Genetic association analysis of KCNQ3 and juvenile myoclonic epilepsy in a South Indian population

Juvenile myoclonic epilepsy (JME) is a common subtype of idiopathic generalized epilepsy that shows a complex pattern of inheritance. We have tested the association between JME phenotype and an intragenic marker in KCNQ3 by using the transmission disequilibrium test in 119 probands and their parents. Mutations in KCNQ3 are known to cause benign familial neonatal convulsions and are involved in the physiologically important M current in neurons. Our results provide suggestive evidence of allelic association between JME and KCNQ3 (P-value=0.008) and raise an interesting possibility of a genetic contribution to JME, viz., of a gene that causes a monogenic form of human epilepsy.     

Do the different parental 'heteromes' cause genomic shock in newly formed allopolyploids?

Allopolyploidy, the joining of two parental genomes in a polyploid organism with diploid meiosis, is an important mechanism of reticulate evolution. While many successful long-established allopolyploids are known, those formed recently undergo an instability phase whose basis is now being characterized. We describe observations made with the Arabidopsis system that include phenotypic instability, gene silencing and activation, and methylation changes. We present a model based on the epigenetic destabilization of genomic repeats, which in the parents are heterochromatinized and suppressed. We hypothesize that loss of epigenetic suppression of these sequences, here defined as the heterome, results in genomic instability including silencing of single-copy genes.     

QSAR studies in substituted 1,2,3,4,6,7,12,12a-octa-hydropyrazino[2',1':6,1]pyrido[3,4-b]indoles--a potent class of neuroleptics

A series of nineteen substituted 1,2,3,4,6,7,12,12a-octahydropyrazino[2',1':6,1]pyrido[3, 4-b]indoles analogues of neuroleptic drug, Centbutindole have been studied using quantitative structure-activity relationship analysis. The derived models display good fits to the experimental data (r>or=0.75) having good predictive power (r(cv)>or=0.688). The best model describes a high correlation between predicted and experimental activity data (r=0.967). Statistical analysis of the equation populations indicates that hydrophobicity (as measured by pi(R), logP(o/w) and SlogP_VSA8), dipole y and structural parameters in terms of indicator variable, (In(1)) and globularity are important variables in describing the variation in the neuroleptic activity in the series.     

HER-2 DNA and protein vaccines containing potent Th cell epitopes induce distinct protective and therapeutic antitumor responses in HER-2 transgenic mice

Overexpression of the growth factor receptor HER-2 (c-erbB-2, neu) has transforming potential and occurs in approximately 20-30% of breast and ovarian cancers. HER-2 is a self Ag, but Abs and T cells specific for HER-2 have been isolated from cancer patients, suggesting HER-2 may be a good target for active immunotherapy. We constructed rat HER-2 DNA and protein vaccines containing potent Th cell epitopes derived from tetanus toxin and studied their potency in two strains of mice transgenic for the rat HER-2 molecule. Vaccination with HER-2 DNA protected nontransgenic mice from tumor challenge, but induced only moderate protection in one of the tumor models. However, vaccination with the modified HER-2 protein resulted in almost complete protection from tumor challenge in both tumor models. This protection could be mediated by Abs alone. In addition, protein vaccination efficiently eliminated pre-established tumors in both models, even when vaccination occurred 9 days after tumor implantation. These data demonstrate the potential of HER-2-based vaccines as therapeutic agents for the treatment of cancers overexpressing HER-2.     

Intra- and interspecific interactions between Lysiphlebus testaceipes and Lipolexis scutellaris (Hymenoptera: Aphidiidae) reared on Toxoptera citricida (Homoptera: Aphididae)

Classical biological control of the brown citrus aphid Toxoptera citricida Kirkaldy in Florida has involved the release of Lipolexis scutellaris Mackauer. Lysiphlebus testaceipes Cresson is already present in Florida and also parasitizes T. citricida. Because parasitoid-parasitoid interactions may affect the establishment of a newly introduced parasitoid species, intra- and interspecific larval interactions of both parasitoids were studied in the laboratory using T. citricida reared on citrus. Five time intervals were allowed between successive oviposition opportunities. Early developmental times were determined for both parasitoids: eggs of L. testaceipes and L. scutellaris hatched after 54.3 and 61.7 h, while molt to second instar occurred after 73.3 and 87.1 h, respectively, after oviposition at 22 degrees C. At intervals <12 h, both parasitoids had a greater tendency to multiparasitize than to superparasitize, and the tendency to superparasitize or multiparasitize decreased with an increase in time between successive oviposition opportunities. Of the 10 interspecific interactions studied, 5 produced a winning wasp species, 3 of which could be explained by the hypothesis of physical conflict. A combination of development time, age of competing larvae, and oviposition sequence were responsible for the outcomes observed. Neither parasitoid proved to be intrinsically superior when interspecific competition occurs in second- and third-instar T. citricida, indicating there is no reason to suggest that they cannot coexist in Florida citrus groves.     

A role for apolipoprotein(a) in protection of the low-density lipoprotein component of lipoprotein(a) from copper-mediated oxidation

Low-density lipoprotein (LDL) oxidation is stimulated by copper. Addition of a recombinant form of apolipoprotein(a) (apo(a); the distinguishing protein component of lipoprotein(a)) containing 17 plasminogen kringle IV-like domains (17K r-apo(a)) protects LDL against oxidation by copper. Protection is specific to apo(a) and is not achieved by plasminogen or serum albumin. When Cu(2+) is added to 17K r-apo(a), its intrinsic fluorescence is quenched in a concentration-dependent and saturable manner. Quenching is unchanged whether performed aerobically or anaerobically and is reversible by ethylenediaminetetraacetate, suggesting that it is due to equilibrium binding of Cu(2+) and not to oxidative destruction of tryptophan residues. The fluorescence change exhibits a sigmoid dependence on copper concentration, and time courses of quenching are complex. At copper concentrations below 10 microM there is little quenching, whereas above 10 microM quenching proceeds immediately as a double-exponential decay. The affinity and kinetics of copper binding to 17K r-apo(a) are diminished in the presence of the lysine analogue epsilon -aminocaproic acid. We propose that copper binding to the kringle domains of 17K is mediated by a His-X-His sequence that is located about 5A from the closest tryptophan residue of the lysine binding pocket. Copper binding may account for the natural resistance to copper-mediated oxidation of lipoprotein(a) relative to LDL that has been previously reported and for the protection afforded by apo(a) from copper-mediated oxidation of LDL that we describe in the present study.     

