Temperature-sensitive Gbeta mutants discriminate between G protein-dependent and -independent signaling mediated by serpentine receptors

Deletion of the single gene for the Dictyostelium G protein beta-subunit blocks development at an early stage. We have now isolated temperature-sensitive alleles of Gbeta to investigate its role in later development. We show that Gbeta is directly required for adenylyl cyclase A activation and for morphogenetic signaling during the entire developmental program. Gbeta was also essential for induction of aggregative gene expression by cAMP pulses, a process that is mediated by serpentine cAMP receptors (cARs). However, Gbeta was not required for cAR-mediated induction of prespore genes and repression of stalk genes, and neither was Gbeta needed for induction of prestalk genes by the differentiation inducing factor (DIF). cAMP induction of prespore genes and repression of stalk genes is mediated by the protein kinase GSK-3. GSK-3 also determines cell-type specification in insects and vertebrates and is regulated by the wingless/wnt morphogens that are detected by serpentine fz receptors. The G protein-dependent and -independent modes of cAR-mediated signaling reported here may also exist for the wingless/wnt signaling pathways in higher organisms.     

Risk factors for drug-related problems in elderly ambulatory patients

Medication use among elderly patients in ambulatory care, home health care, and long-term care settings has become increasingly complicated during the past decade. Home intravenous therapy, multiple drug regimens to treat multiple chronic diseases, increased acuity of nonhospitalized patients, and increased longevity have placed elderly patients at increased risk for drug-related problems.     

Random mutagenesis of the cAMP chemoattractant receptor, cAR1, of Dictyostelium. Evidence for multiple states of activation

cAMP receptor 1 (cAR1) of Dictyostelium couples to the G protein G2 to mediate activation of adenylyl and guanylyl cyclases, chemotaxis, and cell aggregation. Other cAR1-dependent events, including receptor phosphorylation and influx of extracellular Ca2+, do not require G proteins. To further characterize signal transduction through cAR1, we performed random mutagenesis of the third intracellular loop (24 amino acids), since the corresponding region of other seven helix receptors has been implicated in the coupling to G proteins. Mutant receptors were expressed in car1(-) cells and were characterized for G protein-dependent and -independent signal transduction. Our results demonstrate that cAR1 is remarkably tolerant to amino acid substitutions in the third intracellular loop. Of the 21 positions where amino acid substitutions were observed, one or more replacements were found that retained full biological function. However, certain alterations resulted in receptors with reduced ability to bind cAMP and/or transduce signals. There were specific signal transduction mutants that could undergo cAMP-dependent cAR1 phosphorylation but were impaired either in coupling to G proteins, in G protein-independent Ca2+ influx, or in both pathways. In addition, there were general activation mutants that failed to restore aggregation to car1(-) cells and displayed severe defects in all signal transduction events, including the most robust response, cAMP-dependent cAR1 phosphorylation. Certain of these mutant phenotypes were obtained in a complementary study, where the entire region of cAR1 from the third to the seventh transmembrane helices was randomly mutagenized. Considered together, these studies indicate that the activation cycle of cAR1 may involve a number of distinct receptor intermediates. A model of G protein-dependent and -independent signal transduction through cAR1 is discussed.     

Prognostic value of bacteriological studies in peritonitis

Based on multiple bacteriological assays of lymph and blood, it is concluded that single detection of pathogenic microorganisms in lymph and blood should not be considered an unfavourable prognostic sign. A provisional external drainage of the thoracic duct would reduce intoxication in patients with peritonitis, and it is estimated as a valuable prophylactic method of combating against bacteriemia.     

Myelination in cerebellar slice cultures: development of a system amenable to biochemical analysis

Myelin deposition and maintenance are critical to proper function of the mammalian nervous system. Previous investigations of myelination in the central nervous system (CNS) were hampered by the lack of an in vitro system that can faithfully reproduce in vivo events yet is amenable to biochemical investigation. We have developed a procedure, based on organotypic cultures, which permits efficient preparation of large numbers of cerebellar slice cultures that can be easily manipulated. Cultures have been examined to document myelination biochemically (by incorporation of [35S]sulfate into sulfolipids), immunohistochemically (by labeling the myelin components myelin basic protein and galactocerebroside), and morphologically (by both light and electron microscopy). We tested the effects of biologically active peptides and antibodies on myelination in the thin slices. The results indicate that the cultures provide an in vitro system that can be used to examine specific cellular events that occur during CNS myelination.     

