Cardioprotection mediated by sphingosine-1-phosphate and ganglioside GM-1 in wild-type and PKC epsilon knockout mouse hearts

Sphingosine-1-phosphate (S1P) protects neonatal rat cardiac myocytes from hypoxic damage through unknown signaling pathways. We tested the hypothesis that S1P-induced cardioprotection requires activation by the epsilon-isoform of protein kinase C (PKC epsilon) by subjecting hearts isolated from PKC epsilon knockout mice and wild-type mice to 20 min of global ischemia and 30 min of reperfusion. Pretreatment with a 2-min infusion of 10 nM S1P improved recovery of left ventricular developed pressure (LVDP) in both wild-type and PKC epsilon knockout hearts and reduced the rise in LV end-diastolic pressure (LVEDP) and creatine kinase (CK) release. Pretreatment for 2 min with 10 nM of the ganglioside GM-1 also improved recovery of LVDP and suppressed CK release in wild-type hearts but not in PKC epsilon knockout hearts. Importantly, GM-1 but not S1P, increased the proportion of PKC epsilon localized to particulate fractions. Our results suggest that GM-1, which enhances endogenous S1P production, reduces cardiac injury through PKC epsilon-dependent intracellular pathways. In contrast, extracellular S1P induces equivalent cardioprotection through PKC epsilon-independent signaling pathways.     

Contiguous genomic DNA sequence comprising the 19-kD zein gene family from maize

A new approach has been undertaken to analyze the sequences and linear organization of the 19-kD zein genes in maize (Zea mays). A high-coverage, large-insert genomic library of the inbred line B73 based on bacterial artificial chromosomes was used to isolate a redundant set of clones containing members of the 19-kD zein gene family, which previously had been estimated to consist of 50 members. The redundant set of clones was used to create bins of overlapping clones that represented five distinct genomic regions. Representative clones containing the entire set of 19-kD zein genes were chosen from each region and sequenced. Seven bacterial artificial chromosome clones yielded 1,160 kb of genomic DNA. Three of them formed a contiguous sequence of 478 kb, the longest contiguous sequenced region of the maize genome. Altogether, these DNA sequences provide the linear organization of 25 19-kD zein genes, one-half the number previously estimated. It is suggested that the difference is because of haplotypes exhibiting different degrees of gene amplification in the zein multigene family. About one-half the genes present in B73 appear to be expressed. Because some active genes have only been duplicated recently, they are so conserved in their sequence that previous cDNA sequence analysis resulted in "unigenes" that were actually derived from different gene copies. This analysis also shows that the 22- and 19-kD zein gene families shared a common ancestor. Although both ancestral genes had the same incremental gene amplification, the 19-kD zein branch exhibited a greater degree of far-distance gene translocations than the 22-kD zein gene family.     

A quantitative assay for assessing allelic proportions by iterative gap ligation.

A variety of techniques are currently available for detecting point mutations in DNA. These techniques are frequently not sensitive enough to be applied as quantitative assays in evaluation of relative occurrence of alleles in cases of polymorphism or when variations in allelic gene expression are being evaluated at the level of RNA. We report here the establishment of an iterative gap ligation (IGL) assay that is both quantitative and sensitive. The design of the assay is such that ligation of an upstream to a downstream primer across a single nucleotide gap will only occur if the gap is filled with a deoxynucleotide complementary to the wild-type or mutant sequence. Under conditions in which excess upstream primer saturates the template concurrently with limiting amounts of downstream primer quantitative ligation is absolutely dependent on provision of the appropriate gap filling nucleotide. When gap ligation occurs in a single incubation, or cycle, the amount of ligated product is a linear function of the relative amount of mutant sequence, with a sensitivity and detection limit of approximately 3% over a range of relative concentrations of 0-100%. When the reaction occurs over multiple cycles, or iterations, gap ligation becomes a non-linear function such that small changes in the relative proportions of alleles produce a disproportionately large amount of ligation. As a consequence, the sensitivity and limits of detection of the assay improve to 0.2% after only 8 cycles. The development of this assay provides a unique means of quantifying allelic polymorphisms in both DNA and RNA (after initial amplification by PCR or RT-PCR) and should be applicable to any experimental settings in which nucleic acids from tissues or mixed populations of cells are being evaluated.     

