The effect of chronic ethanol consumption on the fatty acid composition of phosphatidylinositol in rat liver microsomes as determined by gas chromatography and 1H-NMR.

Cell membranes and vesicles composed of extracted phospholipids isolated from rats chronically-fed ethanol develop a resistance to disordering by ethanol in vitro (membrane tolerance) and a decreased partitioning of ethanol into the membranes. The anionic lipid phosphatidylinositol (PtdIns) is the only microsomal phospholipid from the ethanol-fed rats that confers tolerance to vesicles of microsomal phospholipids from control rats in a paradigm where phospholipid classes are sequentially swapped. To investigate the molecular basis of this adaptation, the fatty acid content of microsomal PtdIns extracted from the livers of rats chronically fed ethanol for 5 weeks and their calorically-matched controls was analyzed by gas-liquid chromatography (GLC) and 1H-NMR spectroscopy. Chronic ethanol consumption caused an 8.4% decrease in arachidonic acid [20:4(n - 6)], a 20.0% increase in oleic acid [18: 1(n - 9)] and a 47.1% increase in the quantitatively minor fatty acid [20:3(n - 6)]. 1H-NMR was used to quantitatively assay compositional changes in the delta 5 olefinic moiety of the acyl chains in PtdIns, an approach that should be broadly applicable to other lipid systems. After chronic ethanol feeding PtdIns had decreased delta 5 unsaturates (-7.9% NMR, -8.2% GLC) and a corresponding increase in delta 5 saturates (+5.4% NMR, +5.3% GLC). In the other phospholipids, chronic ethanol feeding caused alterations in the fatty acid compositions specific for each phospholipid. PtdIns was the only microsomal phospholipid that exhibited a significant decrease in both the polyunsaturate pool and the ratio of the total olefinic content to the saturated fatty acid content. The major adaptive response in rat liver microsomal PtdIns to chronic ethanol administration involves a decrease in arachidonic acid [20:4 (n - 6)], which is partly compensated for by increases in oleic acid [18:1(n - 9)] and eicosatrienoic acid [20:3 (n - 6)], resulting in a depressed unsaturation and polyunsaturation index. The decreased unsaturation at the delta 5 position may have special functional relevance, due to the proximity of this position to the membrane surface, where ethanol is believed to reside. Whether these acyl changes are merely coincident with, or causative of, membrane tolerance requires further elucidation.     

Star is required in a subset of photoreceptor cells in the developing Drosophila retina and displays dosage sensitive interactions with rough.

We report that mutations at the Star locus act as dominant enhancers of the eye phenotype displayed by flies carrying a null allele of rough. Our analysis of double mutants at different stages of eye development suggests that this phenotype results from defects in the early stages of photoreceptor cell differentiation in the eye imaginal disc. Complete loss of Star function during retinal development, analyzed in mosaic animals, results in cell death, visible as scars in the adult eye. The requirement for wild-type Star function, however, is confined to only a subset of photoreceptor cells, R8, R2, and R5, which are the first three cells to differentiate neurally in the developing retina. These results suggest an essential role for the Star gene in the initial events of ommatidial cluster formation during the development of the Drosophila compound eye.     

Adrenal activation and the prolactin response to exercise in eumenorrheic and amenorrheic runners.

Adrenocorticotropic hormone (ACTH), cortisol, and prolactin responses following maximal and submaximal (40 min at 80% maximal O2 consumption) running were studied in eumenorrheic (ER; n = 8, 29.0 +/- 1.5 yr) and amenorrheic (AR; n = 8, 24.5 +/- 2.0 yr) runners. ER were studied in the early follicular and midluteal phases of the menstrual cycle. Physical, training, and gynecological characteristics were similar, and cardiorespiratory and metabolic responses to the exercises were indistinguishable in the groups. ACTH, cortisol, and prolactin data from the follicular luteal phases in ER were combined for comparison to AR, because no differences were noted between the menstrual phases at rest. Similar preexercise ACTH levels and responses following exercise occurred in both groups, but preexercise cortisol levels were elevated (ER = 293.1 +/- 46.3, AR = 479.6 +/- 42.4 nmol/l) and cortisol responses blunted in AR. Adrenal sensitivity was blunted in AR compared with ER after submaximal (ER = 121.9 +/- 17.4, AR = 51.7 +/- 13.6) and maximal exercise (ER = 27.9 +/- 9.2, AR = 12.1 +/- 3.8). Preexercise prolactin levels were reduced (ER = 16.4 +/- 2.7, AR = 10 +/- 2.3 micrograms/l), and prolactin responses to maximal exercises were blunted in AR, despite high lactate levels (11.4 +/- 0.4 mmol/l). We conclude that 1) control for menstrual phase in ER is important in studies of prolactin responses following exercise but not in studies of ACTH and cortisol responses following exercise, 2) cortisol responses following submaximal and maximal exercise in AR are blunted at the adrenal level, 3) prolactin responses following submaximal and maximal exercise are also blunted in AR, and 4) prolactin responses following exercise may be mediated by adrenal activation.     

