DNA fingerprinting detects genetic variability in the pearl millet downy mildew pathogen (Sclerospora graminicola)

Genetic variability in six host genotype-specific pathotypes of pearl millet downy mildew pathogen S. graminicola was studied at the molecular level using mini- and micro-satellites. Our results indicated that microsatellites (GAA)₆, (GACA)₄, and especially (GATA)₄ were quite informative and showed high levels of polymorphism among the pathotypes. The six pathotypes could be classified into five groups based on the cluster analysis of their genetic similarities, thereby confirming the existence of distinct host genotype-specific virulence in S. graminicola pathotypes. We demonstrate, for the first time, the use of DNA fingerprinting to detect genetic variation in downy mildew fungus of pearl millet.     

Calcium-activated potassium conductance in presynaptic terminals at the crayfish neuromuscular junction

Membrane potential changes that typically evoke transmitter release were studied by recording intracellularly from the excitor axon near presynaptic terminals of the crayfish opener neuromuscular junction. Depolarization of the presynaptic terminal with intracellular current pulses activated a conductance that caused a decrease in depolarization during the constant current pulse. This conductance was identified as a calcium-activated potassium conductance, gK(Ca), by its disappearance in a zero-calcium/EGTA medium and its block by cadmium, barium, tetraethylammonium ions, and charybdotoxin. In addition to gK(Ca), a delayed rectifier potassium conductance (gK) is present in or near the presynaptic terminal. Both these potassium conductances are involved in the repolarization of the membrane during a presynaptic action potential.     

Subunit interactions in myosin subfragment 1. Subunit scrambling is independent of catalytic center involvement

Evidence is presented that, under conditions of 4.7 M NH4Cl and 10 mM Mg-ATP where no subunit dissociation can be detected by transport methods, a dynamic equilibrium exists in subfragment 1 between the associated and dissociated subunits. This is readily discerned by the formation of hybrid subfragment 1 species when a subfragment 1 isozyme is incubated with excess free light chains of the alternate isozyme. A similar process occurs with p-N,N'-phenylenedimaleimide (pPDM)-modified subfragment 1 containing [14C]Mg-ADP, but in this case, although extensive amounts of hybrid are formed, no loss of the trapped nucleotide is observed. Subunit scrambling without loss of the trapped nucleotide is apparent from incubating pPDM-SF1(A2)-[14C]Mg-ADP with unmodified SF1(A1) under similar conditions since the mixture subsequently contains SF1(A1), SF1(A2)h, pPDM-SF1(A1)h-[14C]Mg-ADP and pPDM-SF1(A2)-[14C]Mg-ADP. These data show that the nucleotide trapped in the presumptive active site does not escape during the dissociation-reassociation cycle, and suggest that the ATPase site resides solely on the heavy chain.     

Studies on the subunit interactions of skeletal muscle myosin subfragment 1. Evidence for subunit exchange between isozymes under physiological ionic strength and temperature

Evidence is presented that under physiological conditions of ionic strength and temperature, where myosin Subfragment 1 is hydrolyzing MgATP, the interaction between its subunits is extremely labile. Incubation of [3H]N-ethylmaleimide-SF1(A1) with N-ethylmaleimide-SF1(A2) in the presence of 10 mM MgATP at 37 degrees C resulted in the exchange of subunits between these isozymes. This is readily discernible from the subunit composition and distribution of the 3H label after separation of the isozymes by ion exchange chromatography. Moreover, incubation of unmodified SF1(A1) or SF1(A2) with the free Alkali light chains A2 and A1, respectively, under the same conditions led to the formation of significant amounts of the hybrid species. These findings suggest that in vivo the Alkali light chain-heavy chain interaction of Subfragment 1 is in a state of dynamic equilibrium between associated and dissociated states.     

