Genetic analysis of functional domains within the Drosophila LARK RNA-binding protein.

LARK is an essential Drosophila RNA-binding protein of the RNA recognition motif (RRM) class that functions during embryonic development and for the circadian regulation of adult eclosion. LARK protein contains three consensus RNA-binding domains: two RRM domains and a retroviral-type zinc finger (RTZF). To show that these three structural domains are required for function, we performed a site-directed mutagenesis of the protein. The analysis of various mutations, in vivo, indicates that the RRM domains and the RTZF are required for wild-type LARK functions. RRM1 and RRM2 are essential for viability, although interestingly either domain can suffice for this function. Remarkably, mutation of either RRM2 or the RTZF results in the same spectrum of phenotypes: mutants exhibit reduced viability, abnormal wing and mechanosensory bristle morphology, female sterility, and flightlessness. The severity of these phenotypes is similar in single mutants and double RRM2; RTZF mutants, indicating a lack of additivity for the mutations and suggesting that RRM2 and the RTZF act together, in vivo, to determine LARK function. Finally, we show that mutations in RRM1, RRM2, or the RTZF do not affect the circadian regulation of eclosion, and we discuss possible interpretations of these results. This genetic analysis demonstrates that each of the LARK structural domains functions in vivo and indicates a pleiotropic requirement for both the LARK RRM2 and RTZF domains.

Circadian regulation of the lark RNA-binding protein within identifiable neurosecretory cells.

Molecular genetic analysis indicates that rhythmic changes in the abundance of the Drosophila lark RNA-binding protein are important for circadian regulation of adult eclosion (the emergence or ecdysis of the adult from the pupal case). To define the tissues and cell types that might be important for lark function, we have characterized the spatial and developmental patterns of lark protein expression. Using immunocytochemical or protein blotting methods, lark can be detected in late embryos and throughout postembryonic development, from the third instar larval stage to adulthood. At the late pupal (pharate adult) stage, lark protein has a broad pattern of tissue expression, which includes two groups of crustacean cardioactive peptide (CCAP)-containing neurons within the ventral nervous system. In other insects, the homologous neurons have been implicated in the physiological regulation of ecdysis. Whereas lark has a nuclear distribution in most cell types, it is present in the cytoplasm of the CCAP neurons and certain other cells, which suggests that the protein might execute two different RNA-binding functions. Lark protein exhibits significant circadian changes in abundance in at least one group of CCAP neurons, with abundance being lowest during the night, several hours prior to the time of adult ecdysis. Such a temporal profile is consistent with genetic evidence indicating that the protein serves a repressor function in mediating the clock regulation of adult ecdysis. In contrast, we did not observe circadian changes in CCAP neuropeptide abundance in late pupae, although CCAP amounts were decreased in newly-emerged adults, presumably because the peptide is released at the time of ecdysis. Given the cytoplasmic localization of the lark RNA-binding protein within CCAP neurons, and the known role of CCAP in the control of ecdysis, we suggest that changes in lark abundance may regulate the translation of a factor important for CCAP release or CCAP cell excitability.

The collaboration between psychiatry and primary care in managed care.

Primary care physicians have an important historical role in the delivery of mental health care despite the evolution of psychiatry as a specialty. Collaboration between primary care physicians and psychiatrists has been limited by problems of access to psychiatric care or consultation. Although managed care, in some forms, has been successful in addressing this issue, it has largely served as a new barrier to effective collaboration and to meeting the mental health care needs of patients.

Maternal function of a retroviral-type zinc-finger protein is essential for Drosophila development.