Neonatal capsaicin treatment alters basal pulmonary mechanics and response to methacholine in F344 rats.

Full methacholine dose-response curves were performed on anesthetized tracheostomized Fischer 344 adult rats treated neonatally with capsaicin (50 mg/kg) or with vehicle alone. Capsaicin, the hot extract of pepper, releases substance P (SP) from nonmyelinated sensory nerve endings and causes acute bronchoconstriction and airway microvascular leakiness. Chronic treatment with capsaicin leads to depletion of SP and other tachykinins from afferent C-fibers and can therefore be used as a tool to investigate the contribution of SP innervation to airway responses. The rats (9 controls and 6 treated with capsaicin) were paralyzed with succinylcholine and mechanically ventilated at a constant tidal volume and frequency. Airway resistance (RL) and dynamic compliance (Cdyn) were determined at each dose of methacholine from measurements of volume, flow, and transpulmonary pressure. Capsaicin-treated rats were found to have a significantly reduced baseline RL [0.150 +/- 0.039 (SD) vs. 0.225 +/- 0.050 cmH2O.ml-1.s, P = 0.009] and a correspondingly significantly elevated Cdyn (0.371 +/- 0.084 vs. 0.268 +/- 0.053 ml/cmH2O, P = 0.012). There was no significant difference in sensitivity to methacholine, but the maximal response to methacholine was significantly greater in the capsaicin-treated rats. In terms of RL, the maximal response for capsaicin-treated rats was 6.03 x baseline +/- 0.98 vs. 4.30 x baseline +/- 1.80 (P = 0.05) for controls, and for Cdyn changes the maximal decrease was 5.75 x baseline +/- 1.22 vs. 3.83 +/- 0.69 (P = 0.002). The observed differences in RL and Cdyn coupled with the differences in maximal responses can be attributed to the selective destruction of a subpopulation of pulmonary afferent C-fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Drosophila roughened mutation: activation of a rap homolog disrupts eye development and interferes with cell determination

Roughened is a dominant mutation of D. melanogaster that disrupts eye development. The majority of the ommatidia in the adult eye lack a single photoreceptor cell, which is most commonly the R7 cell. The Roughened mutation disrupts the early stages of photoreceptor cell determination. Roughened is a dominant gain-of-function mutation that results from a single amino acid change (Phe157 to Leu) in the Drosophila Rap1 protein. Loss of function Rap1 mutations are lethal. Drosophila Rap1 protein is 88% identical to human rap1A/K-rev1 protein, a putative antagonist of ras action.     

Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase

We have conducted a genetic screen for mutations that decrease the effectiveness of signaling by a protein tyrosine kinase, the product of the Drosophila melanogaster sevenless gene. These mutations define seven genes whose wild-type products may be required for signaling by sevenless. Four of the seven genes also appear to be essential for signaling by a second protein tyrosine kinase, the product of the Ellipse gene. The putative products of two of these seven genes have been identified. One encodes a ras protein. The other locus encodes a protein that is homologous to the S. cerevisiae CDC25 protein, an activator of guanine nucleotide exchange by ras proteins. These results suggest that the stimulation of ras protein activity is a key element in the signaling by sevenless and Ellipse and that this stimulation may be achieved by activating the exchange of GTP for bound GDP by the ras protein.     

Expression of liver fatty acid-binding protein/human growth hormone fusion genes within the enterocyte and enteroendocrine cell populations of fetal transgenic mice.