Unveiling steady-state multiplicity in hybridoma cultures: the cybernetic approach

Mammalian cells grown in suspension produce waste metabolites such as lactate, alanine, and ammonia, which reduce the yield of cell mass and the desired product on the nutrients supplied. Previous studies (Cruz et al., 1999; Europa et al., 2000; Follstad et al., 1999) have shown that the cells can be made to alter their metabolism by starving them on their nutrients in continuous cultures at low dilution rates or starting the culture as a fed-batch. This leads to multiple steady states in continuous reactors, with some states being more favorable than others. Mathematical models that take into account the metabolic regulation that leads to these multiple steady states are invaluable tools for bioreactor control. In this article we present a cybernetic modeling strategy in which Metabolic Flux Analysis (MFA) is used to guide the cybernetic formulation. The hybridoma model presented as a result of this strategy considers the partially substitutable, partially complementary nature of glucose and glutamine. The choice of competitions within the network is guided by MFA and the model is successful in explaining the three multiple steady states observed. The cybernetic model though identified for the hybridoma experiments of Hu and others (Europa et al., 2000) seem generally applicable to mammalian systems as it captures the pathways that are common to mammalian cells grown in suspension. The model presented here could be used for start-up strategies for continuous reactors and model-based feedback control for maintaining high productivity of the reactor.     

Synthesis and localization of the Salmonella SPI-1 type III secretion needle complex proteins PrgI and PrgJ

An essential component of type III secretion systems (TTSS) is a supramolecular structure termed the needle complex. In Salmonella enterica, at least four proteins make up this structure: InvG, PrgH, PrgK, and PrgI. Another protein, PrgJ, is thought to play a role in the assembly of this structure, but its function is poorly understood. We have analyzed the expression and localization of PrgJ and the needle protein PrgI in different S. enterica serovar Typhimurium mutant strains. We found that the levels of PrgI and PrgJ were significantly reduced in a TTSS-deficient invA mutant strain and that the decreased levels were due to protein instability. In addition, we found that PrgJ, although associated with the needle complex in wild-type S. enterica serovar Typhimurium, was absent from needle complexes obtained from an invJ mutant strain, which exhibits very long needle substructures. We suggest that PrgJ is involved in capping the needle substructure of the needle complex.     

The calcium sensor protein visinin-like protein-1 modulates the surface expression and agonist sensitivity of the alpha 4beta 2 nicotinic acetylcholine receptor

The calcium sensor protein visinin-like protein-1 (VILIP-1) was isolated from a brain cDNA yeast two-hybrid library using the large cytoplasmic domain of the alpha4 subunit as a bait. VILIP-1 is a myristoylated calcium sensor protein that contains three functional calcium binding EF-hand motifs. The alpha4 subunit residues 302-339 were found to be essential for the interaction with VILIP-1. VILIP-1 coimmunopurified with detergent-solubilized recombinant alpha4beta2 acetylcholine receptors (AChRs) expressed in tsA201 cells and with native alpha4 AChRs isolated from brain. Coexpression of VILIP-1 with recombinant alpha4beta2 AChRs up-regulated their surface expression levels approximately 2-fold and increased their agonist sensitivity to acetylcholine approximately 3-fold. The modulation of the recombinant alpha4beta2 AChRs by VILIP-1 was attenuated in VILIP-1 mutants that lacked the ability to be myristoylated or to bind calcium. Collectively, these results suggest that VILIP-1 represents a novel modulator of alpha4beta2 AChRs that increases their surface expression levels and agonist sensitivity in response to changes in the intracellular levels of calcium.     

Improved performance of preordered fungal protease-stearic acid biocomposites: enhanced catalytic activity,reusability, and temporal stability

In an earlier report on fungal protease (F-prot)-fatty acid biocomposite film formation [Gole et al. Anal. Chem. 2000, 72, 4301], it was observed that the biocatalytic activity of the immobilized enzyme was comparable to that of the free enzyme in solution. However, a somewhat negative aspect of the protocol was the steady loss in activity during reuse and storage of the biocomposite film. In this paper, we address the latter issues and demonstrate successful attempts toward the realization of efficient biocomposite films with enhanced biological activity, temporal stability, and excellent reusability. The improved performance of the F-prot-stearic acid biocomposite is accomplished by preordering the fatty acid film by incorporation of Pb(2+) ions into the lipid matrix prior to enzyme immobilization. The lead cation induces lamellar ordering in the lipid film and thus facilitates diffusion of the F-prot molecules into the lipid matrix and accessibility of the substrate molecules (hemoglobin, Hb) to the entrapped F-prot enzyme molecules. The preordering consequently leads to effective control of the "mass transport" problem and might be responsible for the enhanced biological activity ( approximately 36%) of the enzyme molecules in the biocomposite in comparison with the free enzyme in solution, as well the excellent reusability of the composite film. In addition to biocatalytic activity measurements, the formation and characterization of the F-prot-lead stearate biocomposite films was done by quartz crystal microgravimetry and X-ray diffraction.