Substrate dependent production of extracellular biosurfactant by a marine bacterium

The potential of a marine microorganism to utilize different carbon substrates for the production of an extracellular biosurfactant was evaluated. Among the several carbon substrates tested for this purpose, production of the crude biosurfactant was found to be highest with glycerol (2.9+/-0.11 g L(-1)) followed by starch (2.5+/-0.11 g L(-1)), glucose (1.16+/-0.11 g L(-1)) and sucrose (0.94+/-0.07 g L(-1)). The crude biosurfactant obtained from glycerol, starch and sucrose media had significantly higher antimicrobial action than those obtained from glucose containing medium. RP-HPLC resolved the crude biosurfactants into several fractions one of which had significant antimicrobial action. The antimicrobial fraction was found in higher concentrations in biosurfactant obtained using glycerol, starch and sucrose as compared to the biosurfactants from glucose medium, thereby explaining higher antimicrobial activity. The carbon substrate was thus found to affect biosurfactant production both in a qualitative and quantitative manner.     

Tumor suppressor protein Pdcd4 inhibits translation of p53 mRNA

The tumor suppressor protein Pdcd4 is thought to suppress translation of mRNAs containing structured 5'-UTRs by interacting with translation initiation factor eIF4A and inhibiting its helicase activity. However, natural target mRNAs regulated by Pdcd4 so far are mostly unknown. Here, we identified p53 mRNA as a translational target of Pdcd4. We found that Pdcd4 is associated with p53 mRNA and suppresses its translation. The inhibitory effect of Pdcd4 on the translation of p53 mRNA depends on the ability of Pdcd4 to interact with eIF4A and is mediated by the 5'-UTR of p53 mRNA, which is able to form a stable stem-loop structure. We show that treatment of cells with DNA-damaging agents decreases the expression of Pdcd4. This suggests that translational suppression by Pdcd4 plays a role in maintaining a low level of p53 in unstressed cells and that this suppression is abrogated due to low levels of Pdcd4 after DNA damage. Overall, our work demonstrates for the first time that Pdcd4 is directly involved in translational suppression of a natural mRNA with a 5'-structured UTR and provides novel insight into the translational control of p53 expression.     

ARF6: a newly appreciated player in G protein-coupled receptor desensitization

The luteinizing hormone/choriogonadotropin hormone receptor (LH/CG R) signals to regulate ovulation, corpus luteum formation, and fetal survival during pregnancy. Agonist binding to the LH/CG R is poorly reversible, emphasizing the importance of a cellular mechanism to temper signaling by a potentially persistently active receptor. Like other G protein-coupled receptors (GPCRs), signaling by this receptor is modulated by its binding of an arrestin. We have identified ADP ribosylation factor 6 (ARF6) as a protein whose activation state is regulated by the LH/CG R and which functions to regulate the availability of plasma membrane-docked arrestin 2 to this receptor. We hypothesize that ARF6 might also serve GPCRs other than the LH/CG R to regulate the availability of arrestin 2 for receptor desensitization.     

Influence of Long Term Irrigation with Pulp and Paper Mill Effluent on the Bacterial Community Structure and Catabolic Function in Soil

Microbial communities play a vital role in maintaining soil health. A multiphasic approach to assess the effect of pulp and paper mill effluent on both the structure and function of microbial soil communities is taken. Bacterial communities from agricultural soils irrigated with pulp and paper mill effluent were compared to communities form soils irrigated with well water. Samples were taken from fields in the state of Uttarakhand, India, where pulp and paper mill effluent has been used for irrigation for over 25 years. Comparisons of bacterial community structure were conducted using sequencing of the 16S rRNA gene from both isolates and clone libraries attained from the soil. Community-level physiological profiling was used to characterize the functional diversity and catabolic profile of the bacterial communities. The multiphasic approach using both physiological and molecular techniques proved to be a powerful tool in evaluating the soil bacterial community population and population differences therein. A significant and consistent difference in the population structure and function was found for the bacterial communities from soil irrigated with effluent in comparison to fields irrigated with well water. The diversity index parameters indicated that the microbial community in pulp and paper mill effluent irrigated fields were more diverse in both structure and function. This suggests that the pulp and paper mill effluent is not having a negative effect on the soil microbial community, but in fact may have a positive influence. In terms of soil health, this finding supports the continued use of pulp and paper mill effluent for irrigation. This is however only one aspect of soil health which was evaluated. Further studies on soil resistance and robustness could be undertaken to holistically evaluate soil health in this situation.     

Additions to Gliocephalotrichum species (anamorphic Hypocreales) from fruit litter of the medicinal plant Terminalia chebula in the Western Ghats, India

Two species of Gliocephalotrichum were isolated from fallen fruits of the medicinal plant Terminalia chebula collected from the forests of Western Ghats, India. On the basis of morphological characters and ITS1–5.8S–ITS2 sequence similarity, the fungi have been identified as Gliocephalotrichum longibrachium and G. bulbilium. For the former species, this is the first report of its occurrence from India, whereas the latter, showing significant morphological variability, has been known previously from India.     