Lysine residues of IGF-I are substrates for transglutaminases and modulate downstream IGF-I signalling

Numerous studies have reported associations between IGF-I and other extra cellular matrix (ECM) proteins, including fibronectin (FN), integrins, IGF-binding proteins (IGFBPs) and through IGFBPs, with vitronectin (VN). Nevertheless, the precise nature and mechanisms of these interactions are still being characterised. In this paper, we discuss transglutaminases (TGases) as a constituent of the ECM and provide evidence for the first time that IGF-I is a lysine (K)-donor substrate to TGases. When IGF-I was incubated with an alpha-2 plasmin inhibitor-derived Q peptide in the presence of tissue transglutaminase (TG2), an IGF-I:Q peptide cross-linked species was detected using Western immunoblotting and confirmed by mass spectrometry. Similar findings were observed in the presence of Factor XIIIa (FXIIIa) TGase. To identify the precise location of this K-donor TGase site/s on IGF-I, all the three IGF-I K-sites, individually and collectively (K27, K65 and K68), were substituted to arginine (R) using site-directed mutagenesis. Incubation of these K→R IGF-I analogues with Q peptide in the presence of TG2 or FXIIIa resulted in the absence of cross-linking in IGF-I analogues bearing arginine substitution at site 68. This established that K68 within the IGF-I D-domain was the principal K-donor site to TGases. We further annotated the functional significance of these K→R IGF-I analogues on IGF-I mediated actions. IGF-I analogues with K→R substitution within the D-domain at K65 and K68 hindered migration of MCF-7 breast carcinoma cells and correspondingly reduced PI3-K/AKT activation. Therefore, this study also provides first insights into a possible functional role of the previously uncharacterised IGF-I D-domain.     

In silico discovery of potential drug molecules to improve the treatment of isoniazid-resistant Mycobacterium tuberculosis

The emergence of multidrug-resistant Mycobacterium tuberculosis (M.tb) has become one of the major hurdles in the treatment of tuberculosis (TB). Drug-resistant M.tb has evolved with various strategies to avoid killing by the anti-tubercular drugs. Thus, there is a rising need to develop effective anti-TB drugs to improve the treatment of these strains. Traditional drug design approach has earned little success due to time and the cost involved in the process of development of anti-infective drugs. Numerous reports have demonstrated that several mutations in the drug target sites cause emergence of drug-resistant M.tb strains. In this study, we performed computational mutational analysis of M.tb inhA, fabD, and ahpC genes, which are the primary targets for first-line isoniazid (INH) drug. In silico virtual drug screening was performed to identify the potent drugs from a ChEMBL compound library to improve the treatment of INH-resistant M.tb. Further, these compounds were analyzed for their binding efficiency against active drug binding cavity of M.tb wild-type and mutant InhA, FabD and AhpC proteins. The drug efficacy of predicted lead compounds was verified by molecular docking using M.tb wild-type and mutant InhA, FabD and AhpC protein template models. Different in silico and pharmacophore analysis predicted three potent lead compounds with better drug-like properties against both M.tb wild-type and mutant InhA, FabD, and AhpC proteins as compared to INH drug, and thus may be considered as effective drugs for the treatment of INH-resistant M.tb strains. We hypothesize that this work may accelerate drug discovery process for the treatment of drug-resistant TB.

Communicated by Ramaswamy H. Sarma     

Influence of host plant on growth and reproduction of Aphis nerii and feeding and prey utilization of its predator Menochilus sexmaculatus

Influence of different physiological stages (young, mature and senescent) of Calotropis gigantea leaves on growth and reproductive ability of A. nerii and feeding, prey utilization, fecundity and lipid content of its predator M. sexmaculatus were investigated. Increased reproductive period, total life span and reproduction of nymphs per female of A. nerii were observed when reared on mature leaves. This relative preference of A. nerii and maximum utilization of mature leaves as compared to other physiological aged leaves are mainly due to changes in the chemical composition such as protein, carbohydrate, lipid, amino acid, nitrogen and phenolic of C. gigantea. Further, aphids reared on mature leaves influenced its predator's (M. sexmaculatus) growth, prey utilization and reproductive performances. Fecundity and longevity were high, while developmental time of predator was shorter on mature leaves fed aphid. Maximum prey utilization and increased efficiency of ingested and digested food of predator was observed on mature leaves reared aphid. The results are interpreted and discussed in relation to plant aphid and predator interaction (tritrophic).     

