Faculty Bibliography

We have conducted longitudinal studies focused on the expression profiles of signaling pathways and gene networks in children with septic shock. Genome-level expression profiles were generated from whole blood-derived RNA of children with septic shock (n=30) corresponding to day one and day three of septic shock, respectively. Based on sequential statistical and expression filters, day one and day three of septic shock were characterized by differential regulation of 2,142 and 2,504 gene probes, respectively, relative to controls (n=15). Venn analysis demonstrated 239 unique genes in the day one dataset, 598 unique genes in the day three dataset, and 1,906 genes common to both datasets. Functional analyses demonstrated time-dependent, differential regulation of genes involved in multiple signaling pathways and gene networks primarily related to immunity and inflammation. Notably, multiple and distinct gene networks involving T cell- and MHC antigen-related biology were persistently downregulated on both day one and day three. Further analyses demonstrated large scale, persistent downregulation of genes corresponding to functional annotations related to zinc homeostasis. These data represent the largest reported cohort of patients with septic shock subjected to longitudinal genome-level expression profiling. The data further advance our genome-level understanding of pediatric septic shock and support novel hypotheses

Human septic shock involves multiple genome-level perturbations. We have conducted microarray analyses in children with septic shock within 24 h of intensive care unit admission, using whole blood-derived RNA. Based on sequential statistical and expression filters, there were 2,482 differentially regulated gene probes (1,081 upregulated and 1,401 downregulated) between patients with septic shock (n = 42) and controls (n = 15). Both gene lists encompassed several biologically relevant gene ontologies and canonical pathways. Notably, many of the genes downregulated in the patients with septic shock, relative to the controls, participate in gene ontologies related to metal or zinc homeostasis. Comparison of septic shock survivors (n = 33) and nonsurvivors (n = 9) demonstrated differential regulation of 63 gene probes. Among the 63 gene probes differentially regulated between septic shock survivors and nonsurvivors, two isoforms of metallothionein (MT) demonstrated increased expression in the nonsurvivors. Consistent with the ability of MT to sequester zinc in the intracellular compartment, nonsurvivors had lower serum zinc levels compared with survivors. In a corroborating study of murine sepsis, MT-null mice demonstrated a survival advantage compared with wild-type mice. These data represent the largest reported cohort of pediatric patients with septic shock that has undergone genome-level expression profiling based on microarray. The data are biologically plausible and demonstrate that genome-level alterations of zinc homeostasis may be prevalent in clinical pediatric septic shock

The aim of the present study was to investigate the physiological role and the expression pattern of heterologous gap junctions during Xenopus laevis vitellogenesis. Dye transfer experiments showed that there are functional gap junctions at the oocyte/follicle cell interface during the vitellogenic process and that octanol uncouples this intercellular communication. The incubation of vitellogenic oocytes in the presence of biotinylated bovine serum albumin (b-BSA) or fluorescein dextran (FDX), showed that oocytes develop stratum of newly formed yolk platelets. In octanol-treated follicles no sign of nascent yolk sphere formation was observed. Thus, experiments in which gap junctions were downregulated with octanol showed that coupled gap junctions are required for endocytic activity. RT-PCR analysis showed that the expression of connexin 43 (Cx43) was first evident at stage II of oogenesis and increased during the subsequent vitellogenic stages (III, IV and V), which would indicate that this Cx is related to the process that regulates yolk uptake. No expression changes were detected for Cx31 and Cx38 during vitellogenesis. Based on our results, we propose that direct gap junctional communication is a requirement for endocytic activity, as without the appropriate signal from surrounding epithelial cells X. laevis oocytes were unable to endocytose VTG

It is well-established that the endoplasmic reticulum is the major site of phosphatidylinositol (PtdIns) synthesis. The PtdIns synthetic ability of other organelles, such as plasma membrane and nucleus, remains controversial. In the present study, we re-examine this question by comparing PtdIns synthesis in isolated cytoplasts (enucleated cells) with that in corresponding karyoplasts (nuclei surrounded by plasma membrane but lacking most cytoplasmic components). We report that cytoplasts are competent to carry out both basal and stimulated PtdIns synthesis as well as polyphosphoinositide hydrolysis, while karyoplasts can neither synthesize PtdIns nor hydrolyze phosphoinositides in response to agonists. The karyoplasts are, however, capable of synthesizing phosphatidylcholine (PtdCho), as previously reported. From these data, we conclude that PtdIns synthesis is limited to cytoplasmic components, and cannot be sustained by either plasma membrane or nucleus under conditions that permit robust PtdCho synthesis

