Antiproliferative and antioxidant properties of nematocysts crude venom from jellyfish Acromitus flagellatus against human cancer cell lines

This study aimed to investigate the antiproliferative and antioxidant properties of crude venom from the nematocyst of Jellyfish Acromitus flagellates on human lung cancer (A549) and liver cancer (HepG2) cell lines. The prepared crude venom was subjected to analyses of the biochemical constituents, protein profiles, antioxidant and anticancer activities by standard methods. The extracted venom was pale-yellow in color and viscous/sticky. The biochemical composition such as, protein (1.547 mg/ml), lipid (0.039 mg/ml) and carbohydrate (0.028 mg/ml) was estimated. Protein profiles were determined by SDS PAGE, the result revealed that the molecular weight range from 205 ? 3.5 kDa. The free radical scavenging activity was analyzed by the reducing potential (56.36%), DPPH (72.47%), hydroxyl (68.50%), superoxide anion (65.75%), and nitric oxide (33.04%). The cell viability was observed by using different concentrations (20 to 100 µg/ml) of crude venom on A549 and HepG2 cancer cell lines and the IC₅₀ values were recorded in (60 μg/ml and 40 μg/ml) respectively, while it had none cytotoxic effects on Vero cell line up to the concentration of 90 μg/ml. These results suggest that crude venom from nematocyst of A. flagellatus possesses anti-cancer activity and able to develop novel drugs on marine-derived compounds.     

Seasonal changes in oleosomic lipids and fatty acids of perennial root nodules of beach pea

Seasonal changes in the fatty acid composition of phospholipids (PL), monoglycerides (MG), diglycerides (DG), free fatty acids (FA) and triglycerides (TG) separated from oleosomes (lipid bodies) of perennial root nodules of beach pea (Lathyrus maritimus) were analysed. Thin layer chromatography (TLC) revealed that PL and MG are the major lipids in nodule oleosomes. The fatty acid profile and overall double bond index (DBI) varied among lipid classes depending upon the season. High DBI in PL and MG found during late winter and early spring indicated that they may play a major role in winter survival and regeneration of perennial nodules. The DBI of DG was high at the end of the fall season and the DBI of FA and TG was high in summer months. The dominant fatty acids are C16:0 followed by C18:0 and C18:1. The levels of many unsaturated fatty acids such as C18:1, C18:2 and C18:3 increased while saturated fatty acid C18:0 decreased during winter. These unsaturated fatty acids possibly play an important role in the protection of nodule cells from cold stress. Nodules seem to retain some fatty acids and selectively utilize specific fatty acids to survive the winter and regenerate in spring.     

Synthesis and characterization of tritylthioethanamine derivatives with potent KSP inhibitory activity

Assembly of a bipolar mitotic spindle requires the action of class 5 kinesins, and inhibition or depletion of this motor results in mitotic arrest and apoptosis. S-Trityl-l-cysteine is an allosteric inhibitor of vertebrate Kinesin Spindle Protein (KSP) that has generated considerable interest due to its anti-cancer properties, however, poor pharmacological properties have limited the use of this compound. We have modified the triphenylmethyl and cysteine groups, guided by biochemical and cell-based assays, to yield new cysteinol and cysteamine derivatives with increased inhibitory activity, greater efficacy in model systems, and significantly enhanced potency against the NCI60 tumor panel. These results reveal a promising new class of conformationally-flexible small molecules as allosteric KSP inhibitors for use as research tools, with activities that provide impetus for further development as anti-tumor agents.     

Normal rat hepatic stellate cells respond to endotoxin in LBP-independent manner to produce inhibitor(s) of DNA synthesis in hepatocytes

Endotoxin is implicated in the pathology of acute liver failure. The mechanisms of its actions on quiescent hepatic stellate cells (qHSCs) and their implications in hepatocyte injury are incompletely understood. We investigated effects of endotoxin (bacterial lipopolysaccharide; LPS) on qHSCs and subsequently on hepatocytes. After overnight culture following their isolation, qHSCs were incubated with or without endotoxin for 24 h. The cells and the culture supernatant were analyzed for cytokines and nitric oxide (NO) synthesis. The effects of qHSC-conditioned media on hepatocytes were then determined. LPS increased inducible NO synthase expression, stimulated NO synthesis, and inhibited DNA synthesis in qHSCs. qHSC-conditioned medium inhibited DNA synthesis in hepatocytes without affecting NO synthesis, while LPS (1-1,000 ng/ml)-conditioned qHSC medium stimulated NO synthesis and caused further inhibition of DNA synthesis and apoptosis. These effects of LPS were more pronounced when qHSCs were incubated with serum, but not with LPS-binding protein (LBP) although CD14 (a receptor for LPS-LBP complex) was found in qHSCs. LPS stimulated the synthesis of TNF-alpha, interleukin (IL)-6, and IL-1beta but not of TGF-beta in qHSCs. Individually or together, L-N(G)-monomethylarginine and antibodies to IL-1beta, IL-6, and TNF-alpha only partly reversed qHSC + LPS-conditioned medium-induced inhibition of DNA synthesis in hepatocytes. These results suggest that the effects of LPS on qHSCs are novel, occurring without the aid of LBP/CD14. They also indicate that other factors, in addition to NO, TGF-beta, TNF-alpha, IL-1beta, and IL-6 are involved in the mechanisms of the growth inhibitory effects of qHSCs on hepatocytes.     