Interactions Between Augmentatively Released Diachasmimorpha longicaudata (Hymenoptera: Braconidae) and a Complex of Opiine Parasitoids in a Commercial Guava Orchard

A study was conducted to determine the eVect of augmentatively releasing mass-reared Diachasmimorpha longicaudata (Ashmead) on a complex of four co-existing parasitoid species which attack the oriental fruit fly, Bactrocera dorsalis (Hendel). The species belonging to this complex are the egg-pupal parasitoid, Biosteres arisanus (Sonan) and the larval-pupal parasitoids, Biosteres vandenboschi (Fullaway), D. longicaudata and Psyttalia incisi (Silvestri). The study site was a 160-ha commercial orchard of common guava, Psidium guajava L. (cv. Beaumont) located on Kauai island. One year before the release of D. longicaudata, B. arisanus was the dominant parasitoid, accounting for 91.1% of the parasitoids recovered. Despite releases of large numbers of D. longicaudata (600000-800 000 parasitized puparia/ week), B. arisanus continued to account for 90% of all parasitoids recovered from the oriental fruit fly. The larval parasitoid P. incisi may have been reduced as a result of D. longicaudata releases. D. longicaudata was significantly more abundant in over-ripe and rotting fruits compared with freshly fallen fruits. However, no increase in the overall percentage parasitism was observed in any fruit ripeness category. B. arisanus was eY cient at colonizing hosts when fruit fly densities and fruit abundance were relatively low in the orchard. The implications for future augmentative release programmes with D. longicaudata are discussed.     

Primary structure of a genomic zein sequence of maize.

The nucleotide sequence of a genomic clone (termed Z4 ) of the zein multigene family was compared to the nucleotide sequence of related cDNA clones of zein mRNAs. A tandem duplication of a 96-bp sequence is found in the genomic clone that is not present in the related cDNA clones. When the duplication is disregarded, the nucleotide sequence homology between Z4 and its related cDNAs was approximately 97%. The nucleotide sequence is also compared to other isolated cDNAs. No introns in the coding region of the zein gene are detected. The first nucleotide of a putative TATA box, TATAAATA , was located 88 nucleotides upstream of the first nucleotide of the first ATG codon which initiated the open reading frame. The first nucleotide of a putative CCAAT box, CAAAAT , appeared 45 nucleotides upstream of the first nucleotide of the zein cDNA clones in the 3' non-coding region also appeared in the genomic sequence at the same locations. The amino acid composition of the polypeptide specified by the Z4 nucleotide sequence is similar to the known composition of zein proteins.     

Isolation and sequence of a gene encoding a methionine-rich 10-kDa zein protein from maize

We have isolated the gene encoding a methionine-rich 10-kDa zein protein from a lambda EMBL3 maize genomic 'mini' library of the inbred line BSSS-53 and determined its nucleotide sequence. The sequence matches perfectly with a cDNA clone from the inbred line W22 (which has the same restriction fragment length polymorphism as many inbred lines tested) indicating that we have isolated a functional storage protein gene that is very conserved in maize. This comparison also excludes any splicing of any precursor mRNA and therefore any presence of introns. A number of potential regulatory sequences have been located in the flanking regions. The 10-kDa-zein gene represents the last size class in the zein multigene family to be characterized. Its structure allows us now to re-examine the relationship of all the zein proteins and also to compare the structure of a new class of storage proteins that are rich in methionine, an essential amino acid in livestock fodder.     

Chronic ethanol exposure induces an N-type calcium channel splice variant with altered channel kinetics

Chronic ethanol exposure increases the density of N-type calcium channels in brain. We report that ethanol increases levels of mRNA for a splice variant of the N channel specific subunit alpha1 2.2 that lacks exon 31a. Whole cell recordings demonstrated an increase in N-type current with a faster activation rate and a shift in activation to more negative potentials after chronic alcohol exposure, consistent with increased abundance of channels containing this variant. These results identify a novel mechanism whereby chronic ethanol exposure can increase neuronal excitability by altering levels of channel splice variants.     