Race Profiling and Molecular Diversity Analysis of Fusarium oxysporum f.sp. ciceris Causing Wilt in Chickpea

Seventy isolates of Fusarium oxysporum f.sp. ciceris (Foc) causing chickpea wilt representing 13 states and four crop cultivation zones of India were analysed for their virulence and genetic diversity. The isolates of the pathogen showed high variability in causing wilt incidence on a new set of differential cultivars of chickpea, namely C104, JG74, CPS1, BG212, WR315, KWR108, GPF2, DCP92‐3, Chaffa and JG62. New differential cultivars for each race were identified, and based on differential responses, the isolates were characterized into eight races of the pathogen. The same set of isolates was used for molecular characterization with four different molecular markers, namely random amplified polymorphic DNA, universal rice primers, simple sequence repeats and intersimple sequence repeats. All the four sets of markers gave 100% polymorphism. Unweighted paired group method with arithmetic average analysis grouped the isolates into eight categories at genetic similarities ranging from 37 to 40%. The molecular groups partially corresponded to the states of origin/chickpea‐growing region of the isolates as well as races of the pathogen characterized in this study. The majority of southern, northern and central Indian populations representing specific races of the pathogen were grouped separately into distinct clusters along with some other isolates, indicating the existence of variability in population predominated by a single race of the pathogen. The present race profiling for the Indian population of the pathogen and its distribution pattern is entirely new. The knowledge generated in this study could be utilized in resistance breeding programme. The existence of more than one race, predominated by a single one, in a chickpea cultivation zone as supported by the present molecular findings is also a new information.     

Constrained peptides mimic a viral suppressor of RNA silencing

The design of high-affinity, RNA-binding ligands has proven very challenging. This is due to the unique structural properties of RNA, often characterized by polar surfaces and high flexibility. In addition, the frequent lack of well-defined binding pockets complicates the development of small molecule binders. This has triggered the search for alternative scaffolds of intermediate size. Among these, peptide-derived molecules represent appealing entities as they can mimic structural features also present in RNA-binding proteins. However, the application of peptidic RNA-targeting ligands is hampered by a lack of design principles and their inherently low bio-stability. Here, the structure-based design of constrained α-helical peptides derived from the viral suppressor of RNA silencing, TAV2b, is described. We observe that the introduction of two inter-side chain crosslinks provides peptides with increased α-helicity and protease stability. One of these modified peptides (B3) shows high affinity for double-stranded RNA structures including a palindromic siRNA as well as microRNA-21 and its precursor pre-miR-21. Notably, B3 binding to pre-miR-21 inhibits Dicer processing in a biochemical assay. As a further characteristic this peptide also exhibits cellular entry. Our findings show that constrained peptides can efficiently mimic RNA-binding proteins rendering them potentially useful for the design of bioactive RNA-targeting ligands.     

A natural osmolyte trimethylamine N-oxide promotes assembly and bundling of the bacterial cell division protein, FtsZ and counteracts the denaturing effects of urea

Assembly of FtsZ was completely inhibited by low concentrations of urea and its unfolding occurred in two steps in the presence of urea, with the formation of an intermediate [Santra MK & Panda D (2003) J Biol Chem278, 21336-21343]. In this study, using the fluorescence of 1-anilininonaphthalene-8-sulfonic acid and far-UV circular dichroism spectroscopy, we found that a natural osmolyte, trimethylamine N-oxide (TMAO), counteracted the denaturing effects of urea and guanidium chloride on FtsZ. TMAO also protected assembly and bundling of FtsZ protofilaments from the denaturing effects of urea and guanidium chloride. Furthermore, the standard free energy changes for unfolding of FtsZ were estimated to be 22.5 and 28.4 kJ.mol(-1) in the absence and presence of 0.6 M TMAO, respectively. The data are consistent with the view that osmolytes counteract denaturant-induced unfolding of proteins by destabilizing the unfolded states. Interestingly, TMAO was also found to affect the assembly properties of native FtsZ. TMAO increased the light-scattering signal of the FtsZ assembly, increased sedimentable polymer mass, enhanced bundling of FtsZ protofilaments and reduced the GTPase activity of FtsZ. Similar to TMAO, monosodium glutamate, a physiological osmolyte in bacteria, which induces assembly and bundling of FtsZ filaments in vitro[Beuria TK, Krishnakumar SS, Sahar S, Singh N, Gupta K, Meshram M & Panda D (2003) J Biol Chem278, 3735-3741], was also found to counteract the deleterious effects of urea on FtsZ. The results together suggested that physiological osmolytes may regulate assembly and bundling of FtsZ in bacteria and that they may protect the functionality of FtsZ under environmental stress conditions.     

Discovery of novel, highly potent and selective beta-hairpin mimetic CXCR4 inhibitors with excellent anti-HIV activity and pharmacokinetic profiles

Novel highly potent CXCR4 inhibitors with good pharmacokinetic properties were designed and optimized starting from the naturally occurring β-hairpin peptide polyphemusin II. The design involved incorporating important residues from polyphemusin II into a macrocyclic template-bound β-hairpin mimetic. Using a parallel synthesis approach, the potency and ADME properties of the mimetics were optimized in iterative cycles, resulting in the CXCR4 inhibitors POL2438 and POL3026. The inhibitory potencies of these compounds were confirmed in a series of HIV-1 invasion assays in vitro. POL3026 showed excellent plasma stability, high selectivity for CXCR4, favorable pharmacokinetic properties in the dog, and thus has the potential to become a therapeutic compound for application in the treatment of HIV infections (as an entry inhibitor), cancer (for angiogenesis suppression and inhibition of metastasis), inflammation, and in stem cell transplant therapy.