Annual variation in floral phenology and pollen production in a 25‐year‐old plantation of Tectona grandis

Teak is a timber tree that is widely distributed in the tropics. Several studies on pollination and reproductive biology have been conducted, but generally information on flowering phenology and annual variation in total pollen production per tree is lacking. The reproductive phenology as well as flower‐ and pollen grain production of individuals in a population is important to theoreticians, field biologists and plant breeders, as they determine the distribution of genotypes within populations and influences the degree of differentiation among populations. This study reports flowering phenology and variation in total flower, fruit‐ and pollen production per tree in teak in a 25‐year‐old plantation across three consecutive years (2006–2008). The results show that the date of onset and end of flowering was highly variable across years. The longest flowering period of 93 days was observed in 2006. There was an asynchrony in the number of open flowers due to differences in time of anthesis among individuals (± 2 days) and inflorescences within individuals (± 6 h). The production of pollen grains per tree in 2007 was 33%, i.e. 16% more compared to 2006 and 2008. The fruit production per tree was 42% and 27% higher in 2007 compared to 2006 and 2008. Concentration of pollen grains (both on jelly‐coated microscopic slides and stigmas) were highest between noon and 2 pm. At this time, the stigmatic pollen load ranged between 4–8 pollen grains per stigma, which is sufficient for fruit development. The study concludes that the asynchronization of the flower opening might give rise to a high amount of self‐pollination in the stand, ultimately leading to poor fruit setting. Also, the large production of flowers and pollen per tree induced geitonogamy and decreased female fitness, as T. grandis is preferentially an out‐crossing species.     

Impact of low and high fluence rates of UV-B radiation on growth and oxidative stress in Phormidium foveolarum and Nostoc muscorum under copper toxicity: differential display of antioxidants system

In the present study, impact of low (UV-B_L) and high (UV-B_H) fluence rates of UV-B on growth, oxidative stress and antioxidant system was studied in two cyanobacteria i.e. Phormidium foveolarum and Nostoc muscorum under Cu (2 and 5???M) toxicity after 24 and 72?h of experiments. UV-B_H and Cu treatment decreased growth of both the cyanobacteria and Cu induced decrease in growth was accompanied by a significant increase in Cu accumulation. Levels of reactive oxygen species (ROS), i.e. superoxide radicals (SOR; $ \text O_{2}^{\cdot\,-} $ ) and hydrogen peroxide (H₂O₂) were significantly increased by Cu and UV-B_H exposure which in turn accelerated lipid peroxidation (malondialdehyde: MDA) and protein oxidation (reactive carbonyl groups: RCG). Activities of enzymatic antioxidants, such as superoxide dismutase (SOD), peroxidase (POD) and glutathione-S-transferase (GST) were increased by both doses of Cu as well as UV-B. Conversely, Cu and UV-B_H drastically decreased catalase (CAT) activity. After the commencement of 24?h of treatment with Cu alone and together with UV-B_H, non-protein thiols (NP-SH) contents were decreased while after 72?h, a reverse trend was noticed. Unlike NP-SH, cysteine content decreased appreciably during the treatments. In contrast to this, low dose (UV-B_L) of UV-B did not influence growth, SOR, H₂O₂, MDA and RCG contents. An improvement in CAT activity and NP-SH content was observed under Cu and UV-B_L treatment; hence, UV-B_L treatment resulted into certain degree of protection against Cu toxicity in both the organisms. Thus, the results showed that UV-B_H and UV-B_L exerted differential effects on both the organisms under Cu toxicity, and compared to N. muscorum, P. foveolarum was less affected by Cu and UV-B_H.     

3-Formylchromone interacts with cysteine 38 in p65 protein and with cysteine 179 in IκBα kinase, leading to down-regulation of nuclear factor-κB (NF-κB)-regulated gene products and sensitization of tumor cells

3-Formylchromone (3-FC) has been associated with anticancer potential through a mechanism yet to be elucidated. Because of the critical role of NF-κB in tumorigenesis, we investigated the effect of this agent on the NF-κB activation pathway. Whether activated by inflammatory agents (such as TNF-α and endotoxin) or tumor promoters (such as phorbol ester and okadaic acid), 3-FC suppressed NF-κB activation. It also inhibited constitutive NF-κB expressed by most tumor cells. This activity correlated with sequential inhibition of IκBα kinase (IKK) activation, IκBα phosphorylation, IκBα degradation, p65 phosphorylation, p65 nuclear translocation, and reporter gene expression. We found that 3-FC inhibited the direct binding of p65 to DNA, and this binding was reversed by a reducing agent, thus suggesting a role for the cysteine residue. Furthermore, mutation of Cys38 to Ser in p65 abolished this effect of the chromone. This result was confirmed by a docking study. 3-FC also inhibited IKK activation directly, and the reducing agent reversed this inhibition. Furthermore, mutation of Cys179 to Ala in IKK abolished the effect of the chromone. Suppression of NF-κB activation led to inhibition of anti-apoptotic (Bcl-2, Bcl-xL, survivin, and cIAP-1), proliferative (cyclin D1 and COX-2), invasive (MMP-9 and ICAM-1), and angiogenic (VEGF) gene products and sensitization of tumor cells to cytokines. Thus, this study shows that modification of cysteine residues in IKK and p65 by 3-FC leads to inhibition of the NF-κB activation pathway, suppression of anti-apoptotic gene products, and potentiation of apoptosis in tumor cells.     