Use of octreotide acetate for control of symptoms in patients with islet cell tumors

Gut tumor syndromes are rare, occurring in less than two cases per million population per year: Insulinomas are most common and gastrinomas are less common; all the others are extremely rare. Conventional treatment of the symptoms caused by these tumors has included surgery, hepatic arterial embolization, and chemotherapy; some patients with Zollinger-Ellison syndrome (ZES) have been treated with specific agents such as gastric antisecretory drugs. The development of octreotide, a synthetic, long-acting analogue of the natural peptide somatostatin, has offered an alternative to such therapies. Octreotide has a half life of > 100 minutes and inhibits both physiological- and tumor release of many peptides. It also has direct effects on the gut that modify secretion and motility. Octreotide has been shown to be particularly useful for the symptoms of tumors producing vasoactive intestinal peptide (VIP), and of the carcinoid syndrome. It is also useful in patients with glucagonomas, with growth hormone-releasing hormone producing tumors, and in some patients with Cushing's syndrome and unresectable insulinomas. Octreotide is effective in patients with ZES, but alternative therapies such as omeprazole are more effective, safer, and more convenient for those patients. Side effects of octreotide have not been troublesome in these patients, but the incidence of long term effects is still not entirely clear. Octreotide has proved to be a significant advance in the treatment of patients with islet cell tumors.     

Identification of a novel actin binding site within the Dp71 dystrophin isoform

A method is described by which the diffusion coefficients of electrically charged micelles can be determined using micellar electrokinetic chromatography (MEKC). The determination is based on a theory for the dispersion, at low electric field strengths, of analytes that are solubilized by only the micellar phase. The dispersion is represented by contributions from instrumental sources and from longitudinal diffusion. The latter depends on the micellar diffusion coefficient. The theory is used to determine the micellar diffusion coefficients of the surfactant sodium dodecyl sulfate in three solutions having different surfactant concentrations. These diffusion coefficients compare very favorably with those determined by diffusion-ordered two-dimensional NMR spectroscopy. An argument is presented justifying that the diffusion coefficients determined by MEKC are self-diffusion coefficients.     

Apolipoprotein E polymorphism influences lipid phenotypic expression, but not the low density lipoprotein subfraction distribution in familial combined hyperlipidemia

The impact of apo E polymorphism on interindividual variation in plasma lipid, lipoprotein concentrations, and LDL subfraction profiles was studied in 201 well-defined patients (88 men and 103 women) with familial combined hyperlipidemia (FCH). When corrected for the concomitant influences of age, gender and obesity, the allelic variation in the apo E gene was shown to explain a statistically significant portion of the variability in lipid and (apo)lipoprotein concentrations. Carriers of the apo epsilon 2 allele exhibited a substantially higher plasma triglyceride concentration and a lower low density lipoprotein (LDL) cholesterol level, while subjects with the apo epsilon 4 allele had significant higher total plasma cholesterol and LDL cholesterol levels. In line with this observation, our FCH population was characterized by an over-representation of the apo E4 allele as compared with a Dutch standard population (chi 2 = 55.2, P < 0.0001). The contribution of apo E polymorphism to trait variability was different between sexes for plasma triglyceride, VLDL cholesterol, VLDL triglycerides, and high density lipoprotein (HDL) cholesterol levels. Apo E polymorphism had no impact on chemical composition of VLDL; for LDL particles the apo epsilon 2 allele was associated with a lower cholesterol to protein (C/P) ratio, whereas the opposite was true for the apo epsilon 4 allele. Despite the demonstrated impact of apo E polymorphism on plasma lipids and LDL chemical composition, in all phenotypic groups a dense LDL subfraction profile predominated. Thus, apo E polymorphism contributes to the lipid phenotypic expression in FCH, whereas further evidence was obtained that a dense LDL subfraction profile is an integral feature of FCH.