Computational Design and Biological Testing of Highly Cytotoxic Colchicine Ring A Modifications

Microtubules are the primary target for many anti-cancer drugs, the majority of which bind specifically to β-tubulin. The existence of several β-tubulin isotypes, coupled with their varied expression in normal and cancerous cells provides a platform upon which to construct selective chemotherapeutic agents. We have examined five prevalent human β-tubulin isotypes and identified the colchicine-binding site as the most promising for drug design based on specificity. Using this binding site as a template, we have designed several colchicine derivatives and computationally probed them for affinity to the β-tubulin isotypes. These compounds were synthesized and subjected to cytotoxicity assays to determine their effectiveness against several cancerous cell lines. We observed a correlation between computational-binding predictions and experimentally determined IC₅₀ values, demonstrating the utility of computational screening in the design of more effective colchicine derivatives. The most promising derivative exhibited an IC₅₀ approximately threefold lower than values previously reported for either colchicine or paclitaxel, demonstrating the utility of computational design and assessment of binding to tubulin.     

Effect of phosphamidon on convulsive behavior and biochemical parameters: modulation by progesterone and 4′-chlorodiazepam in rats

Phosphamidon (PHOS) has been shown to affect nervous system adversely. The present study was designed to explore the modulation of the effects of PHOS on convulsions by neurosteroids, progesterone (PROG), and 4′-chlorodiazepam (4′-CD), in both acute and chronic seizure models. In acute study, seizures were induced by either pentylenetetrazole (PTZ) injection or maximal electroshock seizures, while in the chronic study, kindling was induced by injecting PTZ (30 mg/kg, s.c.) on alternate days three times in a week. Oxidative stress was assessed in the brain by measuring the levels of malondialdehyde (MDA), acetylcholinesterase (AChE), and non-protein thiol (NP-SH). PROG and 4′-CD were able to modulate the PHOS-induced convulsions in acute PTZ convulsions as well as in chronic kindling model. However, they failed to reverse the derangements in oxidative stress parameters of MDA and NP-SH produced by PHOS in kindled animals. PROG significantly increased the AChE activity in untreated rats, while PROG and 4′-CD reversed the AChE activity inhibition induced by PHOS. The study indicates a possible anticonvulsive mechanism of neurosteroids, since both PROG and 4′-CD reversed PHOS-induced inhibition of AChE activity. The neurosteroids seem to play a protective role in PHOS-induced convulsions besides their antioxidant property.     

Comparison of viral load and duration of virus shedding in symptomatic and asymptomatic neonatal rotavirus infections

A single rotavirus strain causing asymptomatic infections as well as severe gastrointestinal disease has been described in the neonatal nurseries of the Christian Medical College, Vellore. In this study, quantitative real‐time RT‐PCR was used to determine the association of viral load with the presence of gastrointestinal symptoms in neonates. Viral load was estimated in terms of the crossing point [C(t) value] at which the amplicon could be detected in the real‐time PCR assay. The study was carried out on 103 neonates, including 33 asymptomatic neonates and 70 neonates with different gastrointestinal symptoms. The duration of virus shedding was also compared between five symptomatic and four asymptomatic neonates using real‐time RT‐PCR. There was no significant difference in viral load between symptomatic and asymptomatic neonates (P = 0.087). Among neonates with different gastrointestinal symptoms, those presenting with feed intolerance and abdominal distension had a significantly higher viral load than those with other gastrointestinal symptoms (P = 0.02). For the study on virus shedding, nine neonates were followed up for a median duration of 53 days, with a median of 31 samples tested per child. Extended shedding of low copies of rotavirus was found, with no significant differences in pattern of shedding between symptomatic and asymptomatic neonates. The lack of correlation between viral load and gastrointestinal disease demonstrates yet another difference between neonatal rotavirus infection and infection in older children where higher viral load correlates with severe disease. J. Med. Virol. 82:1803–1807, 2010. © 2010 Wiley‐Liss, Inc.     

Risk of noncardiac surgery after coronary drug-eluting stent implantation

We examined the records of 38 patients who underwent 41 major and 18 minor noncardiac surgeries after successful drug-eluting stent (DES) implantation (57% sirolimus-eluting stents and 43% paclitaxel-eluting stents) at the Dallas Veterans Affairs Medical Center from April 2003 to January 2006. The mean patient age was 62 +/- 9 years, and all patients were men. A total of 41 major noncardiac surgeries (34% abdominal, 22% vascular, 17% genitourinary, and 27% other) were performed in 28 patients a median of 260 days after DES implantation. Also, 18 minor noncardiac surgeries (44% skin surgery, 44% injections, and 12% other) were performed in 10 patients a median of 297 days after DES implantation. No major adverse cardiac events or death occurred during or after the 41 major (0%, 95% confidence interval 0% to 9%) and 18 minor noncardiac (0%, 95% confidence interval 0% to 19%) surgeries. In conclusion, although our data were limited by the small sample size, they suggest a low risk of major cardiac complications in patients undergoing noncardiac surgery after coronary DES implantation.     