Expression, purification and evaluation of diagnostic potential and immunogenicity of dengue virus type 3 domain III protein

Dengue hemorrhagic fever and dengue shock syndrome are the severe manifestations of dengue infection. The quest for reliable dengue diagnostics and a dengue vaccine remained elusive for decades. Domain III of dengue virus envelope contains multiple conformation dependant neutralizing epitopes, thus making it an attractive diagnostic and vaccine candidate. In this report we show the expression of dengue virus type 3 envelope domain III protein (D3EDIII) and demonstrate its potential as a diagnostic and vaccine candidate. Accordingly, D3EDIII was expressed to high levels in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified protein was used to develop an in-house plate ELISA and was further tested with a panel of 40 dengue infected serum samples previously characterized by commercially available serological tests. The in-house results were in excellent agreement with the commercial kits. D3EDIII was refolded by rapid dilution method and the refolded monomer protein was purified by Ion exchange chromatography. Further, the recombinant protein was biologically functional and found to inhibit dengue virus type 3 plaque formation on LLC-MK2 cells demonstrating its function of receptor interaction. Furthermore, D3EDIII in combination with Freund's complete adjuvant induced high antibody titers in BALB/c mice and these antibodies efficiently neutralized dengue 3 virus. Additionally, D3EDIII induced expression of Th1 cytokines that can inhibit the intracellular viral infections. Thus, our results demonstrate that D3EDIII protein has tremendous potential both in diagnosis of dengue infections and in vaccine development.     

Non-enzymatic antioxidative defence in drought-stressed mulberry (<Emphasis Type="Italic">Morus indica</Emphasis> L.) genotypes

The present study investigated drought-induced responses of non-enzymatic antioxidants in four diverse mulberry genotypes (Morus indica L. S-36, M-5, MR-2 and V-1). Inside the glasshouse, potted plants were subjected to four water regimes for 75 days: (a) control: pots maintained at 100% pot water holding capacity (PC) (b) low water stress: 75% PC (c) medium water stress: 50% PC and (d) high water stress: 25% PC. Photosynthetic leaf gas exchange and non-enzymatic antioxidants including α-tocopherol, ascorbic acid (AA), glutathione, proline and total carotenoids were measured in leaves at regular intervals. Amongst all, V-1 was relatively drought tolerant and showed exceeded accumulation of α-tocopherol and AA-glutathione pool in association with higher carotenoids and proline contents. Susceptible S-36, M-5 and MR-2 could not induce any significant up-regulation in AA-glutathione pool leading to endogenous loss of α-tocopherol and more lipid peroxidation. Reactive oxygen species (ROS) like hydrogen peroxide (H₂O₂) and superoxide (O₂ ^{· ?}) showed apparent accumulation in water-stressed leaves and significantly contributed to lipid peroxidation in susceptible genotypes when compared to V-1. Our study demonstrated that proline, AA and glutathione were the major non-enzymatic antioxidants in mulberry with α-tocopherol and carotenoids as good additional indicators for drought stress tolerance. These non-enzymatic antioxidants can cumulatively render effective protection against oxidative damage and can be considered as reliable markers for screening drought-tolerant mulberry genotypes.     

The saponin-rich fraction of a Gymnema sylvestre R. Br. aqueous leaf extract reduces cafeteria and high-fat diet-induced obesity

We examined the antiobesity effect of a saponin-rich fraction of a Gymnema sylvestre R. Br. aqueous leaf extract (SGE) using cafeteria and high-fat diet-induced obese rats for a period of eight weeks. SGE was orally administered at a dose of 100 mg/kg body weight once a day to the treatment group. It significantly decreased the body weight, food consumption, visceral organs weight, and the levels of triglycerides, total cholesterol, low-density lipoproteins, very low-density lipoproteins, atherogenic index, glucose, and increased the levels of high-density lipoproteins. There was no significant difference with respect to all parameters of the study in case of normal (N) diet and N diet + SGE rats. In vitro, SGE inhibited the pancreatic lipase activity. The present study gave clear evidence that the SGE has a significant antiobese action, supporting its use in traditional medicine, and can be used as a substitute for synthetic drugs.     

