Allocation and specification of the genital disc precursor cells in Drosophila

The adult structures of Drosophila melanogaster are derived from larval imaginal discs, which originate as clusters of cells within the embryonic ectoderm. The genital imaginal disc is composed of three primordia (female genital, male genital, and anal primordia) that originate from the embryonic tail segments A8, A9, and A10, respectively, and produce the sexually dimorphic genitalia and analia. We show that the genital disc precursor cells (GDPCs) are first detectable during mid-embryogenesis as a 22-cell cluster in the ventral epidermis. Analysis of mutant and double mutant phenotypes of embryonic patterning genes in the GDPCs, together with their expression patterns in these cells, revealed the following with respect to the origins and specification of the GDPCs. The allocation of the GDPCs from the ventral epidermis requires the function of ventral patterning genes, including the EGF receptor and the spitz group of genes. The ventral localization of the GDPCs is further restricted by the action of dorsal patterning genes. Along the anterior-posterior axis, several segment polarity genes (wingless, engrailed, hedgehog, and patched) are required for the proper allocation of the GDPCs. These segment polarity genes are expressed in some, but not all of the GDPCs, indicating that anterior and posterior compartments are not fully established in the GDPCs. In addition, we found that the three primordia of the larval genital disc have already been specified in the GDPCs by the coordinated actions of the homeotic (Hox) genes, abdominal-A, Abdominal-B, and caudal. By identifying how these different patterning networks regulate the allocation and primordial organization of the 22 embryonic precursors of the compound genital disc, we demonstrate that at least some of the organization of the larval disc originates as positional information in the embryo, thus providing a context for further studies on the development of the genital disc.     

Fatty acid utilisation and metabolism in caecal enterocytes of rainbow trout (Oncorhynchus mykiss) fed dietary fish or copepod oil

A combined fatty acid metabolism assay was employed to determine fatty acid uptake and relative utilisation in enterocytes isolated from the pyloric caeca of rainbow trout. In addition, the effect of a diet high in long-chain monoenoic fatty alcohols present as wax esters in oil derived from Calanus finmarchicus, compared to a standard fish oil diet, on caecal enterocyte fatty acid metabolism was investigated. The diets were fed for 8 weeks before caecal enterocytes from each dietary group were isolated and incubated with [1-14C]fatty acids: 16:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:1n-9, 20:4n-6, 20:5n-3, and 22:6n-3. Uptake was measured over 2 h with relative utilisation of different [1-14C]fatty acids calculated as a percentage of uptake. Differences in uptake were observed, with 18:1n-9 and 18:2n-6 showing the highest rates. Esterification into cellular lipids was highest with 16:0 and C18 fatty acids, accounting for over one-third of total uptake, through predominant incorporation in triacylglycerol (TAG). The overall utilisation of fatty acids in phospholipid synthesis was low, but highest with 16:0, the most prevalent fatty acid recovered in intracellular phosphatidylcholine (PC) and phosphatidylinositol (PI), although exported PC exhibited higher proportions of C20/C22 polyunsaturated fatty acids (PUFA). Other than 16:0, incorporation into PC and PI was highest with C20/C22 PUFA and 20:4n-6 respectively. Recovery of labelled 18:1n-9 in exported TAG was 3-fold greater than any other fatty acid which could be due to multiple esterification on the glycerol 'backbone' and/or increased export. Approximately 20-40% of fatty acids taken up were beta-oxidised, and was highest with 20:4n-6. Oxidation of 20:5n-3 and 22:6n-3 was also surprisingly high, although 22:6n-3 oxidation was mainly attributed to retroconversion to 20:5n-3. Metabolic modification of fatty acids by elongation-desaturation was generally low at <10% of [1-14C]fatty acid uptake. Dietary copepod oil had generally little effect on fatty acid metabolism in enterocytes, although it stimulated the elongation and desaturation of 16:0 and elongation of 18:1n-9, with radioactivity recovered in longer n-9 monoenes. The monoenoic fatty acid, 20:1n-9, abundant in copepod oil as the homologous alcohol, was poorly utilised with 80% of uptake remaining unesterified in the enterocyte. However, the fatty acid composition of pyloric caeca was not influenced by dietary copepod oil.     

Dengue fever in humanized NOD/SCID mice

The increased transmission and geographic spread of dengue fever (DF) and its more severe presentation, dengue hemorrhagic fever (DHF), make it the most important mosquito-borne viral disease of humans (50 to 100 million infections/year) (World Health Organization, Fact sheet 117, 2002). There are no vaccines or treatment for DF or DHF because there are no animal or other models of human disease; even higher primates do not show symptoms after infection (W. F. Scherer, P. K. Russell, L. Rosen, J. Casals, and R. W. Dickerman, Am. J. Trop. Med. Hyg. 27:590-599, 1978). We demonstrate that nonobese diabetic/severely compromised immunodeficient (NOD/SCID) mice xenografted with human CD34+ cells develop clinical signs of DF as in humans (fever, rash, and thrombocytopenia), when infected in a manner mimicking mosquito transmission (dose and mode). These results suggest this is a valuable model with which to study pathogenesis and test antidengue products.     