Anatomical and Biochemical Changes Associated with In Vitro Rhizogenesis in Dendrocalamus giganteus

Successful micropropagation protocol of a difficult-to-root bamboo species, Dendrocalamus giganteus (10–15 years old) along with the analysis of anatomical and biochemical changes during in vitro rhizogenesis was accomplished. Proliferated axillary shoots from nodal segments of 10–15 years old field culms exhibited shoot necrosis during multiple shoot formation phase and was controlled by subculturing in modified MS liquid medium having 825 mg ^l?1 NH₄NO₃, 3800 mg ^l?1 KNO₃, 740 mg ^l?1 MgSO₄ and 9% coconut water, 26.64 μM 6-benzylaminopurine (BA) and 0.46 μM kinetin. These multiple shoots proliferated from field grown culms, failed to root and hence callus was induced on MS solid medium containing 4.44 μM BA, 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.37 μM naphthalene acetic acid (NAA). Organogenesis from the callus was achieved upon transfer to MS medium with 11.10 μM BA and 2.32 pM kinetin. The callus-derived shoots multiplied on modified MS medium were rooted the best (91%) by culturing 3 days on MS medium having glucose (0.5%), sucrose (2.5%) and 98.41 μM indolebutyric acid (IBA) and subsequently to IBA-free MS medium containing 3% sucrose. Studies on peroxidase and IAA oxidase activity and endogenous free- and bound-IAA content showed that IAA oxidase and peroxidase oxidize endogenous IAA resulting in root initials formation. Anatomical studies confirmed the root primordia formation from 3^rd day of IBA treatment and primordia were visible over the surface on 8^th to 10^th day. However, the shoot necrosis symptoms which started on 6^th day of treatment intensified by 10^th day leading to the death of the whole shoot system by 12^th–15^th day. Nevertheless, on the root formation medium with 9.84 μM IBA, new shoot buds were emerged and showed shoot growth in 60% of the rooted cultures, which were successfully acclimatized in shade-house with 100% survival. The present study establishes rooting of callus-derived shoots as the best way for the successful propagation of the difficult-to-root bamboo, D. giganteus when compared to axillary bud proliferated shoots.     

Computational kinetic studies of pyruvate metabolism in Carboxydothermus hydrogenoformans Z-2901 for improved hydrogen production

Hydrogen is considered as a renewable energy source and it is also regarded as future fuel. Currently, hydrogen production through a biotechnological approach is a research priority. Hydrogenogens, a microbial species, are of significant interest to researchers because of their ability to produce biological hydrogen. Carboxydothermus hydrogenoformans Z-2901 is one among the hydrogenogens that can grow anaerobically by utilizing pyruvate as a carbon source, and can produce molecular hydrogen. In the present study, we performed an in silico kinetic simulation using the available Kyoto Encyclopedia of Genes and Genomes (KEGG) model and reconstructed pyruvate metabolism in C. hydrogenoformans Z-2901. During this metabolism, dissimilation of pyruvate leads to the formation of energy co-factors, such as ATP and NAD⁺/ NADH, and the level of these co-factors influences the specific growth rate of organism and hydrogen production. Our strategy for improving hydrogen production involves maximizing the ATP and NAD⁺ yield by modification of kinetic properties and adding new reactions in pyruvate metabolism through metabolic pathway reconstruction. Moreover, the influence of phosphoenol pyruvate carboxylase and pyruvate dehydrogenase enzyme concentration on cofactor productions was also simulated. The theoretical molar yield of ATP and NAD⁺ were obtained as 2.32 and 1.83 mM, respectively, from 1 mM/mg of phosphoenol pyruvate (PEP) utilization. A higher yield of ATP is achieved when the PEP level reaches 5 mM/mg. This work also suggests that PEP can be considered as an alternative substrate. In conclusion, the simulation results reported in this paper can be applied to design and evaluate strategies of strain construction for optimal hydrogen yield in C. hydrogenoformans.     

Prevalence of Diabetes and Pre-Diabetes and Associated Risk Factors among Tuberculosis Patients in India

Background

Diabetes mellitus (DM) is recognised as an important risk factor to tuberculosis (TB). India has high TB burden, along with rising DM prevalence. There are inadequate data on prevalence of DM and pre-diabetes among TB cases in India. Aim was to determine diabetes prevalence among a cohort of TB cases registered under Revised National Tuberculosis Control Program in selected TB units in Tamil Nadu, India, and assess pattern of diabetes management amongst known cases.

Methods

827 among the eligible patients (n = 904) underwent HbA1c and anthropometric measurements. OGTT was done for patients without previous history of DM and diagnosis was based on WHO criteria. Details of current treatment regimen of TB and DM and DM complications, if any, were recorded. A pretested questionnaire was used to collect information on sociodemographics, habitual risk factors, and type of TB.