The intestinal epithelium establishes and maintains a precise spatial organization despite its continuous and rapid renewal. We have used transgenic mice containing liver fatty acid-binding protein/human growth hormone (L-FABP/hGH) fusion genes to begin to define the molecular mechanisms which are responsible for appropriate regional and cell-specific expression of genes in the gut. Multilabel immunocytochemical methods were employed to characterize the patterns of expression of two transgenes in the enteroendocrine and enterocytic populations of late gestation fetal mice at the time of initial cytodifferentiation of the gastrointestinal epithelium (fetal days 16-19). Surveys of the enteroendocrine cell population using a panel of antibodies directed against 11 neuroendocrine products revealed that these cells are scarce prior to fetal day 17, show a progressive increase in number through day 19, and while the relative proportion of subpopulations (defined by their principal peptide product) are somewhat different than in adults, their geographic distribution along the duodenal to colonic and intervillus(crypt) to villus axes are very similar to that encountered in adult (2-5 month old) mice. Immunoreactive L-FABP is first detectable at fetal day 17 and at this time of first appearance shows an adult pattern of regional enterocytic expression: i.e. it is present in cells overlying nascent villi but not those in the intervillus zone, it is highest in proximal small bowel, declines distally, and is absent from colonocytes. Colocalization studies indicate that L-FABP is not present in enteroendocrine cells during fetal life. Mapping studies indicate that nucleotides -596 to +21 of the rat L-FABP gene are sufficient to reproduce an appropriate temporal, cellular, and regional pattern of reporter (hGH) expression in fetal transgenic mice (with the exception that a subset(s) of enteroendocrine cells, typically containing immunoreactive gastric inhibitory peptide, support transgene but not L-FABP expression). This is in marked contrast to adult transgenic mice where inappropriate hGH accumulation occurs in crypt-associated epithelial cells, in colonocytes, and in many enteroendocrine populations. These studies indicate the importance of considering developmental stage when interpreting the results of any mapping study of cis-acting elements that regulate cell-specific and regional expression of genes in the perpetually renewing intestinal epithelium. Moreover, they also raise the possibility of using transgenes to define fundamental temporal changes in the gut's epithelial cell populations.     

Molecular characterization of bovine brain P75, a high affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II beta

In mammalian brain, physiological signals carried by cAMP seem to be targeted to intraneuronal sites by the association of cAMP-dependent protein kinase II beta with anchoring proteins that bind the regulatory subunit (RII beta) of the enzyme. Previously, an RII beta-binding domain was characterized in a large (Mr approximately 150,000) candidate anchor protein, rat brain P150 (Bregman, D. B., Bhattacharyya, N., and Rubin, C. S. (1989) J. Biol. Chem. 264, 4648-4656). RII beta-binding proteins with Mr values of 65,000-80,000 were detected in the brains of other species. Since little was known about the structural features of these lower Mr proteins, we undertook the characterization of bovine brain P75 as a prototype. A cDNA encoding 258 amino acid residues at the C terminus of P75 was cloned by probing a lambda gt11 expression library with 32P-RII beta. The cDNA insert was ligated into the pET-3b expression plasmid, and large amounts of the partial P75 polypeptide (designated P47) were produced in Escherichia coli. A purification scheme that yielded 9 mg of soluble P47 from a 1-liter bacterial culture was devised. Antibodies directed against the P47 polypeptide revealed that P75 is expressed almost exclusively in brain. The sequence of 117 amino acid residues at the C terminus of P75 contains the RII beta-binding site and is 80% identical to the corresponding region of P150. In contrast, a lower level of identity (36%) between P75 and P150 at a more N-terminal region indicates that the two RII beta-binding proteins are related, but distinct proteins. P75 is not homologous to microtubule-associated protein 2, an RII alpha-selective binding protein, or any other previously studied proteins. C-terminal truncation analysis disclosed that the final 26 residues in P75 are essential for binding RII beta.     

Experimental visceral leishmaniasis: role of endogenous IFN-gamma in host defense and tissue granulomatous response

The capacity of BALB/c mice to acquire resistance to and eliminate intracellular visceral Leishmania donovani is T cell dependent, associated with a granulomatous tissue reaction, and correlates with the ability to secrete the macrophage-activating lymphokine, IFN-gamma. These responses appear by 4 wk after infection and are fully established by 8 wk. To examine the role of endogenous IFN-gamma, BALB/c mice were injected with anti-IFN-gamma mAb before and for 8 wk after infection. At 4 wk, mAb treatment inhibited the acquisition of resistance to L. donovani and abolished mature granuloma formation. Although liver parasite burdens in mAb-treated mice were fivefold higher than in controls at 8 wk, continually treated mice nevertheless began for form tissue granulomas and decreased their parasite loads by 50% from peak values. The levels of anti-IFN-gamma antibody in the serum of mice injected for 8 wk were appreciably reduced, thus raising the possibilities of either insufficient neutralization of endogenous IFN-gamma at this time point or a pathway independent of IFN-gamma. Although the role of IFN-gamma and the potential effect of an IFN-gamma-independent mechanism in the resolution of visceral infection remain to be defined, these results indicate that IFN-gamma plays a critical role in the early immune response that both optimally controls L. donovani infection and induces the tissue granuloma.