A truncated RHAMM protein for discovering novel therapeutic peptides

The receptor for hyaluronan mediated motility (RHAMM, gene name HMMR) belongs to a group of proteins that bind to hyaluronan (HA), a high-molecular weight anionic polysaccharide that has pro-angiogenic and inflammatory properties when fragmented. We propose to use a chemically synthesized, truncated version of the protein (706–767), 7?kDa RHAMM, as a target receptor in the screening of novel peptide-based therapeutic agents. Chemical synthesis by Fmoc-based solid-phase peptide synthesis, and optimization using pseudoprolines, results in RHAMM protein of higher purity and yield than synthesis by recombinant protein production. 7?kDa RHAMM was evaluated for its secondary structure, ability to bind the native ligand, HA, and its bioactivity. This 62-amino acid polypeptide replicates the HA binding properties of both native and recombinant RHAMM protein. Furthermore, tubulin-derived HA peptide analogues that bind to recombinant RHAMM and were previously reported to compete with HA for interactions with RHAMM, bind with a similar affinity and specificity to the 7?kDa RHAMM. Therefore, in terms of its key binding properties, the 7?kDa RHAMM mini-protein is a suitable replacement for the full-length recombinant protein.     

Screening of plant extracts for organic management of downy mildew of sorghum

Antipathogenic potential of 38 plants was evaluated in the form of aqueous extracts against Peronoclerospora sorghi, causing downy mildew of sorghum. Conidial suspension and plant extracts were mixed individually and allowed to stand for 5 min and then used to inoculate the host by sprout-dip method. The sprouts thus inoculated were grown in pots, and the disease incidence was observed. Eight plant extracts (Cicer areatinum, Datura metel, Croton sparsiflorus, Parthenium hysterophorus, Nerium oleander, Chromolaena odorata, Duranta repens and Oxalis latifolia) at 20% concentration performed at par with chemical fungicide (Mancozeb 75%) by exhibiting total suppression of disease incidence to 0%, when compared with 64.1% of negative control. Organic management of air-borne inoculum of downy mildew of sorghum is feasible and preferable when compared with chemical control methods, considering human and environmental health concerns. The use of water extract keeps the technology simple so that it can be directly prepared and used by the farmers. Short-listing of eight most effective water extracts would help in self-reliance of farmers, reducing their dependence on commercial products.     

Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Avena shoots

While two monoclonal antibodies directed to phytochrome from etiolated oat (Avena sativa L.) shoots can precipitate up to about 30% of the photoreversible phytochrome isolated from green oat shoots, most precipitate little or none at all. These results are consistent with a report by J.G. Tokuhisa and P.H. Quail (1983, Plant Physiol. 72, Suppl., 85), according to which polyclonal rabbit antibodies directed to phytochrome from etiolated oat shoots bind only a small fraction of the phytochrome obtained from green oat shoots. The immunoprecipitation data reported here indicate that essentially all phytochrome isolated from green oat shoots is distinct from that obtained from etiolated oat shoots. The data indicate further that phytochrome from green oat shoots might itself be composed of two or more immunochemically distinct populations, each of which is distinct from phytochrome from etiolated shoots. Phytochrome isolated from light-grown, but norflurazon-bleached oat shoots is like that isolated from green oat shoots. When light-grown, green oat seedlings are kept in darkness for 48 h, however, much, if not all, of the phytochrome that reaccumulates is like that from etiolated oat shoots. Neither modification during purification from green oat shoots of phytochrome like that from etiolated oat shoots, nor non-specific interference by substances in extracts of green oat shoots, can explain the inability of antibodies to recognize phytochrome isolated from green oat shoots. Immunopurified polyclonal rabbit antibodies to phytochrome from etiolated pea (Pisum sativum L.). shoots precipitate more than 95% of the photoreversible phytochrome obtained from etiolated pea shoots, while no more than 75% of the pigment is precipitated when phytochrome is isolated from green pea shoots. These data indicate in preliminary fashion that an immunochemically unique pool of phytochrome might also be present in extracts of green pea shoots.Abbreviation ELISA enzyme-linked immunosorbent assay - mU milliunit - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome     

On-line monitoring of enzymes in downstream processing by flow injection analysis (FIA)

Flow injection analysis (FIA) has been employed to automate enzyme assays for formate dehydrogenase (FDH) and l-leucine dehydrogenase (l-LeuDH). Coupled to a special sampling device the FIA assays were used to monitor on-line downstream processes, e.g. disintegration of microbial cells and cross-flow filtration of cell homogenates.     

Discovery of 3-(4-sulfamoylnaphthyl)pyrazolo[1,5-a]pyrimidines as potent and selective ALK2 inhibitors

The pyrazolo[1,5-a]pyrimidine LDN-193189 is a potent inhibitor of activin receptor-like kinase 2 (ALK2) but is nonselective for highly homologous ALK3 and shows only modest kinome selectivity. Herein, we describe the discovery of a novel series of potent and selective ALK2 inhibitors by replacing the quinolinyl with a 4-(sulfamoyl)naphthyl, yielding ALK2 inhibitors that exhibit not only excellent discrimination versus ALK3 but also high kinome selectivity. In addition, the optimized compound 23 demonstrates good ADME and in vivo pharmacokinetic properties.     