Evidence for Extracellular ATP as a Stress Signal in a Single-Celled Organism

ATP is omnipresent in biology and acts as an extracellular signaling molecule in mammals. Information regarding the signaling function of extracellular ATP in single-celled eukaryotes is lacking. Here, we explore the role of extracellular ATP in cell volume recovery during osmotic swelling in the amoeba Dictyostelium. Release of micromolar ATP could be detected during cell swelling and regulatory cell volume decrease (RVD) phases during hypotonic challenge. Scavenging ATP with apyrase caused profound cell swelling and loss of RVD. Apyrase-induced swelling could be rescued by 100 μM βγ-imidoATP. N-Ethylmalemide (NEM), an inhibitor of vesicular exocytosis, caused heightened cell swelling, loss of RVD, and inhibition of ATP release. Amoebas with impaired contractile vacuole (CV) fusion (drainin knockout [KO] cells) displayed increased swelling but intact ATP release. One hundred micromolar Gd³⁺ caused cell swelling while blocking any recovery by βγ-imidoATP. ATP release was 4-fold higher in the presence of Gd³⁺. Cell swelling was associated with an increase in intracellular nitric oxide (NO), with NO-scavenging agents causing cell swelling. Swelling-induced NO production was inhibited by both apyrase and Gd³⁺, while NO donors rescued apyrase- and Gd³⁺-induced swelling. These data suggest extracellular ATP released during cell swelling is an important signal that elicits RVD. Though the cell surface receptor for ATP in Dictyostelium remains elusive, we suggest ATP operates through a Gd³⁺-sensitive receptor that is coupled with intracellular NO production.     

The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser359 Mediate the Redox-Dependent Increase in Protein Kinase C-δ Activity

The diverse roles of protein kinase C-δ (PKCδ) in cellular growth, survival, and injury have been attributed to stimulus-specific differences in PKCδ signaling responses. PKCδ exerts membrane-delimited actions in cells activated by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Tyr³¹³-phosphorylated enzyme that displays a high level of lipid-independent activity and altered substrate specificity during oxidative stress. This study identifies an interaction between PKCδ''s Tyr³¹³-phosphorylated hinge region and its phosphotyrosine-binding C2 domain that controls PKCδ''s enzymology indirectly by decreasing phosphorylation in the kinase domain ATP-positioning loop at Ser³⁵⁹. We show that wild-type (WT) PKCδ displays a strong preference for substrates with serine as the phosphoacceptor residue at the active site when it harbors phosphomimetic or bulky substitutions at Ser^359. In contrast, PKCδ-S359A displays lipid-independent activity toward substrates with either a serine or threonine as the phosphoacceptor residue. Additional studies in cardiomyocytes show that oxidative stress decreases Ser³⁵⁹ phosphorylation on native PKCδ and that PKCδ-S359A overexpression increases basal levels of phosphorylation on substrates with both phosphoacceptor site serine and threonine residues. Collectively, these studies identify a C2 domain-pTyr³¹³ docking interaction that controls ATP-positioning loop phosphorylation as a novel, dynamically regulated, and physiologically relevant structural determinant of PKCδ catalytic activity.     

Novel diarylheptanoids as inhibitors of TNF-α production

Synthesis and anti-inflammatory activity of novel diarylheptanoids [5-hydroxy-1-phenyl-7-(pyridin-3-yl)-heptan-3-ones and 1-phenyl-7-(pyridin-3-yl)hept-4-en-3-ones] as inhibitors of tumor necrosis factor-α (TNF-α) production is described in the present article. The key reactions involve the formation of a β-hydroxyketone by the reaction of substituted 4-phenyl butan-2-ones with pyridine-3-carboxaldehyde in presence of LDA and the subsequent dehydration of the same to obtain the α,β-unsaturated ketones. Compounds 4i, 5b, 5d, and 5g significantly inhibit lipopolysaccharide (LPS)-induced TNF-α production from human peripheral blood mononuclear cells in a dose-dependent manner. Of note, the in vitro TNF-α inhibition potential of 5b and 5d is comparable to that of curcumin (a naturally occurring diarylheptanoid). Most importantly, oral administration of 4i, 5b, 5d, and 5g (each at 100 mg/kg) but not curcumin (at 100 mg/kg) significantly inhibits LPS-induced TNF-α production in BALB/c mice. Collectively, our findings indicate that these compounds may have potential therapeutic implications for TNF-α-mediated auto-immune/inflammatory disorders.