Mechanisms that function to regulate the rate of de novo phosphatidylinositol (PtdIns) synthesis in mammalian cells have not been elucidated. In this study, we characterize the effect of phorbol ester treatment on de novo PtdIns synthesis in C3A human hepatoma cells. Incubation of cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA) initially (1-6 h) results in a decrease in precursor incorporation into PtdIns; however, at later times (18-24 h), a marked increase is observed. TPA-induced glucose uptake from the medium is not required for observation of the stimulation of PtdIns synthesis, because the effect is apparent in glucose-free medium. Inhibition of the activation of arachidonic acid substantially blocks the synthesis of PtdIns but has no effect on the synthesis of phosphatidylcholine (PtdCho). Increasing the concentration of cellular phosphatidic acid by blocking its conversion to diacylglycerol, on the other hand, enhances the synthesis of PtdIns and inhibits the synthesis of PtdCho. The TPA-induced stimulation of PtdIns synthesis is not the result of the concomitant TPA-induced G1 arrest, because G1 arrest induced by mevastatin has no effect on PtdIns synthesis. Inhibition of protein kinase C activity blocks the stimulatory action of TPA on de novo synthesis of PtdIns but has no effect on TPA-induced inhibition. Potential sites of enzymatic regulation are discussed

Somatostatin analogs (SAs) treat acromegaly by lowering pituitary GH secretion, which, in turn, lowers systemic IGF-I. The profound systemic effect is often greater than expected in the face of only partial GH suppression. Here we report that the SA SOM230 can also act by a nonpituitary-mediated inhibition of IGF-I action. SOM230 inhibited mammary development in intact and hypophysectomized female rats, a process requiring IGF-I. IGF-I overcame this inhibition. SOM230 also inhibited other actions of IGF-I (inhibition of apoptosis, phosphorylation of insulin receptor substrate-1, and cell division). SOM230 did not reduce IGF-I mRNA abundance in mammary gland but did stimulate IGF binding protein 5 (IGFBP5). IGFBP5 was 3.75 times higher in mammary epithelium of SOM230 than in placebo animals (P < 0.001). Administration of IGFBP-5 also inhibited GH-induced mammary development (P < 0.001). Measurement of sstr(1-5) (somatostatin subtype receptor) by real-time RT-PCR revealed that the mammary glands had an abundance of sstr(3) and lower amounts of sstr(4) and sstr(5) but no sstr(1) or sstr(2.) That mammary development was also inhibited to a lesser degree than SOM230 by octreotide, whose main action is through sstr(2), strongly suggests that sstr(3) is at least in part mediating the effects of the SAs. We conclude that 1) SAs inhibit IGF-I action in the mammary gland through a novel nonpituitary mechanism; 2) IGFBP-5, here shown to inhibit pubertal mammary development, might mediate the effect; and 3) Measurement of available sstr receptors in the mammary gland suggests that sstr(3) mediates the SA activity, but sstr(5) is also a possible mediator

Progestins have been implicated in breast cancer development, yet a role for progesterone (Pg) in ductal morphogenesis (DM) has not been established. To determine whether Pg could cause DM, we compared relative effects of Pg, estradiol (E2) and IGF-I on anatomical and molecular biological parameters of IGF-I-related DM in oophorectomized female IGF-I(-/-) mice. Pg had little independent effect on mammary development, but together with IGF-I, in the absence of E2, Pg stimulated an extensive network of branching ducts, occupying 92% of the gland vs. 28.3% with IGF-I alone, resembling pubertal development (P < 0.002). Its major effect was on enhancing duct length and branching (P < 0.002). Additionally, Pg enhanced phosphorylation of IRS-1, increased cell division, and increased the antiapoptotic effect of IGF-I. Pg action was inhibited by RU486 (P < 0.01). E2 also stimulated DM by enhancing IGF-I action but had a greater effect on terminal end bud formation and side branching (P < 0.002). In contrast to previous findings, long-term exposure to E2 alone, without IGF-I, caused formation of ducts and side branches, a novel finding. Both IGF-I and E2 were found necessary for Pg-induced alveolar development. In conclusion, Pg, through Pg receptor can enhance IGF-I action in DM, and E2 acts through a similar mechanism; E2 alone caused formation of ducts and side branches; there were differences in the actions of Pg and E2, the former largely affecting duct formation and extension, and the latter side branching; and both IGF-I and E2 were necessary for Pg to form mature alveoli

