Genomic regions influencing resistance to the parasitic weed <Emphasis Type="Italic">Striga hermonthica</Emphasis> in two recombinant inbred populations of sorghum

Molecular markers for resistance of sorghum to the hemi-parasitic weed Striga hermonthica were mapped in two recombinant inbred populations (RIP-1, -2) of F_3:5 lines developed from the crosses IS9830 × E36-1 (1) and N13 × E36-1 (2). The resistant parental lines were IS9830 and N13; the former is characterized by a low stimulation of striga seed germination, the latter by mechanical resistance. The genetic maps of RIP-1 and RIP-2 spanned 1,498 cM and 1,599 cM, respectively, with 137 and 157 markers distributed over 11 linkage groups. To evaluate striga resistance, we divided each RIP into set 1 (116 lines tested in 1997) and set 2 (110 lines evaluated in 1998). Field trials were conducted in five environments per year in Mali and Kenya. Heritability estimates for area under the striga number progress curve (ASNPC) in sets 1 and 2 were respectively 0.66 and 0.74 in RIP-1 and 0.81 and 0.82 in RIP-2. Across sites, composite interval mapping detected 11 QTL (quantitative trait loci) and nine QTL in sets 1 and 2 of RIP-1, explaining 77% and 80% of the genetic variance for ASNPC, respectively. The most significant RIP-1 QTL corresponded to the major-gene locus lgs (low stimulation of striga seed germination) in linkage group I. In RIP-2, 11 QTL and nine QTL explained 79% and 82% of the genetic variance for ASNPC in sets 1 and 2, respectively. Five QTL were common to both sets of each RIP, with the resistance alleles deriving from IS9830 or N13. Since their effects were validated across environments, years and independent RIP samples, these QTL are excellent candidates for marker-assisted selection.     

Phorbol esters affect skeletal muscle glucose transport in a fiber type-specific manner

Recent evidence has shown that activation of lipid-sensitive protein kinase C (PKC) isoforms leads to skeletal muscle insulin resistance. However, earlier studies demonstrated that phorbol esters increase glucose transport in skeletal muscle. The purpose of the present study was to try to resolve this discrepancy. Treatment with the phorbol ester 12-deoxyphorbol-13-phenylacetate 20-acetate (dPPA) led to an approximately 3.5-fold increase in glucose transport in isolated fast-twitch epitrochlearis and flexor digitorum brevis muscles. Phorbol ester treatment was additive to a maximally effective concentration of insulin in fast-twitch skeletal muscles. Treatment with dPPA did not affect insulin signaling in the epitrochlearis. In contrast, phorbol esters had no effect on basal glucose transport and inhibited maximally insulin-stimulated glucose transport approximately 50% in isolated slow-twitch soleus muscle. Furthermore, dPPA treatment inhibited the insulin-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the threonine and serine phosphorylation of PKB by approximately 50% in the soleus. dPPA treatment also caused serine phosphorylation of IRS-1 in the slow-twitch soleus muscle. In conclusion, our results show that phorbol esters stimulate glucose transport in fast-twitch skeletal muscles and inhibit insulin signaling in slow-twitch soleus muscle of rats. These findings suggest that mechanisms other than PKC activation mediate lipotoxicity-induced whole body insulin resistance.     

Enzymes of adenosine metabolism in the brain: diurnal rhythm and the effect of sleep deprivation