Findings

DM prevalence was 25.3% (95% CI 22.6–28.5) and that of pre-diabetes 24.5% (95% CI 20.4–27.6). Risk factors associated with DM among TB patients were age (31–35, 36–40, 41–45, 46–50, >50 years vs <30 years) [OR (95% CI) 6.75 (2.36–19.3); 10.46 (3.95–27.7); 18.63 (6.58–52.7); 11.05 (4.31–28.4); 24.7 (9.73–62.7) (p<0.001)], positive family history of DM [3.08 (1.73–5.5) (p<0.001)], sedentary occupation [1.69 (1.10–2.59) (p = 0.016)], and BMI (18.5–22.9, 23–24.9 and ≥25 kg/m² vs <18.5 kg/m²) [2.03 (1.32–3.12) (p = 0.001); 0.87 (0.31–2.43) (p = 0.78); 1.44 (0.54–3.8) (p = 0.47)]; for pre-diabetes, risk factors were age (36–40, 41–45, 46–50, >50 years vs <30 years) [2.24 (1.1–4.55) (p = 0.026); 6.96 (3.3–14.7); 3.44 (1.83–6.48); 4.3 (2.25–8.2) (p<0.001)], waist circumference [<90 vs. ≥90 cm (men), <80 vs. ≥80 cm (women)] [3.05 (1.35–6.9) (p = 0.007)], smoking [1.92 (1.12–3.28) (p = 0.017)] and monthly income (5000–10,000 INR vs <5000 INR) [0.59 (0.37–0.94) (p = 0.026)]. DM risk was higher among pulmonary TB [3.06 (1.69–5.52) (p<0.001)], especially sputum positive, than non-pulmonary TB.

Interpretation

Nearly 50% of TB patients had either diabetes or pre-diabetes.     

Probing the role of aromatic residues at the secondary saccharide-binding sites of human salivary alpha-amylase in substrate hydrolysis and bacterial binding

Human salivary α-amylase (HSAmy) has three distinct functions relevant to oral health: (1) hydrolysis of starch, (2) binding to hydroxyapatite (HA), and (3) binding to bacteria (e.g., viridans streptococci). Although the active site of HSAmy for starch hydrolysis is well-characterized, the regions responsible for bacterial binding are yet to be defined. Since HSAmy possesses several secondary saccharide-binding sites in which aromatic residues are prominently located, we hypothesized that one or more of the secondary saccharide-binding sites harboring the aromatic residues may play an important role in bacterial binding. To test this hypothesis, the aromatic residues at five secondary binding sites were mutated to alanine to generate six mutants representing either single (W203A, Y276A, and W284A), double (Y276A/W284A and W316A/W388A), or multiple [W134A/W203A/Y276A/W284A/W316A/W388A; human salivary α-amylase aromatic residue multiple mutant (HSAmy-ar)] mutations. The crystal structure of HSAmy-ar as an acarbose complex was determined at a resolution of 1.5 Å and compared with the existing wild-type acarbose complex. The wild-type and the mutant enzymes were characterized for their abilities to exhibit enzyme activity, starch-binding activity, HA-binding activity, and bacterial binding activity. Our results clearly showed that (1) mutation of aromatic residues does not alter the overall conformation of the molecule; (2) single or double mutants showed either moderate or minimal changes in both starch-binding activity and bacterial binding activity, whereas HSAmy-ar showed significant reduction in these activities; (3) starch-hydrolytic activity was reduced by 10-fold in HSAmy-ar; (4) oligosaccharide-hydrolytic activity was reduced in all mutants, but the action pattern was similar to that of the wild-type enzyme; and (5) HA binding was unaffected in HSAmy-ar. These results clearly show that the aromatic residues at the secondary saccharide-binding sites in HSAmy play a critical role in bacterial binding and in starch-hydrolytic functions of HSAmy.     

Synthesis of [beta-(4-pyridyl-1-oxide)-L-alanine4]-angiotensin I as a potential suicide substrate for protein-tyrosine kinases

An asymmetric synthesis of [beta-(4-pyridyl-1-oxide)-L-alanine4]-angiotensin I (1a), which is a potential suicide substrate (mechanism-based inhibitor) for protein-tyrosine kinases, has been performed. Deprotonation of 6 with n-butyllithium in THF gave the anion 7, which was alkylated with 4-(chloromethyl)pyridine-1-oxide to afford intermediate 9 as a crystalline solid. Hydrolysis of 9 afforded a mixture of 11 and 12 in a ratio of 96:4 as estimated by conversion to the diastereomeric dipeptides 13 and 14 followed by HPLC analysis. The 96:4 mixture of 11 and 12 was used in the solid phase synthesis of the target angiotensin analog 1a and its diastereomer 1b, which were separated and tested for inhibitory activity against two thymocyte protein-tyrosine kinases: p40 and p56lck. Neither peptide displayed significant inhibitory activity toward p40 and both served as weak competitive inhibitors of p56lck.