized form (2), PT, which is present in solutions of PTH2, was shown to be the actual inhibitory species which irreversibly inactivates the protease; recycling of PTH2 by dissolved oxygen results in complete inhibition of the protease at substoichiometric amounts of compound. No evidence for a covalent adduct between the protease and the inhibitor was obtained, and protease activity was restored by incubation of the inactivated enzyme with the reducing agent bismercaptoethyl sulfone, suggesting that disulfide bond formation was responsible for the observed inhibition. The five cysteines of the protease are normally in the reduced state; analysis of tryptic peptides from inhibited protease indicated that disulfide bonds Cys84-Cys87 and Cys138-Cys161 were formed. Using site-directed mutagenesis, the disulfide pair induced between Cys138 and Cys161 disulfide is dependent upon interaction of PT with the protease and does not form spontaneously, unlike that of the Cys84-Cys87 pair which can form in the absence of inhibitor. The inhibitor's redox chemistry is analogous to that of flavin, and, in fact, flavin inhibits the protease by the same mechanism, causing formation of a disulfide bond between Cys138 and Cys161. That the cysteines are dispensable, but can regulate protease activity by formation of a unique disulfide pair, suggests a plausible mechanism for control of proteolysis during the viral life cycle.

Base Sequence↗
PubMed1995

Salt dependence of calicheamicin-DNA site-specific interactions.

Calicheamicin gamma 1I site-specifically binds and cleaves three closely spaced tetranucleotide sequences embedded in an AT-rich region of a 142 base pair DNA restriction fragment. Cleavage is observed predominantly at the TCCT, TTGT, and ATCT sequences, of which TCCT is the primary cleavage site. The Gibbs free energies required to bind calicheamicin to these sequences within the DNA restriction fragment have been determined as a function of NaCl concentration at pH 8.1 and 0 degrees C and at pH 7.5 and 23 and 0 degrees C. Between 150 mM and 1 M NaCl, calicheamicin binding to all three sequences is insensitive to salt. The insensitivity of calicheamicin binding to salt continues to 50 mM NaCl for the TTGT and ATCT sequences; the delta G values for calicheamicin binding to these sequences are on the order of -7.8 to -7.9 kcal mol-1 over the entire range of NaCl concentrations studied. However, between 150 and 125 mM NaCl, the TCCT sequence displays a sharp transition in the delta G of calicheamicin binding from -7.6 to -8.9 kcal mol-1. Below 125 mM NaCl, the delta G values for calicheamicin binding to the TCCT sequence again are invariant. An analysis of the data in terms of polyelectrolyte theory suggests that counterion release from DNA does not contribute significantly to the energetics of the association and that the association of calicheamicin with specific DNA sequences is dominated by nonionic rather than electrostatic forces. Our results further suggest that some calicheamicin binding/cleavage sites are dependent on flanking sequences.

Aminoglycosides↗
PubMed1995

Defective lymphoproliferative responses & interleukin-2 production in chronic renal failure patients.

Cellular immune responses were evaluated in 12 early renal failure (ERF) patients who were not on maintenance haemodialysis, 43 end stage renal disease (ESRD) patients on haemodialysis (HD) and 25 healthy volunteers. Peripheral blood mononuclear cells (PBMC) of ERF and ESRD patients on HD had a significantly diminished lymphoproliferative responses to phytohaemagglutinin and a monoclonal antibody to the CD3 (anti-CD3) receptor on T-cells as compared to normals. The interleukin-2 (IL-2) production by the PBMC was also significantly reduced in renal failure patients as compared to normals. These data suggest that both IL-2 dependent and IL-2 independent T-cell functions are defective in renal failure patients.

Adult↗
PubMed1990

A photochemical method to map ethidium bromide binding sites on DNA: application to a bent DNA fragment.

It is shown that, when irradiated in the visible, ethidium bromide (EB) engages in direct photochemistry with its DNA binding site. At the photochemical end point, an average of one single-strand break is produced per bound EB molecule in a reaction which also bleaches the dye chromophore. Using high-resolution electrophoresis, we have mapped the distribution of EB photocleavage sites on DNA, at one-base resolution. It is argued that because the photocleavage is stoichiometric, the resulting pattern is similar to, if not identical with, the local distribution of EB binding affinity. When interpreted in the context of the extensive thermodynamic and structural data which are available for EB, a binding distribution of that kind can be used to infer details of DNA structure variation within the underlying helix. As a first application of the method, we have used EB to probe the structure of a 265 bp fragment of DNA, which had been described as being bent as the result of a periodic array of oligo(A) segments [Kitchin et al. (1986) J. Biol. Chem. 261, 11302]. The EB mapping data provide evidence that the oligo(A) elements in this fragment assume a local secondary structure which is different than that assumed by isolated ApA nearest neighbors and that the ends of the oligo(A) elements comprise a junctional domain with EB binding properties which differ from those of the oligo(A) element or of random-sequence DNA.

Adenine Nucleotides↗
PubMed1987

Reproducibility of rest and exercise left ventricular ejection fraction and volumes in chronic aortic regurgitation.

To assess the variability of rest and exercise radionuclide ventriculography in patients with aortic regurgitation (AR), 22 patients in stable condition with chronic AR underwent radionuclide ventriculography at rest and during exercise for measurement of left ventricular volumes and ejection fraction (EF) on 2 occasions 3 months apart. For the group, there were no significant differences between the findings of the 2 studies except for a slightly lower exercise end-systolic volume on the second study. The inter-study differences and variabilities (expressed as the standard deviation of differences) for rest and exercise EF and change in EF were 0 +/- 0.04, +0.02 +/- 0.05 and +0.01 +/- 0.05, respectively. Thus, although mean differences were minor, considerable individual variability occurred, the magnitude of which was similar to that reported for other patient populations. In 3 patients who had an initial normal response to exercise (increase in EF greater than or equal to 0.05), the response became abnormal and in 2 who initially had an abnormal response, the response normalized. This variability must be considered in clinical decision-making or in research study design for patients with chronic AR.

Adult↗
PubMed1985

Association between the exercise ejection fraction response and systolic wall stress in patients with chronic aortic insufficiency.

We studied the exercise ejection fraction response in 56 patients with chronic aortic insufficiency. All had left ventricular dilatation but preserved resting ejection fraction and minimal or no symptoms. The exercise ejection fraction increased by 0.05 units or greater in 18 (32%) patients (group I), remained within 0.05 units of the resting value in 18 (32%) patients (group II), and fell by 0.05 units or greater in 20 (36%) patients (group III). There were no significant differences among the groups in left ventricular end-diastolic dimension, end-systolic dimension, or fractional shortening by echocardiography or in resting left ventricular volumes and ejection fraction by radionuclide angiography. Left ventricular end-systolic wall stress was significantly higher in group III than in either group I or group II (89 +/- 20 vs 70 +/- 18 and 69 +/- 17 X 10(3) dyne/cm2; p less than .005). At peak exercise there were no differences among groups in systolic blood pressure. However, end-systolic volume increased from 65 +/- 28 to 77 +/- 36 ml/m2 in group III and fell from 50 +/- 21 to 28 +/- 18 ml/m2 in group I during exercise. Thus, at peak exercise end-systolic volume was nearly three times greater in group III than in group I. Although stress could not be determined directly during exercise, the directional changes in its determinants suggest that it also would have been higher in group III patients. A highly significant inverse correlation was present between the ejection fraction response and the change in end-systolic volume (r = -.87, p less than .0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