Identification and functional characterization of four novel aldo/keto reductases in <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC 7120 by integrating wet lab with in silico approaches

Aldo/keto reductases (AKRs) constitute a multitasking protein family that catalyzes diverse metabolic transformations including detoxification of stress generated reactive aldehydes. Yet this important protein family is poorly understood particularly in cyanobacteria, the ecologically most diverse and significant group of micro-organisms. Present study is an attempt to characterize all putative AKRs of Anabaena sp. PCC 7120. In silico analysis, it revealed the presence of at least four putative AKRs in Anabaena PCC7120 genome. All four proteins share less than 40% sequence identity with each other and also with the identified members of AKR superfamily and hence deserve to be assigned in new families. Dissimilarity in sequences is also reflected through their substrate specificity. While reduction of trans-2-nonenal, a LPO-derived reactive aldehyde was common across the four proteins, these proteins were found to be activated during heat, salt, Cd, As, and butachlor treatments, and their ectopic expression in Escherichia coli conferred tolerance to the above abiotic stresses. These findings affirm the role of AKRs in providing a broad tolerance to environmental stresses conceivably by detoxifying the stress-generated reactive aldehydes.     

Genetic diversity and population structure detection in sponge gourd ( Luffa cylindrica ) using ISSR,SCoT and morphological markers

Investigation of genetic diversity is essential for the selection of parents for crop breeding and conservation of genetic resources. To estimate the genetic variability and population structure in the midst of 45 accessions of sponge gourd brought together from different geographical areas of India, morphological traits and two molecular markers, ISSR and SCoT markers were compared. Principal components analysis of 20 morphological traits showed 72.70% variability and significant positive correlations between fruit traits. All three marker techniques clustered all accessions into two groups with few outgroups. High level of polymorphism was observed among ISSR (74.6%) and SCoT (71.5%) primers. The Bayesian model revealed the hidden grouping and showed admixture type of population. The diversity pattern is influenced by genetic marker used, as different molecular markers have different polymorphism evaluation efficiency. This study can be helpful in amplifying the genetic base and selection of specific traits for breeding. Thus, ISSR and SCoT markers are potential marker for identification in sponge gourd and provide valuable data on its genetic correlation and structure.     

Detecting amyloid-beta aggregation with fiber-based fluorescence correlation spectroscopy

Soluble aggregates critically influence the chemical and biological aspects of amyloid protein aggregation, but their population is difficult to measure, especially in vivo. We take an optical fiber-based fluorescence correlation spectroscopy (FCS) approach to characterize a solution of aggregating amyloid-beta molecules. We find that this technique can easily resolve aggregate particles of size 100 nm or greater in vitro, and the size distribution of these particles agrees well with that obtained by conventional FCS techniques. We propose fiber FCS as a tool for studying aggregation in vivo.     

Identification of the Chemotypes of Ocimum forskolei and Ocimum basilicum by NMR Spectroscopy

The chemotypes of Ocimum forskolei Benth and Ocimum basilicum L. growing wild in Oman have been established by ¹³C‐NMR analyses of the vegetative and floral oils of the plants. The chemotypes, estragole for O. forskolei and linalool for O. basilicum, suggested by ¹³C‐NMR fingerprinting were also confirmed by GC‐FID and GC/MS analyses. The oil of O. forskolei demonstrated better activities against bacteria and dermatophytes. The significance of the presence of estragole and linalool in the volatile oils of plants whose fragrances are traditionally inhaled, added to food, or rubbed on the skin are discussed.     