The importance of leaf surface wax as short-range attractant and oviposition stimulant in a generalist Lepidoptera

Green gram, Vigna radiata (L.) Wilczek, is an important pulse crop of Asia. Severe attack by the larvae of Spilosoma obliqua Walker (Lepidoptera: Arctiidae) causes defoliation of green gram and reduces seed yield. Females lay eggs on the leaf surface, and therefore, surface wax plays an important role as short-range attractant and oviposition stimulant. So, we have attempted to find out whether leaf surface wax compounds (alkanes and free fatty acids) from three green gram cultivars (PDM 54, PUSA BAISAKHI and SAMRAT) could act as short-range attractant and oviposition stimulant in females. The TLC, GC-MS and GC-FID analyses of n-hexane extracts revealed 20 n-alkanes from n-C₁₅ to n-C₃₆ and 13 free fatty acids from C12:0 to C21:0, whilst linoleic acid was unique in SAMRAT. Pentacosane was the predominant amongst n-alkanes in the leaf surface waxes of three cultivars. Heneicosanoic acid and palmitoleic acid were the predominant free fatty acids in the leaf surface waxes of PDM 54, and PUSA BAISAKHI and SAMRAT, respectively. Females were attracted towards one leaf equivalent surface wax of three green gram cultivars against solvent controls (n-hexane) in Y-tube olfactometer bioassays. A synthetic blend of pentacosane, heptacosane, nonacosane, hexatriacontane, palmitoleic acid, linolenic acid and stearic acid, a synthetic blend of pentacosane, hexatriacontane and stearic acid, and a synthetic blend of hexatriacontane, linolenic acid and stearic acid resembling in amounts present in one leaf equivalent surface wax of PDM 54, PUSA BAISAKHI and SAMRAT, respectively, served as short-range attractant and oviposition stimulant in females. Females showed equal preference for egg laying towards the above three synthetic blends when these blends were tested against each other, and hence, these blends could be employed in development of baited traps in pest management strategies.     

A simple and sensitive ribonucleotide reductase assay

Ribonucleotide reductase (RR) is a key regulatory enzyme in the DNA synthesis pathway and is the target of the cancer chemotherapeutic agent hydroxyurea. The study of RR is significantly hindered by the tedious and labor-intensive nature of enzymatic assay. In this report, we present a novel RR assay in which detection of the deoxyribonucleotides produced by RR occurs via coupling to the DNA polymerase reaction, and is enhanced by using RNase to degrade endogenous RNA. Cell extracts from various cell lines were treated with RNase and then reacted with ATP and radioactive ribonucleotide diphosphate as the substrate. Incorporation of the radioactive substrate [¹⁴C]CDP into DNA was linear over 30 min and was linear with the amount of extract, which provided RR activity. The reaction was inhibited by hydroxyurea and required Mg²⁺ and ATP, suggesting that the assay is specific to RR activity. While RR activities determined by our method and by a conventional method were comparable, this novel method proved to be simpler, faster, more sensitive and less expensive. In addition, assay of the RR activity for multiple samples can easily be performed simultaneously. It is superior to other RR assays in all aspects.     

Measurement of bacterial random motility and chemotaxis coefficients: I. Stopped-flow diffusion chamber assay

Bacterial chemotaxis, the directed movement of a cell population in response to a chemical gradient, plays a critical role in the distribution and dynamic interaction of bacterial populations in nonmixed systems. Therefore, in order to make reliable predictions about the migratory behavior of bacteria within the environment, a quantitative characterization of the chemotactic response in terms of intrinsic cell properties is needed.The design of the stopped-flow diffusion chamber (SFDC) provides a well-characterized chemical gradient and reliable method for measuring bacterial migration behavior. During flow through the chamber, a step change in chemical concentration is imposed on a uniform suspension of bacteria. Once flow is stopped, diffusion causes a transient chemical gradient to develop, and bacteria respond by forming a band of high cell density which travels toward higher concentrations of the attractant. Changes in bacterial spatial distributions observed through light scattering are recorded on photomicrographs during a 10-min period. Computer-aided image analysis converts absorbance of the photographic negatives to a digital representation of bacterial density profiles. A mathematical model (part II) is used to quantitatively characterize these observations in terms of intrinsic cell parameters: a chemotactic sensitivity coefficient, mu(0), from the aggregate cell density accumulated in the band and a random motility coefficient, mu, from population dispersion in the absence of a chemical gradient.Using the SFDC assay and an individual-cell-based mathematical model, we successfully determined values for both of these population parameters for Escherichia coli K12 responding to fucose. The values obtained were mu = 1.1 +/- 0. 4 x 10(-5) cm(2)/s and chi(o) = 8 +/- 3 +/- 10(-5) cm(2)/s. We have demonstrated a method capable of determining these parameter values from the now validated mathematical model which will be useful for predicting bacterial migration in application systems.