Previous studies have shown that zygotic expression of the Drosophila lark gene, which encodes an RNA-binding protein, is essential for embryogenesis. We now show that lark mRNA is abundant in preblastoderm (0-2 h) embryos, prior to zygotic transcription, indicative of maternal inheritance. Lark can also be detected within the nurse cells of developing egg chambers, suggesting a function for the protein during oogenesis. To test the hypothesis that the maternal inheritance of lark is required for oogenesis or early development, we employed the "FLP-DFS" technique to eliminate the lark maternal component within the germ line. Unfertilized and fertilized eggs lacking the lark maternal component exhibit a "fragile" phenotype, indicating that the protein functions during oogenesis. Furthermore, differentiation of the fertilized eggs is abnormal-most embryos arrest prior to blastoderm formation and exhibit morphological phenotypes that might reflect underlying defects in syncytial nuclear cycling or cellularization. Mutational analysis of a retroviral-type zinc finger within the lark protein indicates that it is required for the maternal function of the protein: females are completely sterile when their only source of lark protein contains a zinc-finger mutation. The aggregate of our studies shows that the germ-line expression of lark is essential for development, and suggests that the retroviral-type zinc finger mediates important RNA-binding functions during oogenesis and/or early development.

An extraretinally expressed insect cryptochrome with similarity to the blue light photoreceptors of mammals and plants.

Photic entrainment of insect circadian rhythms can occur through either extraretinal (brain) or retinal photoreceptors, which mediate sensitivity to blue light or longer wavelengths, respectively. Although visual transduction processes are well understood in the insect retina, almost nothing is known about the extraretinal blue light photoreceptor of insects. We now have identified and characterized a candidate blue light photoreceptor gene in Drosophila (DCry) that is homologous to the cryptochrome (Cry) genes of mammals and plants. The DCry gene is located in region 91F of the third chromosome, an interval that does not contain other genes required for circadian rhythmicity. The protein encoded by DCry is approximately 50% identical to the CRY1 and CRY2 proteins recently discovered in mammalian species. As expected for an extraretinal photoreceptor mediating circadian entrainment, DCry mRNA is expressed within the adult brain and can be detected within body tissues. Indeed, tissue in situ hybridization demonstrates prominent expression in cells of the lateral brain, which are close to or coincident with the Drosophila clock neurons. Interestingly, DCry mRNA abundance oscillates in a circadian manner in Drosophila head RNA extracts, and the temporal phasing of the rhythm is similar to that documented for the mouse Cry1 mRNA, which is expressed in clock tissues. Finally, we show that changes in DCry gene dosage are associated predictably with alterations of the blue light resetting response for the circadian rhythm of adult locomotor activity.

A molecular rhythm mediating circadian clock output in Drosophila.

Analysis of the Drosophila lark gene indicates that it encodes an RNA-binding protein that functions as a regulatory element of the circadian clock output pathway controlling adult eclosion. We now demonstrate that the lark RNA-binding protein oscillates in abundance during the circadian cycle; importantly, the phasing of the lark rhythm is consistent with gene-dosage studies, which indicate that the protein behaves as a repressor molecule. The lark protein rhythm persists in constant conditions (continuous darkness and constant temperature) and is eliminated by period gene null mutations, confirming that it is under clock control and suggesting that it acts as an output mechanism that mediates the temporal regulation of adult eclosion. We also show that lark protein oscillates in abundance within a defined group of neuropeptide (CCAP) -containing neurons of the ventral nervous system (VNS), which in other insects are thought to comprise cellular elements of the clock output pathway regulating eclosion.

Phenotypic variants of meningococci and their potential in phagocytic interactions: the influence of opacity proteins, pili, PilC and surface sialic acids.

In previous studies we have examined the roles of meningococcal surface structures (capsule, lipopolysaccharides, pili and opacity proteins: Opa and Opc) in bacterial interactions with human epithelial, endothelial and mononuclear phagocytic cells. In the current investigations, using defined derivatives of a serogroup A strain C751 and a serogroup B strain MC58, we studied the roles of these structures with human polymorphonuclear phagocytes (PMN). In addition, we examined the potential influence of the pilus-associated protein, PilC, previously known to affect epithelial cell interactions. The data show, that, as with monocytes, opacity proteins affect bacterial interactions with PMN and require surface sialic acids (on capsule and LPS) to be down-modulated in order to function. Also, in contrast to their role in human epithelial and endothelial adherence, neither pili nor PilC expression had any effect on phagocytic cell interactions with respect to induction of chemiluminescence as well as phagocytic killing. Examination of the relative influence of Opa and Opc indicated that Opa proteins are more effective than Opc in PMN interactions whereas the reverse was the case with monocytes. These results suggest that Opa and Opc mediate interactions with phagocytic cells via distinct mechanisms. Observations presented here and reported previously collectively show that the structural requirements of meningococci for interacting with phagocytes, in the absence of opsonins, are present in the phenotype which is often isolated from the nasopharynx (asialylated, piliated, Opa/Opc+) whereas the phenotype prevalent in the blood (sialyted, piliated, Opa/Opc+) retains the ability to adhere to endothelial cells (via pili) but appears to be refractory to interactions with phagocytic cells.

