Toxicity and anti-angiogenicity evaluation of Pak1 inhibitor IPA-3 using zebrafish embryo model.

p21-activated kinase 1 (Pak1)-a key node protein kinase regulating various cellular process including angiogenesis-has been recognised to be a therapeutic target for multitude of diseases, and hence, various small molecule inhibitors targeting its activity have been tested. However, the direct toxic and anti-angiogenic effects of these pharmacologic agents have not been examined. In this study, we evaluate the translational efficacy of Pak1 inhibitor IPA-3 using zebrafish toxicity model system to stratify its anti-angiogenic potential and off-target effects to streamline the compound for further therapeutic usage. The morphometric analysis has shown explicit delay in hatching, tail bending, pericardial sac oedema and abnormal angiogenesis. We provide novel evidence that Pak1 inhibitor could act as anti-angiogenic agents by impeding the development of sub-intestinal vessel (SIV) and intersegmental vessels (ISVs) by suppressing the expression of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), neurophilin 1 (NRP1) and its downstream genes matrix metalloproteinase (MMP)-2 and MMP-9. Knockdown studies using 2-O-methylated oligoribonucleotides targeting Pak1 also revealed similar phenotypes with inhibition of angiogenesis accompanied with deregulation of major angiogenic factor and cardiac-specific genes. Taken together, our findings indicate that Pak1 signalling facilitates enhanced angiogenesis and also advocated the design and use of small molecule inhibitors of Pak1 as potent anti-angiogenic agents and suggest their utility in combinatorial therapeutic approaches targeting anomalous angiogenesis.

"Role of the adipocyte hormone leptin in cardiovascular diseases - a study from Chennai based Population".

BACKGROUND: Obesity is currently regarded as a pro-inflammatory condition during which leptin (Ob gene product) might act as a risk factor for Cardiovascular Diseases (CVD) including Acute Myocardial Infarction (AMI). There is a marked increase in circulating leptin concentrations and inflammatory markers such as Tumor Necrosis Factor-α (TNF-α) in AMI patients but still the association of leptin with inflammation during AMI is not known. The present study suggest that elevated levels of leptin might elicit the risk for CVD by signaling for the secretion of inflammatory cytokines especially, TNF-α. METHODS: Blood samples were collected from 100 CVD subjects diagnosed for AMI immediately after their admission to the hospital and serum leptin, insulin, glucose, lipids and inflammatory marker such as TNF-α were measured. 5 ml random (non-fasting) blood was collected from 100 non-CVD (control) subjects and the results obtained in case of AMI subjects were compared with that of the control subjects. The subjects under study included both men and women belonging to the age group of 35 - 75 and they were classified based on their BMI as normal weight, overweight and obese. RESULTS: Circulating levels of leptin are found to be elevated in obese control subjects and in patients with AMI irrespective of their Body Mass Index (BMI). In addition, leptin is also found to be positively correlated to serum triglycerides, insulin and TNF-α in AMI subjects. MANOVA analysis suggests that leptin might influence the synthesis of insulin and TNF-α. This is the first report relating leptin to TNF-α in Chennai based population, India. CONCLUSIONS: Hyperleptinemia might act as a risk marker for AMI. The present study suggests that at elevated levels, leptin may favor atherosclerosis by promoting the synthesis of TNF-α and insulin. However, our report warrants further investigation both in vitro and in vivo to determine the exact mechanism behind the pro-atherogenic role of leptin. The observed positive correlation between leptin and BMI in both AMI and control subjects suggests that obese subjects manifest leptin resistance and hence, they possess a greater risk for the incidence of CVD.

Modulation of DNA intercalation by resveratrol and genistein.

Time correlated Single Photon Counting study (TCSPC) was performed for the first time to evaluate the effect of resveratrol (RES) and genistein (GEN) at 10-100 microM and 10-150 microM respectively, in modulating the DNA conformation and the variation induced due to intercalation by the dyes, ethidium bromide (EtBr) and acridine orange (AO). It is demonstrated using UV-absorption and fluorescence spectroscopy that RES and GEN, at 50 microM and 100 microM respectively can bind to DNA resulting in significant de-intercalation of the dyes, preventing their further intercalation within DNA. Hyperchromicity with red/blue shifts in DNA when bound to dyes was reduced upon addition of RES and GEN. DNA-dependent fluorescence of EtBr and AO was quenched in the presence of RES by 87.97% and 79.13% respectively, while similar quenching effect was observed for these when interacted with GEN (85.52% and 83.85%). It is found from TCSPC analysis that the higher lifetime component or constituent of intercalated dyes (tau(2), A (2)) decreased with the subsequent increase in smaller component or constituent of free dye (tau(1), A (1)) after the interaction of drugs with the intercalated DNA. Thus these findings signify that RES and GEN can play an important role in modulating DNA intercalation, leading to the reduction in DNA-directed toxicity.