Three redundant murein endopeptidases catalyse an essential cleavage step in peptidoglycan synthesis of Escherichia coli K12

Bacterial peptidoglycan (PG or murein) is a single, large, covalently cross‐linked macromolecule and forms a mesh‐like sacculus that completely encases the cytoplasmic membrane. Hence, growth of a bacterial cell is intimately coupled to expansion of murein sacculus and requires cleavage of pre‐existing cross‐links for incorporation of new murein material. Although, conceptualized nearly five decades ago, the mechanism of such essential murein cleavage activity has not been studied so far. Here, we identify three new murein hydrolytic enzymes in Escherichia coli, two (Spr and YdhO) belonging to the NlpC/P60 peptidase superfamily and the third (YebA) to the lysostaphin family of proteins that cleave peptide cross‐bridges between glycan chains. We show that these hydrolases are redundantly essential for bacterial growth and viability as a conditional mutant lacking all the three enzymes is unable to incorporate new murein and undergoes rapid lysis upon shift to restrictive conditions. Our results indicate the step of cross‐link cleavage as essential for enlargement of the murein sacculus, rendering it a novel target for development of antibacterial therapeutic agents.     

Kinetic and mechanistic differences in the interactions between caldendrin and calmodulin with AKAP79 suggest different roles in synaptic function

The kinetic and mechanistic details of the interaction between caldendrin, calmodulin and the B‐domain of AKAP79 were determined using a biosensor‐based approach. Caldendrin was found to compete with calmodulin for binding at AKAP79, indicating overlapping binding sites. Although the AKAP79 affinities were similar for caldendrin (K_D = 20 n m ) and calmodulin (K_D = 30 n m ), their interaction characteristics were different. The calmodulin interaction was well described by a reversible one‐step model, but was only detected in the presence of Ca²⁺. Caldendrin interacted with a higher level of complexity, deduced to be an induced fit mechanism with a slow relaxation back to the initial encounter complex. It interacted with AKAP79 also in the absence of Ca²⁺, but with different kinetic rate constants. The data are consistent with a similar initial Ca²⁺‐dependent binding step for the two proteins. For caldendrin, a second Ca²⁺‐independent rearrangement step follows, resulting in a stable complex. The study shows the importance of establishing the mechanism and kinetics of protein–protein interactions and that minor differences in the interaction of two homologous proteins can have major implications in their functional characteristics. These results are important for the further elucidation of the roles of caldendrin and calmodulin in synaptic function. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of chemical and microbial vitamin B<Subscript>12</Subscript> analogues on production of vitamin B<Subscript>12</Subscript>

Strain improvement by genetic manipulation or optimization of fermentation conditions for overproduction of vitamin B₁₂ has a drawback due to feed back inhibition. To resist the feed back inhibition by analogues of vitamin B₁₂ in Propionibacterium freudenrechii subsps. shermanii (OLP-5), we have tested with microbially separated B₁₂ analogues from three different strains. Microbial analogues were differentiated from commercially available vitamin B₁₂ by high pressure liquid chromatography and spectrophotometric method. An analogue isolated from NRRL-B-4327 was shown to increase vitamin B₁₂ concentration from 18.53 ± 0.15 to 31.67 ± 0.58 mg/l in OLP-5 strain. The presence of chemical analogue (ICH₂ Co(DH)₂ (H₂Py)₄) increased vitamin B₁₂ production from 16.13 ± 0.15 to 18.53 ± 0.15 mg/l in OLP-5. These findings revealed that addition of B₁₂ analogues in fermentation media have developed strain resistance to feed back inhibition by vitamin B₁₂.     

Cu-mediated N-arylation of 1,2,3-triazin-4-ones: synthesis of fused triazinone derivatives as potential inhibitors of chorismate mutase

A rapid and direct access to N-aryl substituted fused triazinone derivatives has been accomplished via N-arylation of 1,2,3-triazin-4-one ring involving a Cu-mediated coupling between triazinone derivatives and aryl boronic acids. A combination of Cu(OAc)(2)-Et(3)N in 1,2-dichloroethane was found to be effective and various fused triazinone derivatives have been prepared by using this methodology. Molecular structure of a representative compound was confirmed by single crystal X-ray diffraction study. The scope and limitations of this reaction is discussed. Some of the compounds synthesized were tested for chorismate mutase inhibitory properties in vitro. The in vitro dose response study of an active compound is presented.