A note on the use of cross-linked starch in microbiology with special reference to detecting amylase production

Anilogel-E, a cross-linked starch, can be used with distinct advantages where native starch or soluble starch are conventionally used, e.g. in scoring for amylolytic organisms, as an ingredient of fermentation media, and in enhancing protoplast regeneration. It is particularly useful for the direct visualization of amylase producing micro-organisms on solid media, making prior replication of colonies unnecessary.     

Tunicamycin-resistant mutants of Bacillus amyloliquefaciens are deficient in amylase, protease and penicillinase synthesis and have altered sensitivity to antibiotics and autolysis

Mutants of Bacillus amyloliquefaciens resistant to at least 10 μg/ml of tunicamycin were isolated and shown to be pleiotropic. The mutants were more resistant to streptomycin, chloramphenicol, kanamycin and neomycin than was the parent strain but less resistant to penicillin G and tetracycline. They were more autolytic, presumably due to an altered cell wall. The mutants produced reduced levels of amylase, penicillinase and both metal and serine protease besides having an enhanced sporulation frequency and being more motile.     

Substituted thiophene-anthranilamides as potent inhibitors of human factor Xa

A series of thiophene-containing non-amidine factor Xa inhibitors is described. Simple methyl-substituted thiophene analogs were relatively weak inhibitors. However, introduction of hydrophilic substituents at C-4 or C-5 of the thiophene afforded inhibitors with low nanomolar potency. Optimization of the thiophene substituent at C-4 afforded subnanomolar inhibitors with improved in vitro anticoagulant activity. Incorporating basic amine substituents on the thiophene increased hydrophilicity and improved anticoagulant activity. The pharmacokinetic profile of one inhibitor was evaluated in dogs, and the X-ray crystal structure of this compound bound to factor Xa provides insight into the observed SAR for binding to factor Xa.     

CCR5 receptor antagonists: discovery and SAR of novel 4-hydroxypiperidine derivatives

The guanylhydrazone of 2-(4-chlorobenzyloxy)-5-bromobenzaldehyde, 1, with an IC(50) of 840 nM against the CCR5 receptor was identified using high-throughput screening. Optimization efforts led to the discovery of a novel piperidine series of CCR5 antagonists. In particular, the 4-hydroxypiperidine derivative, 6k, had improved potency against CCR5, and was a starting point for further optimization. SAR elaboration using parallel synthesis led to the identification of 10h, a potent CCR5 antagonist with an IC(50) of 11 nM.     

Structural and functional studies of fatty acyl adenylate ligases from E. coli and L. pneumophila

Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 Å, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.     

Structure of human dual specificity protein phosphatase 23, VHZ, enzyme-substrate/product complex

Protein phosphorylation plays a crucial role in mitogenic signal transduction and regulation of cell growth and differentiation. Dual specificity protein phosphatase 23 (DUSP23) or VHZ mediates dephosphorylation of phospho-tyrosyl (pTyr) and phospho-seryl/threonyl (pSer/pThr) residues in specific proteins. In vitro, it can dephosphorylate p44ERK1 but not p54SAPK-beta and enhance activation of c-Jun N-terminal kinase (JNK) and p38. Human VHZ, the smallest of the catalytically active protein-tyrosine phosphatases (PTP) reported to date (150 residues), is a class I Cys-based PTP and bears the distinctive active site signature motif HCXXGXXRS(T). We present the crystal structure of VHZ determined at 1.93A resolution. The polypeptide chain adopts the typical alphabetaalpha PTP fold, giving rise to a shallow active site cleft that supports dual phosphorylated substrate specificity. Within our crystals, the Thr-135-Tyr-136 from a symmetry-related molecule bind in the active site with a malate ion, where they mimic the phosphorylated TY motif of the MAPK activation loop in an enzyme-substrate/product complex. Analyses of intermolecular interactions between the enzyme and this pseudo substrate/product along with functional analysis of Phe-66, Leu-97, and Phe-99 residues provide insights into the mechanism of substrate binding and catalysis in VHZ.     

Assessment of the efficacy of amino acids and polyamines on regeneration of watermelon (<Emphasis Type="Italic">Citrullus lanatus</Emphasis> Thunb.) and analysis of genetic fidelity of regenerated plants by SCoT and RAPD markers

A simple and efficient regeneration protocol was developed for watermelon from cotyledonary node explants excised from 7-day-old in vitro grown seedlings. This study describes the effect of amino acids and polyamines (PAs) along with plant growth regulators (PGRs) on multiple shoot induction and rooting. The highest number of multiple shoots (46.43 shoots/explant) was obtained from cotyledonary node and they were also elongated (6.3 cm/shoot) on MS medium supplemented with 1 mg l^??1 N ⁶ –Benzyladenine (BA), 5 mg l^??1 leucine, and 10 mg l^??1 spermidine. The elongated shoots developed profuse roots (23.03 roots/shoot) in MS medium containing 1 mg l^??1 indole-3-butyric acid (IBA), 5 mg l^??1 isoleucine, and 10 mg l^??1 putrescine. All the rooted plantlets were successfully hardened and acclimatized in the greenhouse with a survival rate of 98%. The present study described an efficient method to obtain a 1.5-fold increase in the number of shoots, compared with the available regeneration protocols for watermelon. The plants developed in this study showed fivefold higher photosynthetic pigments compared to the control plants. The genetic fidelity of the regenerated plants was evaluated by SCoT and RAPD marker analyses, and banding patterns confirmed the true-to-type nature of in vitro regenerated plants.     

Effectiveness of spinosad on four classes of wheat against five stored-product insects

Spinosad is a commercial reduced-risk pesticide that is naturally derived. Spinosad's performance was evaluated on four classes of wheat (hard red winter, hard red spring, soft red winter, and durum wheats) against adults of the lesser grain borer, Rhyzopertha dominica (F.); rice weevil, Sitophilus oryzae (L.); sawtoothed grain beetle, Oryzaephilus surinamensis (L.); red flour beetle, Tribolium castaneum (Herbst); and larvae of the Indianmeal moth, Plodia interpunctella (Hübner). Beetle adults (25) or P. interpunctella eggs (50) were exposed to untreated wheat and wheat treated with spinosad at 0.1 and 1 mg (AI)/kg of grain. On all untreated wheat classes, adult beetle mortality ranged from 0 to 6%, and P. interpunctella larval mortality ranged from 10 to 19%. The effects of spinosad on R. dominica and P. interpunctella were consistent across all wheat classes. Spinosad killed all exposed R. dominica adults and significantly suppressed progeny production (84-100%) and kernel damage (66-100%) at both rates compared with untreated wheat. Spinosad was extremely effective against P. interpunctella on all wheat classes at 1 mg/kg, based on larval mortality (97.6-99.6%), suppression of egg-to-adult emergence (93-100%), and kernel damage (95-100%), relative to similar effects on untreated wheats. The effects of spinosad on S. oryzae varied among wheat classes and between spinosad rates. Spinosad was effective against S. oryzae, O. surinamensis and T. castaneun only on durum wheat at 1 mg/kg. Our results suggest spinosad to be a potential grain protectant for R. dominica and P. interpunctella management in stored wheat.