Podophyllotoxin as a probe for the colchicine binding site of tubulin.

The binding of [3H]podophyllotoxin to tubulin, measured by a DEAE-cellulose filter paper method, occurs with an affinity constant of 1.8 X 10(6) M-1 (37 degrees at pH 6.7). Like colchicine, approximately 0.8 mol of podophyllotixin are bound per mol of tubulin dimer, and the reaction is entropy-driven (43 cal deg-1 mol-1). At 37 degrees the association rate constant for podophyllotoxin binding is 3.8 X 10(6) M-1 h-1, approximtaely 10 times higher than for colchicine; this is reflected in the activation energies for binding which are 14.7 kcal/mol for podophyllotoxin and 20.3 kcal/mol for colchicine. The dissociation rate constant for the tubulin-podophyllotoxin complex is 1.9 h-1, and the affinity constant calculated from the ratio of the rates is close to that obtained by equilibrium measurements. Podophyllotxin and colchicine are mutually competitive inhibitors. This can be ascribed to the fact that both compounds have a trimethoxyphenyl ring and analogues of either compound with bulky substituents in their trimethoxyphenyl moiety are unable to inhibit the the binding of either of the two ligands. Tropolone, which inhibits colchicine binding competitively, has no effect on the podophyllotoxin/tubulin reaction. Conversely, podophyllotoxin does not influence tropolone binding. Moreover, the tropolone binding site of tubulin does not show the temperature and pH lability of the colchicine and podophyllotoxin domains, hence this lability can be ascribed to the trimethoxyphenyl binding region of tubulin. Since podophyllotoxin analogues with a modified B ring do not bind, it is concluded that both podophyllotoxin and colchicine each have at least two points of attachment to tubulin and that they share one of them, the binding region of the trimethoxyphenyl moiety.     

Malaria transmission risk by the mosquito Anopheles baimaii (formerly known as An. dirus species D) at different hours of the night in North-east India

The risk of acquiring malaria transmitted by Anopheles baimaii Sallum & Peyton, 2005, formerly known as An. dirus species D (Sallum et al., 2005) (Diptera: Culicidae), at different hours of the night in a forest-fringed village of Assam, North-east India was assessed through all-night mosquito landing catches during 1995-2000. An estimated overall mean biting rate of 36.1 bites/person/night (95% CI = 26.2-45.8), a sporozoite rate of 1.9% (95% CI = 1.1-2.9%) and a parous rate of 58.7% (95% CI = 55.3-62.0%) were recorded. Parous and sporozoite-positive females tended to be caught mainly before midnight. The effective entomological inoculation rate was the highest (0.249 positive bites/person/night) from 21.00 to 24.00 hours, suggesting that the second quartile of the night is the most risky period for malaria transmission by An. baimaii. Considering that approximately 21% of mean inoculations take place before 21.00 hours, it appears that there is a need for appropriate protective measures during the pre-bed time period to supplement the impact of insecticide-treated nets against An. baimaii in north-east India.     

Chaperone activity and prodan binding at the self-associating domain of erythroid spectrin

Spectrin, the major constituent protein of the erythrocyte membrane skeleton, exhibits chaperone activity by preventing the irreversible aggregation of insulin at 25 degrees C and that of alcohol dehydrogenase at 50 degrees C. The dimeric spectrin and the two subunits, alpha-spectrin and beta-spectrin prevent such aggregation appreciably better, 70% in presence of dimeric spectrin at an insulin:spectrin ratio of 1:1, than that in presence of the tetramer of 25%. Our results also show that spectrin binds to denatured enzymes alpha-glucosidase and alkaline phosphatase during refolding and the reactivation yields are increased in the presence of the spectrin derivatives when compared with those refolded in their absence. The unique hydrophobic binding site on spectrin for the fluorescence probe, 6-propionyl-2-(dimethylamino)naphthalene (Prodan) has been established to localize at the self-associating domain with the binding stoichiometry of one Prodan/both dimeric and tetrameric spectrin. The other fluorescence probe, 1-anilinonaphthalene-8-sulfonic acid, does not show such specificity for spectrin, and the binding stoichiometry is between 3 and 5 1-anilinonaphthalene-8-sulfonic acid/dimeric and tetrameric spectrin, respectively. Regions in alpha- and beta-spectrins have been found to have sequence homology with known chaperone proteins. More than 50% similarities in alpha-spectrin near the N terminus with human Hsp90 and in beta-spectrin near the C terminus with human Hsp90 and Escherichia coli DnaJ have been found, indicating a potential chaperone-like sequence to be present near the self-associating domain that is formed by portions of alpha-spectrin near the N terminus and the beta-spectrin near the C terminus. There are other patches of sequences also in both the spectrin polypeptides, at the other termini as well as in the middle of the rod domain having significant homology with well known chaperone proteins.     