Regulation of plant disease resistance, stress responses, cell death, and ethylene signaling in Arabidopsis by the EDR1 protein kinase

ENHANCED DISEASE RESISTANCE 1 (EDR1) encodes a CTR1-like kinase and was previously reported to function as a negative regulator of disease resistance and ethylene-induced senescence. Here, we report that the edr1 mutant displays enhanced stress responses and spontaneous necrotic lesions under drought conditions in the absence of pathogen, suggesting that EDR1 is also involved in stress response signaling and cell death regulation. Double mutant analysis revealed that these drought-induced phenotypes require salicylic acid but not ethylene signaling pathways. In addition, the edr1-mediated ethylene-induced senescence phenotype was suppressed by mutations in EIN2, but not by mutations in SID2, PAD4, EDS1, or NPR1, suggesting that EDR1 functions at a point of cross talk between ethylene and salicylic acid signaling that impinges on senescence and cell death. Two edr1-associated phenotypes, drought-induced growth inhibition and ethylene-induced senescence, were suppressed by mutations in ORE9, implicating ubiquitin-mediated protein degradation in the regulation of these phenotypes. However, the ore9 mutation did not suppress edr1-mediated enhanced disease resistance to powdery mildew or spontaneous lesions, indicating that these phenotypes are controlled by separate signaling pathways. To investigate the function of the EDR1 kinase domain, we expressed the C-terminal third of EDR1 in wild-type Columbia and edr1 backgrounds under the control of a dexamethasone-inducible promoter. Overexpression of the EDR1 kinase domain in an edr1 background had no obvious effect on edr1-associated phenotypes. However, overexpression of the EDR1 kinase domain in a wild-type Columbia background caused dominant negative phenotypes, including enhanced disease resistance to powdery mildew and enhanced ethylene-induced senescence; thus, the overexpressed EDR1 kinase domain alone does not exert EDR1 function, but rather negatively affects the function of native EDR1 protein.     

Divergence of melanocortin pathways in the control of food intake and energy expenditure

Activation of melanocortin-4-receptors (MC4Rs) reduces body fat stores by decreasing food intake and increasing energy expenditure. MC4Rs are expressed in multiple CNS sites, any number of which could mediate these effects. To identify the functionally relevant sites of MC4R expression, we generated a loxP-modified, null Mc4r allele (loxTB Mc4r) that can be reactivated by Cre-recombinase. Mice homozygous for the loxTB Mc4r allele do not express MC4Rs and are markedly obese. Restoration of MC4R expression in the paraventricular hypothalamus (PVH) and a subpopulation of amygdala neurons, using Sim1-Cre transgenic mice, prevented 60% of the obesity. Of note, increased food intake, typical of Mc4r null mice, was completely rescued while reduced energy expenditure was unaffected. These findings demonstrate that MC4Rs in the PVH and/or the amygdala control food intake but that MC4Rs elsewhere control energy expenditure. Disassociation of food intake and energy expenditure reveals unexpected divergence in melanocortin pathways controlling energy balance.     

Production of extracellular protease and glucose uptake in Bacillus clausii in steady-state and transient continuous cultures

The production of the extracellular alkaline protease Savinase (EC 3.4.21.62) and glucose uptake in a non-sporulating strain of Bacillus clausii were investigated by analysing steady-state and transients during continuous cultivations. The specific production rate was found to have an optimum at a dilution rate between 0.14 and 0.17 h(-1), whereas the yield of Savinase on glucose was found to increase with decreasing specific growth rate. A linear relationship between the ribosomal RNA content and the specific production rate was found, indicating that the translational capacity may be limiting for product formation. The dynamics of the production of Savinase were studied during step changes in the dilution rate. During a step down in the dilution rate the specific production rate decreased immediately until it reached a new steady value. During a step-up an initial cease in the production rate was observed, but when glucose stopped to accumulate the production rate was regained. The glucose uptake was further investigated when chemostat cultures growing at different dilution rates were exposed to glucose pulses. The maximal glucose uptake capacity was found to be dependent on the initial specific growth rate. Furthermore, the adaptation to high glucose concentrations was faster at high dilution rates than at low dilution rates.     

IL-6 and TNF-alpha expression in,and release from,contracting human skeletal muscle

The aim of the present study was to examine whether IL-6 and TNF-alpha are expressed in, and released from, human skeletal muscle during exercise. We hypothesized that the skeletal muscle will release IL-6, but not TNF-alpha, during exercise because of previous observations that TNF-alpha negatively affects glucose uptake in skeletal muscle. Six healthy, male subjects performed 180 min of two-legged knee-extensor exercise. Muscle samples were obtained from the vastus lateralis of one limb. In addition, blood samples were obtained from a femoral artery and vein. Plasma was analyzed for IL-6 and TNF-alpha. We detected both IL-6 and TNF-alpha mRNA in resting muscle samples, and whereas IL-6 increased (P < 0.05) approximately 100-fold throughout exercise, no significant increase in TNF-alpha mRNA was observed. Arterial plasma TNF-alpha did not increase during exercise. Furthermore, there was no net release of TNF-alpha either before or during exercise. In contrast, IL-6 increased throughout exercise in arterial plasma, and a net IL-6 release from the contracting limb was observed after 120 min of exercise (P < 0.05).