Evaluation of guar gum derivatives as gelling agents for microbial culture media

Guar gum, a galactomannan, has been reported to be an inexpensive substitute of agar for microbial culture media. However, its use is restricted probably because of (1) its highly viscous nature even at high temperatures, making dispensing of the media to Petri plates difficult and (2) lesser clarity of the guar gum gelled media than agar media due to impurities present in guar gum. To overcome these problems, three guar gum derivatives, carboxymethyl guar, carboxymethyl hydroxypropyl guar and hydroxypropyl guar, were tested as gelling agents for microbial growth and differentiation. These were also evaluated for their suitability for other routine microbiological methods, such as, enumeration, use of selective and differential media, and antibiotic sensitivity test. For evaluation purpose, growth and differentiation of eight fungi and eight bacteria grown on the media gelled with agar (1.5%), guar gum (4%) or one of the guar gum derivatives (4%), were compared. All fungi and bacteria exhibited normal growth and differentiation on all these media. Generally, growth of most of the fungi was better on guar gum derivatives gelled medium than on agar medium. The enumeration carried out for Serratia sp. and Pseudomonas aeruginosa by serial dilution and pour plate method yielded similar counts in all the treatments. Likewise, the selective succinate medium, specific for P. aeruginosa, did not allow growth of co-inoculated Bacillus sp. even if gelled with guar gum derivatives. The differential medium, Congo red mannitol agar could not differentiate between Agrobacterium tumefaciens and Rhizobium meliloti on color basis, if gelled with guar gum or any of its derivatives However, for antibiotic sensitivity tests for both Gram-positive and -negative bacteria, guar gum and its derivatives were as effective as agar.     

Quantitative imaging of lymph function

Functional lymphatic imaging was demonstrated in the abdomen and anterior hindlimb of anesthetized, intact Yorkshire swine by using near-infrared (NIR) fluorescence imaging following intradermal administration of 100-200 microl of 32 microM indocyanine green (ICG) and 64 microM hyaluronan NIR imaging conjugate to target the lymph vascular endothelial receptor (LYVE)-1 on the lymph endothelium. NIR fluorescence imaging employed illumination of 780 nm excitation light ( approximately 2 mW/cm(2)) and collection of 830 nm fluorescence generated from the imaging agents. Our results show the ability to image the immediate trafficking of ICG from the plexus, through the vessels and lymphangions, and to the superficial mammary, subiliac, and middle iliac lymph nodes, which were located as deep as 3 cm beneath the tissue surface. "Packets" of ICG-transited lymph vessels of 2-16 cm length propelled at frequencies of 0.5-3.3 pulses/min and velocities of 0.23-0.75 cm/s. Lymph propulsion was independent of respiration rate. In the case of the hyaluronan imaging agent, lymph propulsion was absent as the dye progressed immediately through the plexus and stained the lymph vessels and nodes. Lymph imaging required 5.0 and 11.9 microg of ICG and hyaluronan conjugate, respectively. Our results suggest that microgram quantities of NIR optical imaging agents and their conjugates have a potential to image lymph function in patients suffering from lymph-related disorders.     

PAN1/NALP2/PYPAF2, an inducible inflammatory mediator that regulates NF-kappaB and caspase-1 activation in macrophages

Genes encoding proteins with PYRIN/PAAD/DAPIN domains, a nucleotide binding fold (NACHT), and leucine rich repeats have recently been recognized as important mediators in autoimmune inflammatory disorders. Here we characterize the expression and function of a member of the PYRIN and NACHT domain (PAN) family, PAN1 (also known as NALP2 and PYPAF2). PAN1 protein expression is regulated by lipopolysaccharide (LPS) and interferons (IFNbeta and IFNgamma) in THP-1 macrophage cells. In gene transfection studies PAN1 manifests an inhibitory influence on NF-kappaB activation induced by various pro-inflammatory stimuli, including tumor necrosis factor TNFalpha and interleukin-1beta (IL-1beta). Gene transfer-mediated elevations in PAN1 protein also suppressed activation of IkappaB kinases induced by inflammatory cytokines. Conversely, reducing endogenous levels of PAN1 using small interfering RNA enhanced LPS-induced production of ICAM-1 (intercellular adhesion molecule 1), an NF-kappaB-dependent gene. We also show here that PAN1 binds via its PYRIN domain to ASC, an adapter protein involved in caspase-1 activation. This binding is disrupted by mutation of the alpha1 helix of ASC. In gene transfer experiments PAN1 enhances caspase-1 activation and IL-1beta secretion in collaboration with ASC. Conversely, reducing endogenous levels of PAN1 using small interfering RNA significantly reduced LPS-induced secretion of IL-1beta in monocytes. We propose that PAN1 functions as a modulator of the activation of NF-kappaB and pro-caspase-1 in macrophages.