Tumor necrosis factor alpha-induced inflammation is increased but apoptosis is inhibited by common food additive carrageenan

Tumor necrosis factor (TNF)-α, a homotrimeric, pleiotropic cytokine, is secreted in response to inflammatory stimuli in diseases such as rheumatoid arthritis and inflammatory bowel disease. TNF-α mediates both apoptosis and inflammation, stimulating an inflammatory cascade through the non-canonical pathway of NF-κB activation, leading to increased nuclear RelB and p52. In contrast, the common food additive carrageenan (CGN) stimulates inflammation through both the canonical and non-canonical pathways of NF-κB activation and utilizes the adaptor molecule BCL10 (B-cell leukemia/lymphoma 10). In a series of experiments, colonic epithelial cells and mouse embryonic fibroblasts were treated with TNF-α and carrageenan in order to simulate the possible effects of exposure to dietary CGN in the setting of a TNF-α-mediated inflammatory disease process. A marked increase in secretion of IL-8 occurred, attributable to synergistic effects on phosphorylated NF-κB-inducing kinase (NIK) in the non-canonical pathway. TNF-α induced the ubiquitination of TRAF2 (TNF receptor-associated factor 2), which interacts with NIK, and CGN induced phosphorylation of BCL10, leading to increased NIK phosphorylation. These results suggest that TNF-α and CGN in combination act to increase NIK phosphorylation, thereby increasing activation of the non-canonical pathway of NF-κB activation. In contrast, the apoptotic effects of TNF-α, including activation of caspase-8 and PARP-1 (poly(ADP-ribose) polymerase 1) fragmentation, were markedly reduced in the presence of CGN, and CGN caused reduced expression of Fas. These findings demonstrate that exposure to CGN drives TNF-α-stimulated cells toward inflammation rather than toward apoptotic cell death and suggest that CGN exposure may compromise the effectiveness of anti-TNF-α therapy.     

Effects of diesel exhaust, heavy metals and pesticides on various organ systems: possible mechanisms and strategies for prevention and treatment

Environmental pollutants have a significant impact on the ecosystem and disrupt balance between environment, human and non-human components that result in deleterious effects to all forms of life. Identifying environmental factors for potential imbalance are extremely crucial for devising strategies for combating such toxic dysregulation. Automobile exhaust (in air), heavy metals (in food and water) and pesticides (in air, food, soil and water) are the most common environmental pollutants and their short and long-term exposures can cause hazardous effects in humans leading to systemic disorders involving lungs, kidney and immune systems. Mechanisms involved in genesis of such toxic effects have revealed complex, interactive pathways. Strategies for the protection of homeostasis and health, viz., general preventive measures, nutritional supplements and herbal agents have been described, to counter these pollutants induced damaging effects on various body systems.     