Adenosine plays a role in promoting sleep, an effect that is thought to be mediated in the basal forebrain. Adenosine levels vary in this region with prolonged wakefulness in a unique way. The basis for this is unknown. We examined, in rats, the activity of the major metabolic enzymes for adenosine - adenosine deaminase, adenosine kinase, ecto- and cytosolic 5'-nucleotidase - in sleep/wake regulatory regions as well as cerebral cortex, and how the activity varies across the day and with sleep deprivation. There were robust spatial differences for the activity of adenosine deaminase, adenosine kinase, and cytosolic and ecto-5'-nucleotidase. However, the basal forebrain was not different from other sleep/wake regulatory regions apart from the tuberomammillary nucleus. All adenosine metabolic enzymes exhibited diurnal variations in their activity, albeit not in all brain regions. Activity of adenosine deaminase increased during the active period in the ventrolateral pre-optic area but decreased significantly in the basal forebrain. Enzymatic activity of adenosine kinase and cytosolic-5'-nucleotidase was higher during the active period in all brain regions tested. However, the activity of ecto-5'-nucleotidase was augmented during the active period only in the cerebral cortex. This diurnal variation may play a role in the regulation of adenosine in relationship to sleep and wakefulness across the day. In contrast, we found no changes specifically with sleep deprivation in the activity of any enzyme in any brain region. Thus, changes in adenosine with sleep deprivation are not a consequence of alterations in adenosine enzyme activity.     

Ethanol Alters Glutamate but Not Adenosine Uptake in Rat Astrocytes: Evidence for Protein Kinase C Involvement

Glutamate is the primary excitatory neurotransmitter in brain. By stimulating neuronal activity, glutamate increases cellular energy utilization, enhances ATP hydrolysis and promotes the formation of adenosine. Adenosine has receptor-mediated effects that reduce or oppose the excitatory effects of glutamate. As a possible mechanism for ethanol's ability to inhibit excitatory effects of glutamate and enhance inhibitory effects of adenosine, we tested the hypothesis that ethanol promotes [³H]glutamate uptake and inhibits [³H]adenosine uptake. Using primary cultures of rat astrocytes, we found that acute treatment with ethanol (50 mM, 30 min) inhibited [³H]glutamate uptake and reduced protein kinase C (PKC)-induced stimulation of [³H]glutamate uptake. Prolonged treatment (50 mM, 3 day) with ethanol, however, increased both [³H]glutamate uptake and PKC activity. Contrary to other cell types, neither acute or chronic ethanol exposure affected [³H]adenosine uptake in astrocytes. These data indicate that in rat cortical astrocytes ethanol affects [³H]glutamate uptake but not [³H]adenosine uptake by affecting PKC modulation of transporter activity.     

Rhizobial acyl carrier proteins and their roles in the formation of bacterial cell-surface components that are required for the development of nitrogen-fixing root nodules on legume hosts

Acyl carrier protein (ACP) of Escherichia coli is a small acidic protein which functions as carrier of growing acyl chains during their biosynthesis and as donor of acyl chains during transfer to target molecules. This unique ACP of E. coli is expressed constitutively. In more complex bacteria, multiple ACPs are present, indicating a channeling of pools of multi-carbon units into different biosynthetic routes. In rhizobia, for example, besides the constitutive ACP (AcpP) involved in the biosynthesis and transfer of common fatty acids, three specialized ACPs have been reported: (1) the flavonoid-inducible nodulation protein NodF, (2) AcpXL that transfers 27-hydroxyoctacosanoic acid to a sugar backbone during lipid A biosynthesis, and (3) the RkpF protein which is required for the biosynthesis of rhizobial capsular polysaccharides. All three of those specialized rhizobial ACPs are required for the biosynthesis of cell-surface molecules that play a role in establishing the symbiotic relationship between rhizobia and their legume hosts. Surprisingly, the recently sequenced genomes from Mesorhizobium loti and Sinorhizobium meliloti suggest even more candidates for ACPs in rhizobia.     

The X-ray crystallographic structure of the angiogenesis inhibitor angiostatin

Angiogenesis inhibitors have gained much public attention recently as anti-cancer agents and several are currently in clinical trials, including angiostatin (Phase I, Thomas Jefferson University Hospital, Philadelphia, PA). We report here the bowl-shaped structure of angiostatin kringles 1-3, the first multi-kringle structure to be determined. All three kringle lysine-binding sites contain a bound bicine molecule of crystallization while the former of kringle 2 and kringle 3 are cofacial. Moreover, the separation of the kringle 2 and kringle 3 lysiner binding sites is sufficient to accommodate the alpha-helix of the 30 residue peptide VEK-30 found in the kringle 2/VEK-30 complex. Together the three kringles produce a central cavity suggestive of a unique domain where they may function in concert.