Protein kinase C isozymes and the regulation of diverse cell responses

Individual protein kinase C (PKC) isozymes have been implicated in many cellular responses important in lung health and disease, including permeability, contraction, migration, hypertrophy, proliferation, apoptosis, and secretion. New ideas on mechanisms that regulate PKC activity, including the identification of a novel PKC kinase, 3-phosphoinositide-dependent kinase-1 (PDK-1), that regulates phosphorylation of PKC, have been advanced. The importance of targeted translocation of PKC and isozyme-specific binding proteins (like receptors for activated C-kinase and caveolins) is well established. Phosphorylation state and localization are now thought to be key determinants of isozyme activity and specificity. New concepts on the role of individual PKC isozymes in proliferation and apoptosis are emerging. Opposing roles for selected isozymes in the same cell system have been defined. Coupling to the Wnt signaling pathway has been described. Phenotypes for PKC knockout mice have recently been reported. More specific approaches for studying PKC isozymes and their role in cell responses have been developed. Strengths and weaknesses of different experimental strategies are reviewed. Future directions for investigation are identified.     

Activation of protein kinase A and atypical protein kinase C by A(2A) adenosine receptors antagonizes apoptosis due to serum deprivation in PC12 cells

We found in the present study that stimulation of A(2A) adenosine receptors (A(2A)-R) prevents apoptosis in PC12 cells. This A(2A)-protective effect was blocked by protein kinase A (PKA) inhibitors and was not observed in a PKA-deficient PC12 variant. Stimulation of PKA also prevented apoptosis, suggesting that PKA is required for the protective effect of A(2A)-R. A general PKC inhibitor, but not down-regulation of conventional and novel PKCs, readily blocked the protective effect of A(2A)-R stimulation and PKA activation, suggesting that atypical PKCs (aPKCs) serve a critical role downstream of PKA. Consistent with this hypothesis, stimulation of A(2A)-R or PKA enhanced nuclear aPKC activity. In addition, the A(2A)-protective effect was blocked by a specific inhibitor of one aPKC, PKCzeta, whereas overexpression of a dominant-positive PKCzeta enhanced survival. In contrast, inhibitors of MAP kinase and phosphatidylinositol 3-kinase did not modulate the A(2A)-protective effect. Dominant-negative Akt also did not alter the A(2A)-protective effect, whereas it significantly reduced the protective action of nerve growth factor. Collectively, these data suggest that aPKCs can function downstream of PKA to mediate the A(2A)-R-promoted survival of PC12 cells. Furthermore, the results indicate that different extracellular stimuli can employ distinct signaling pathways to protect against apoptosis induced by the same insult.     

Genetic analysis of the mammalian K+ channel beta subunit Kvbeta 2 (Kcnab2)

Kvbeta2 binds to K(+) channel alpha subunits from at least two different families (Kv1 and Kv4) and is a member of the aldo-ketoreductase (AKR) superfamily. Proposed functions for this protein in vivo include a chaperone-like role in Kv1 alpha subunit biogenesis and catalytic activity as an AKR oxidoreductase. To investigate the in vivo function of Kvbeta2, Kvbeta2-null and point mutant (Y90F) mice were generated through gene targeting in embryonic stem cells. In Kvbeta2-null mice, Kv1.1 and Kv1.2 localize normally in cerebellar basket cell terminals and the juxtaparanodal region of myelinated nerves. Moreover, normal glycosylation patterns are observed for Kv1.1 and Kv1.2 in whole brain lysates. Thus, loss of the chaperone-like activity does not appear to account for the phenotype of Kvbeta2-null mice, which include reduced life spans, occasional seizures, and cold swim-induced tremors similar to that observed in Kv1.1-null mice. Mice expressing Kvbeta2, mutated at a site (Y90F) that abolishes AKR-like catalytic activity in other family members, have no overt phenotype. We conclude that Kvbeta2 contributes to regulation of excitability in vivo, although not directly through either chaperone-like or typical AKR catalytic activity. Rather, Kvbeta2 relies upon as yet unidentified mechanisms in the regulation of K(+) channel and/or oxidoreductive functions.