Sequence variants in the melatonin-related receptor gene (GPR50) associate with circulating triglyceride and HDL levels

The gene encoding the melatonin-related receptor (GPR50) is highly expressed within hypothalamic nuclei concerned with the control of body weight and metabolism. We screened GPR50 for mutations in an obese cohort and identified an insertion of four amino acid residues (TTGH) at position 501, two common coding polymorphisms (T528A and V602I), and one noncoding polymorphism (C-16X2GPR50T). Single-nucleotide polymorphisms were then typed in 500 English Caucasian subjects, and associations were sought to intermediate obesity phenotypes. Although no association was seen with body mass index, carriers of two copies of the mutant allele at C-16X2GPR50T, Ins501Del, and A1582G had significantly higher fasting circulating triglyceride levels (P < 0.05). In a separate set of 585 subjects, the associations were replicated, with statistically significant effects of similar magnitude and direction. The association of C-16X2GPR50T with fasting triglycerides was highly significant (P < 0.001). In addition, a significant association between C-16X2GPR50T and circulating HDL levels was observed in the combined population, with C-16X2GPR50T carriers having significantly lower circulating HDL-cholesterol levels (1.39 mM) than wild-type subjects (1.47 mM) (P < 0.01). These findings suggest a previously unexpected role for this orphan receptor in the regulation of lipid metabolism that warrants further investigation.     

Activation of mitochondrial promoter P(H)-binding protein in a radio-resistant Chinese hamster cell strain associated with Bcl-2

The cellular response to ionizing radiation is mediated by a complex interaction of number of proteins involving different pathways. Previously, we have shown that up regulation of mitochondrial genes ND1, ND4, and COX1 transcribed from the heavy strand promoter (P(H)) has been increased in a radio-resistant cell strain designated as M5 in comparison with the parental Chinese hamster V79 cells. These genes are also up regulated in Chinese hamster V79 cells VB13 that express exogenous human Bcl2. In the present study, the expression of the gene ND6 that is expressed from the light strand promoter (P(L)) was found to be similar in both the cell lines, as determined by RT-PCR. To test the possibility that this differential expression of mitochondrial genes under these two promoters was mediated by differences in proteins' affinity to interact with these promoters, we have carried out electrophoretic mobility shift assay (EMSA) using mitochondrial cell extracts from these two cell lines. Our result of these experiments revealed that two different proteins formed complex with the synthetic promoters and higher amount of protein from M5 cell extracts interacted with the P(H) promoter in comparison to that observed with cell extracts from Chinese hamster V79 cells. The promoter-specific differential binding of proteins was also observed in VB13. These results showed that differential mitochondrial gene expression observed earlier in the radio-resistant M5 cells was due to enhanced interaction proteins with the promoters P(H) and mediated by the expression of Bcl2.     

Exploitation of marine bacteria for production of gold nanoparticles

Background

Gold nanoparticles (AuNPs) have found wide range of applications in electronics, biomedical engineering, and chemistry owing to their exceptional opto-electrical properties. Biological synthesis of gold nanoparticles by using plant extracts and microbes have received profound interest in recent times owing to their potential to produce nanoparticles with varied shape, size and morphology. Marine microorganisms are unique to tolerate high salt concentration and can evade toxicity of different metal ions. However, these marine microbes are not sufficiently explored for their capability of metal nanoparticle synthesis. Although, marine water is one of the richest sources of gold in the nature, however, there is no significant publication regarding utilization of marine micro-organisms to produce gold nanoparticles. Therefore, there might be a possibility of exploring marine bacteria as nanofactories for AuNP biosynthesis.

Results

In the present study, marine bacteria are exploited towards their capability of gold nanoparticles (AuNPs) production. Stable, monodisperse AuNP formation with around 10?nm dimension occur upon exposure of HAuCl₄ solution to whole cells of a novel strain of Marinobacter pelagius, as characterized by polyphasic taxonomy. Nanoparticles synthesized are characterized by Transmission electron microscopy, Dynamic light scattering and UV-visible spectroscopy.

Conclusion

The potential of marine organisms in biosynthesis of AuNPs are still relatively unexplored. Although, there are few reports of gold nanoparticles production using marine sponges and sea weeds however, there is no report on the production of gold nanoparticles using marine bacteria. The present work highlighted the possibility of using the marine bacterial strain of Marinobacter pelagius to achieve a fast rate of nanoparticles synthesis which may be of high interest for future process development of AuNPs. This is the first report of AuNP synthesis by marine bacteria.