Mice that are homozygous for the vibrator mutation express 65-85% less phosphatidylinositol transfer protein alpha (PITPalpha) than their wild type litter mates. By postnatal day 10-12 (P10-12) they exhibit signs of neurodegeneration and die prematurely by P40. In the present study, we examine the lipid content of brain, liver, and mammary glands from these animals. Lipid-mediated signal transduction is evaluated in primary fibroblast cultures. With respect to the lipid make-up of brain and liver, we report that there is a significant increase (2- to 4-fold) in the neutral lipids present in the livers of vb/vb animals when compared with wild type (+/+) litter mates. No significant changes are observed in the brains of these animals. The mammary glands of vb/vb mice are underdeveloped with respect to ductal and alveolar structures, and the fat pad is composed of predominantly brown adipose tissue rather than the white adipose tissue characteristic of age-matched wild type litter mates. No differences are observed in any aspect of lipid-mediated signal transduction

The second messenger cAMP exerts powerful stimulatory effects on Ca(2+) signaling and insulin secretion in pancreatic beta-cells. Previous studies of beta-cells focused on protein kinase A (PKA) as a downstream effector of cAMP action. However, it is now apparent that cAMP also exerts its effects by binding to cAMP-regulated guanine nucleotide exchange factors (Epac). Although one effector of Epac is the Ras-related G protein Rap1, it is not fully understood what the functional consequences of Epac-mediated signal transduction are at the cellular level. 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate (8-pCPT-2'-O-Me-cAMP) is a newly described cAMP analog, and it activates Epac but not PKA. Here we demonstrate that 8-pCPT-2'-O-Me-cAMP acts in human pancreatic beta-cells and INS-1 insulin-secreting cells to mobilize Ca(2+) from intracellular Ca(2+) stores via Epac-mediated Ca(2+)-induced Ca(2+) release (CICR). The cAMP-dependent increase of [Ca(2+)](i) that accompanies CICR is shown to be coupled to exocytosis. We propose that the interaction of cAMP and Epac to trigger CICR explains, at least in part, the blood glucose-lowering properties of an insulinotropic hormone (glucagon-like peptide-1, also known as GLP-1) now under investigation for use in the treatment of type-2 diabetes mellitus

Agonist-stimulated phosphoinositide turnover is accompanied by compensatory resynthesis of these lipids. Several lines of evidence suggest that resynthesis of phosphatidylinositol (PtdIns) involves phosphorylation of diacylglycerol (DG) (salvage pathway) rather than acylation of glycerol phosphate (de novo pathway), although a contribution from the de novo pathway has not been ruled out. To determine the relative contribution of the de novo and salvage pathways in stimulated PtdIns resynthesis, an inhibitor of de novo synthesis (Triacsin C) was incubated simultaneously with the hormone agonist. Results indicate that at early times (90 min), hormone-stimulated PtdIns synthesis proceeds predominantly via the salvage pathway, although some de novo synthesis is also taking place. At later times (24 h), stimulated synthesis is solely via the de novo pathway. Increasing cellular DG content by either adding exogenous DG or treating cells with bacterial phospholipase C (bPLC) results in deacylation of the DG rather than phosphorylation; however, inhibition of this deacylation fails to stimulate phosphorylation by DG kinase (DGK), suggesting channeling of the DG substrate between PLC and DG kinase. Receptor activation is not required for activation of DGK, since treatment with a calcium ionophore induces the same Triacsin C-insensitive PtdIns synthesis. Depletion of the polyphosphoinositide pools by treatment with wortmannin prevents both hormone and A23187-induced polyphosphoinositide hydrolysis; however, A23187 is still able to induce hydrolysis of PtdIns and subsequent compensatory resynthesis. The inability of R59949 to inhibit either hormone-induced or ionophore-induced PtdIns resynthesis suggests that the alpha isoform is not involved; however, its possible that the channeling phenomenon prevents the inhibitor from gaining access to the diacylglycerol kinase enzyme. Further study will be required to determine which isoform catalyzes hormone-induced resynthesis of PtdIns