Modulation of the cleavage of glycosylphosphatidylinositol-anchored proteins by specific bacterial phospholipases.

Many enzymes are tethered to the extracellular face of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. These proteins can be released in soluble form by the action of GPI-specific phospholipase. Little is currently known about the factors modulating this release. We investigated the effects of several experimental variables on the cleavage of the GPI-anchored proteins 5'nucleotidase, acetylcholinesterase, and alkaline phosphatase by phospholipases from Bacillus thuringiensis and Staphylococcus aureus. Phospholipase activity was not inhibited by isotonic salt and was relatively unaffected by buffer type and concentration. In both cases, the optimum pH for cleavage was approximately 6.5. Over 80% of 5'-nucleotidase activity present in the lymphocyte plasma membrane was cleaved by the B. thuringiensis enzyme, and the initial rate of release was linear with phospholipase concentration. All three GPI-anchored proteins were released from lymphocyte plasma membrane at comparable phospholipase concentrations, suggesting that they have similar anchor structures. The catalytic activity of 5'-nucleotidase appeared to increase following conversion to the soluble form. The relative surface charge of the host plasma membrane modulated catalytic activity towards GPI-anchored proteins, depending on the net charge of the phospholipase. Studies on purified lymphocyte 5'-nucleotidase reconstituted into bilayers of dimyristoylphosphatidylcholine indicated that the efficiency of phospholipase cleavage was 12- to 50-fold lower when compared with the native plasma membrane. The ability of the phospholipase to cleave the GPI anchor was further reduced when the bilayer was in the gel phase.

Synergistic activation of neurotensin/neuromedin N gene expression by c-Jun and glucocorticoids: novel effects of Fos family proteins.

The cis-regulatory region of the neurotensin/neuromedin N (NT/N) gene integrates diverse environmental signals in the neuroendocrine PC12 cell line, resulting in remarkable synergistic regulation. An AP-1 site appears to play a pivotal role in cooperative NT/N gene activation, as mutations in this site decrease responses to all inducer combinations by at least an order of magnitude. Here we report that c-Jun acts synergistically with glucocorticoids to activate the NT/N promoter, and that Fos family proteins have novel regulatory effects on this interaction. Cotransfection of individual pCMV-AP-1 expression plasmids revealed that c-Jun most potently activates the NT/N promoter and that costimulation with dexamethasone results in a further 6- to 12-fold increase in expression. Unlike its general inhibitory effects on glucocorticoid regulation in other systems, c-Fos potentiated activation by glucocorticoids when coexpressed with c-Jun, and Fos B had a similar, but more limited, positive effect. In contrast, Fra-1 reversed the direction of glucocorticoid regulation, and Fra-2 abolished synergism. AP-1, cAMP response element, and glucocorticoid response element motifs are required for full cooperative activation by either c-Jun or c-Jun/c-Fos and glucocorticoids. These results indicate that NT/N promoter activation involves synergistic interactions between specific AP-1 complexes and ligand-activated glucocorticoid receptor, and similar mechanisms may regulate NT/N gene expression in central neurons.

Characterization of promoter elements required for cell-specific expression of the neurotensin/neuromedin N gene in a human endocrine cell line.

Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine cells (N cells) of the distal small intestine. We have analyzed the NT/N DNA sequences upstream of the RNA start site that direct cell-specific expression using a novel human endocrine cell line, BON, that resembles intestinal N cells in several important aspects, including NT/N precursor protein processing, ratios of different NT/N mRNA forms, and high levels of constitutive expression of the NT/N gene. Transient transfection assays with plasmids with progressive 5' deletions of the rat NT/N promoter identified the proximal 216 bp of 5' flanking sequences as essential for high-level constitutive NT/N expression in BON cells. In addition, a detailed mutational analysis defined multiple regions within the proximal 216 bp that contribute to cell-specific NT/N expression. These elements include a proximal cyclic AMP response element (CRE)/AP-1-like motif (TGACATCA) that binds c-Jun, JunD, CRE-binding (CREB), and ATF proteins, a near-consensus glucocorticoid response element, and a distal consensus AP-1 site that binds c-Fos, Fra-1, and JunD. In addition, elements contained within two 21-bp imperfect direct repeats play an important role in NT/N expression in BON cells and may bind novel factors that act as positive regulators of NT/N expression. DNase I footprinting and gel shift analyses demonstrate that the sites identified by mutational analysis, and at least one additional site, specifically bind BON cell nuclear proteins in vitro. We speculate that a complex pattern of regulation requiring interaction between a proximal CRE/AP-1-like motif and other upstream control elements play an important role in the high-level constitutive expression of NT/N in the human endocrine cell line BON. In addition, the BON cell line provides a unique model to further characterize the factors regulating cell-specific NT/N expression and to better understand the mechanisms responsible for the terminal differentiation of the N-cell lineage in the gut.

Lipid Composition of Cyst Stages of Globodera rostochiensis.

Lipid compositional analysis was conducted on the white, yellow, and brown cyst stages of Globodera rostochiensis (golden cyst nematode). Triacylglycerols were the largest lipid fraction in all stages examined, ranging from 55-75% of total lipid. Ethanolamine phosphoglycerides and choline phosphoglycerides were present in high amounts in all cyst fractions, with a total phospholipid content of 20%, 14.7%, and 12.8% in the white, yellow, and brown cyst stages, respectively. Sterols, steryl esters, sphingomyelin, and cardiolipin were found in minor amounts in all three cyst stages and showed greater changes than other classes of lipids relative to cyst stage. The fatty acid compositions of the three cyst stages were similar. Eicosenoic acid (20:1) and arachidonic acid (20:4) were found in higher concentrations than other fatty acids in all cyst preparations; vaccenic acid (18:1) occurred at the third highest concentration. More than 78% of total fatty acids were unsaturated at all cyst stages, and more than 60% were of C20 or longer chain length. The lipid profile of all three cyst stages is consistent with invertebrate adaptation to low-temperature environments.

Synergistic induction of neurotensin gene transcription in PC12 cells parallels changes in AP-1 activity.

A consensus AP-1 site in the promoter of the rat neurotensin/neuromedin N (NT/N) gene is a critical regulatory element required for synergistic regulation by combinations of nerve growth factor (NGF), lithium, glucocorticoids, and adenylate cyclase activators. A rapid RNase protection assay was developed to examine the kinetics of NT/N gene activation and to determine whether activation requires newly synthesized proteins. Either NGF or lithium in combination with dexamethasone and forskolin transiently activated NT/N gene expression, but with distinct kinetics. Protein synthesis was not required for activation when NGF was used as the permissive inducer, but was required for the rapid down-regulation of the response. In contrast, lithium responses were attenuated in the absence of protein synthesis, consistent with a requirement for newly synthesized AP-1 complexes in activation. In all cases, increases in NT/N gene expression closely paralleled increases in AP-1 binding activity. Lithium in combination with other inducers caused delayed increases in both AP-1 binding activity and c-jun, c-fos and fra-1 gene expression. These results indicate that NGF and lithium exert their effects on NT/N gene expression through distinct pathways. The lithium pathway is active in neuronally-differentiated PC12 cells and could potentially be involved in the regulation of NT/N gene expression in the nervous system.