Interaction of resveratrol and genistein with nucleic acids.

Resveratrol (RES) and genistein (GEN) are the dietary natural products known to possess chemopreventive property and also the ability to repair DNA damage induced by mutagens/carcinogens. It is believed that the therapeutic activity of these compounds could be primarily due to their interaction with nucleic acids but detailed reports are not available. We here explore the interaction of these drugs with nucleic acids considering DNA and RNA as a potential therapeutic target. The interaction of RES and GEN has been analysed in buffered solution with DNA [saline sodium citrate (SSC)] and RNA [tris ethylene diammine tetra acetic acid (TE)] using UV-absorption and Fourier transform infrared (FTIR) spectroscopy. The UV analysis revealed lesser binding affinity with nucleic acids at lower concentration of RES (P/D = 5.00 and 10.00), while at higher drug concentration (P/D = 0.75, 1.00 and 2.50) hyperchromic effect with shift in the lambda(max) is noted for DNA and RNA. A major RES-nucleic acids complexes was observed through base pairs and phosphate backbone groups with K = 35.782 M(-1) and K = 34.25 M(-1) for DNARES and RNA-RES complexes respectively. At various concentrations of GEN (P/D = 0.25, 0.50, 0.75, 1.00 and 2.50) hyperchromicity with shift in the lambda(max) from 260-->263 nm and 260--> 270 nm is observed for DNA-GEN and RNA-GEN complexes respectively. The binding constant (from UV analysis) for GEN-nucleic acids complexes could not be obtained due to GEN absorbance overlap with that of nucleic acids at 260 nm. Nevertheless a detailed analysis with regard to the interaction of these drugs (RES/GEN) with DNA and RNA could feasibly be understood by FTIR.

Cytokines and leptin correlation in patients with polycystic ovary syndrome: Biochemical evaluation in south Indian population.

Background: In polycystic ovary syndrome (PCOS), the leptin (OB protein) is related to reproductive function and inflammatory response. Leptin and cytokines have been thought to be putative local regulators in PCOS. Methods: To examine the relationship between serum leptin and serum interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) levels in underweight, overweight, obese and morbidly obese PCOS and non-PCOS subjects compared with normal weight, regularly menstruating women. Results: Leptin levels are highly correlated with TNF-α, IL-6 and IL-8. There is a significant dependent increase with the degree of obesity, but in underweight PCOS subjects, leptin levels are elevated irrespective of the body mass index. Conclusion: The present study showed that leptin levels were elevated in underweight and morbidly obese PCOS subjects. This could be the result of impaired expression of leptin in PCOS, leading to leptin resistance. As a result of this regulation, TNF-α, IL-6 and IL-8 were also elevated in morbidly obese and underweight PCOS subjects. In obese subjects, where there was an increase in adipose mass, increased levels of leptin were observed and this was attributed to the inflammatory properties while increasing the adipose mass. Serum IL-6 and IL-8 circulate at high levels and are more important systemically. They are, perhaps, the hormonal factors that induce leptin and insulin resistance in underweight PCOS subjects. Therefore, leptin and inflammatory markers were acting at paracrine and endocrine levels in PCOS subjects. (Reprod Med Biol 2005; 4: 247-254).

Mutation analysis of congenital cataracts in Indian families: identification of SNPS and a new causative allele in CRYBB2 gene.

PURPOSE: To study some functional candidate genes in cataract families of Indian descent. METHODS: Nine Indian families, clinically documented to have congenital/childhood cataracts, were screened for mutations in candidate genes such as CRYG (A-->D), CRYBB2, and GJA8 by PCR analyses and sequencing. Genomic DNA samples of either probands or any representative affected member of each family were PCR amplified and sequenced commercially. Documentation of single nucleotide polymorphisms (SNPs) and candidate mutations was done through BLAST SEARCH (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?). RESULTS: Several single nucleotide polymorphisms in CRYG, CRYBB2, and GJA8 genes were observed. Because they do not co-segregate with the phenotype, they were excluded as candidates for the cataract formation in these patients. However, a substitution (W151C in exon 6 of CRYBB2) was identified as the most likely causative mutation underlying the phenotype of central nuclear cataract in all affected members of family C176. Protein structural interpretations demonstrated that no major structural alterations could be predicted and that even the hydrogen bonds to the neighboring Leu166 were unchanged. Surprisingly, hydropathy analysis of the mutant betaB2-crystallin featuring the amino acids at position 147 to 155, further increased the hydrophobicity, which might impair the solubility of the mutant protein. Finally, the Cys residue at position 151 might possibly be involved in intramolecular disulphide bridges with other cysteines during translation, possibly leading to dramatic structural changes. CONCLUSIONS: Exon 6 of CRYBB2 appears to be a critical region susceptible for mutations leading to lens opacity.