S-adenosylmethionine stabilizes cystathionine beta-synthase and modulates redox capacity

The transsulfuration pathway converts homocysteine to cysteine and represents the metabolic link between antioxidant and methylation metabolism. The first and committing step in this pathway is catalyzed by cystathionine beta-synthase (CBS), which is subject to complex regulation, including allosteric activation by the methyl donor, S-adenosylmethionine (AdoMet). In this study, we demonstrate that methionine restriction leads to a >10-fold decrease in CBS protein levels, and pulse proteolysis studies reveal that binding of AdoMet stabilizes the protein against degradation by approximately 12 kcal/mol. These observations predict that under pathological conditions where AdoMet levels are diminished, CBS, and therefore glutathione levels, will be reduced. Indeed, we demonstrate this to be the case in a mouse model for spontaneous steatohepatitis in which the gene for the MAT1A isoenzyme encoding AdoMet synthetase has been disrupted, and in human hepatocellular carcinoma, where MAT1A is silenced. Furthermore, diminished CBS levels are associated with reduced cell viability in hepatoma cells challenged with tert-butyl hydroperoxide. This study uncovers a mechanism by which CBS is allosterically activated by AdoMet under normal conditions but is destabilized under pathological conditions, for redirecting the metabolic flux toward methionine conservation. A mechanistic basis for the coordinate changes in redox and methylation metabolism that are a hallmark of several complex diseases is explained by these observations.     

Use of nanotechnology for the development of novel cancer biomarkers

Novel nanotechnologies can complement and augment existing genomic and proteomic techniques employed to analyze variations across different tumor types, thus offering the potential to distinguish between normal and malignant cells. Sensitive biosensors constructed out of nanoscale components (e.g., nanocantilevers, nanowires and nanochannels) can recognize genetic and molecular events and have reporting capabilities, thereby offering the potential to detect rare molecular signals associated with malignancy. Such signals may then be collected for analysis by nanoscale harvesters that selectively isolate cancer-related molecules from tissues. Another area with near-term potential for the early detection of cancer is the identification of mutations and genomic instability.     

Association of an extended haplotype of HLA class I alleles and their flanking microsatellites with spondyloarthropathies in South Indian patients

Spondyloarthropathy (SpA) is a complex autoimmune disease known to have an association with the HLA system. The aims of the present study were to compare the suballelic association of HLA-B27 and other HLA class I genes with microsatellite markers spanning the HLA class I region in the South Indian population of Kerala. The five microsatellites were C1_2_A (D6S2793), C1_2_5 (D6S2811), C1_4_1 (D6S2927), MIB (D6S2810), and STR-MICA. HLA typing was performed in 67 SpA patients and 77 ethnically matched healthy controls by polymerase chain reaction using sequence-specific primers, whereas fluorescence-labeled microsatellites were analyzed using GeneScan analysis. There was a significant association of HLA-B27 and Cw*02 with SpA, whereas B*44 had a negative association with the disease. Only two HLA-B27 subtypes, B*2704 and B*2705, were observed in the South Indian population. We were able to successfully predict the major B27 subtype B*2705 based on the C1_2_5 microsatellite. A significant association of different alleles of all the microsatellite markers with SpA was observed. An extended six-locus haplotype, B*2705-Cw*02-STR-MICA(A4)-C1_4_1 (213 bp)-C1_2_5 (178 bp)-MIB (340 bp), was significantly associated with SpA.     

Fear of falling more important than pain and depression for functional recovery after surgery for hip fracture in older people

BACKGROUND: Depression and cognitive functioning have a negative impact on functional recovery after hip fracture surgery in older people, and the same has been suggested for pain and fear of falling. These variables, however, have never been studied together, nor has the timing of psychiatric assessment been taken into account. METHOD: Two parallel, randomized controlled trials were undertaken aiming to prevent and treat depression after hip fracture surgery in older people. Multiple logistic regression analyses corrected for age and pre-morbid level of functioning were performed to evaluate the effect of depressive symptoms (15-item Geriatric Depression Scale, GDS), pain (Wong-Baker pain scale), cognitive functioning (Mini-mental State Examination, MMSE) and fear of falling (Modified Falls Efficacy Scale, MFES) within 2 weeks after surgery and 6 weeks later on functional recovery at 6 months. Main outcome measures were performance-based measures (up-and-go test, gait test, functional reach) and the self-report Sickness Impact Profile (SIP) questionnaire to assess the impact of the hip fracture on activities of daily living (ADL). RESULTS: Two hundred and ninety-one patients participated and outcome measures for 187 (64%) patients were available at 6 months. All mental health variables interfered with functional recovery. However, in the final multivariate model, cognitive functioning and fear of falling assessed 6 weeks after surgery consistently predicted functional recovery, whereas pain and depressive symptoms were no longer significant. CONCLUSION: Fear of falling and cognitive functioning may be more important than pain and depression to predict functional recovery after hip fracture surgery. Rehabilitation strategies should take this into account.