Plasma fatty acid levels in infants and adults after myocardial ischemia.

High levels of fatty acids are detrimental during reperfusion of ischemic hearts in part because of an inhibition of myocardial glucose use. We therefore measured plasma fatty acids during and after myocardial ischemia in both adult and pediatric patients. In adult patients undergoing thrombolytic therapy after an acute myocardial infarction, plasma fatty acids levels were elevated on admission to hospital (0.96 +/- 0.06 vs 0.40 +/- 0.01 mmol/L in healthy control subjects) and remained elevated throughout the initial 48 hours of hospitalization. In adult patients undergoing cardiac surgery, plasma fatty acids were markedly increased during surgery and at the time of the release of the aortic cross clamp (2.21 +/- 0.54 and 1.61 +/- 0.32 mmol/L, respectively). In children and infants (mean age 4.33 +/- 0.44 years) who had surgery to correct congenital heart defects, fatty acid levels during surgery increased to 3.27 +/- 0.26 mmol/L and remained elevated during immediate reperfusion (1.91 +/- 0.15 mmol/L) and for 24 hours after surgery (1.67 +/- 0.22 mmol/L). Because experimental studies have shown that high levels of fatty acids are detrimental to recovery of adult animal hearts, we determined the effect of high fatty acid levels on reperfusion recovery of isolated working hearts from 1-day-old rabbits perfused with 0.4 mmol/L palmitate (normal fat) or 1.2 mmol/L palmitate (high fat) and subjected to 50 minutes of global ischemia followed by aerobic reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of Neisseria meningitidis with human monocytes.

The roles of capsule, pili and Class 5 outer-membrane proteins (Opa and Opc) of Neisseria meningitidis (Nm) in bacterial interactions with human monocytes were investigated using several meningococcal isolates of different serogroups. The presence of either Class I or Class II pili in capsulate strains of several serogroups had no significant effect on adherence to and internalisation by monocytes. Using clonal variants derived from a non-piliated serogroup A strain, C751, it was observed that capsulate bacteria (cap+) failed to interact with human monocytes in significant numbers whether or not they expressed outer-membrane proteins. These bacteria were also resistant to phagocytic killing. For capsule-deficient bacteria, expression of the Opc protein or OpaBC751 correlated with high levels of association, while the expression of OpaDC751 or OpaAC751 resulted in comparatively lower levels. Bacteria expressing undetectable levels of Opc or Opa proteins (Opc-, Opa-) failed to interact with monocytes. In phagocytic killing assays, Opc-expressing bacteria (Opc+) as well as Opa-expressing bacteria (Opa+) were killed more readily than Opc-, Opa- bacteria (30% decrease in viability of Opc+ bacteria; 18%, 10% and 8% decrease in viability of OpaB+, OpaD+ and OpaA+ bacteria). A study of intracellular survival showed a gradual decrease in viability of both capsulate and capsule-deficient bacteria. However, proportionately greater numbers of capsule-deficient bacteria were internalized and consequently larger numbers survived over a 4-h period. Prolonged bacterial survival within phagocytic cells may have implications in dissemination of bacteria by carriage within these cells.

Assignment of the nuclear mitotic apparatus protein NuMA gene to human chromosome 11q13.

A monoclonal antibody that was specific for a nuclear matrix protein was obtained and used to screen a human lambda gt11 expression library. Several partial cDNA clones were isolated and sequenced. The sequence for this protein was shown to be identical to that of NuMA, a 236-kDa nuclear mitotic spindle apparatus protein. NuMA has been recently characterized by two independent studies, and is thought to be part of a family of proteins that is required for the completion of mitosis. In this report, the chromosomal localization and copy number of the NuMA gene are analyzed using cDNA clones. High-resolution in situ hybridization reveals a single pair of signals on sister chromatids of human chromosome 11 at band q13. Stringent Southern analysis of human genomic DNA resulted in simple restriction patterns. These results together indicate that the NuMA gene is present as a single copy on human chromosome 11q13.