Differential cargo mobilisation within Weibel-Palade bodies after transient fusion with the plasma membrane.

Inflammatory chemokines can be selectively released from Weibel-Palade bodies (WPBs) during kiss-and-run exocytosis. Such selectivity may arise from molecular size filtering by the fusion pore, however differential intra-WPB cargo re-mobilisation following fusion-induced structural changes within the WPB may also contribute to this process. To determine whether WPB cargo molecules are differentially re-mobilised, we applied FRAP to residual post-fusion WPB structures formed after transient exocytosis in which some or all of the fluorescent cargo was retained. Transient fusion resulted in WPB collapse from a rod to a spheroid shape accompanied by substantial swelling (>2 times by surface area) and membrane mixing between the WPB and plasma membranes. Post-fusion WPBs supported cumulative WPB exocytosis. To quantify diffusion inside rounded organelles we developed a method of FRAP analysis based on image moments. FRAP analysis showed that von Willebrand factor-EGFP (VWF-EGFP) and the VWF-propolypeptide-EGFP (Pro-EGFP) were immobile in post-fusion WPBs. Because Eotaxin-3-EGFP and ssEGFP (small soluble cargo proteins) were largely depleted from post-fusion WPBs, we studied these molecules in cells preincubated in the weak base NH4Cl which caused WPB alkalinisation and rounding similar to that produced by plasma membrane fusion. In these cells we found a dramatic increase in mobilities of Eotaxin-3-EGFP and ssEGFP that exceeded the resolution of our method (∼ 2.4 µm2/s mean). In contrast, the membrane mobilities of EGFP-CD63 and EGFP-Rab27A in post-fusion WPBs were unchanged, while P-selectin-EGFP acquired mobility. Our data suggest that selective re-mobilisation of chemokines during transient fusion contributes to selective chemokine secretion during transient WPB exocytosis. Selective secretion provides a mechanism to regulate intravascular inflammatory processes with reduced risk of thrombosis

Multivesicular exocytosis in rat pancreatic beta cells

AIMS/HYPOTHESIS: To establish the occurrence, modulation and functional significance of compound exocytosis in insulin-secreting beta cells. METHODS: Exocytosis was monitored in rat beta cells by electrophysiological, biochemical and optical methods. The functional assays were complemented by three-dimensional reconstruction of confocal imaging, transmission and block face scanning electron microscopy to obtain ultrastructural evidence of compound exocytosis. RESULTS: Compound exocytosis contributed marginally (<5% of events) to exocytosis elicited by glucose/membrane depolarisation alone. However, in beta cells stimulated by a combination of glucose and the muscarinic agonist carbachol, 15-20% of the release events were due to multivesicular exocytosis, but the frequency of exocytosis was not affected. The optical measurements suggest that carbachol should stimulate insulin secretion by ∼40%, similar to the observed enhancement of glucose-induced insulin secretion. The effects of carbachol were mimicked by elevating [Ca(2+)](i) from 0.2 to 2 μmol/l Ca(2+). Two-photon sulforhodamine imaging revealed exocytotic events about fivefold larger than single vesicles and that these structures, once formed, could persist for tens of seconds. Cells exposed to carbachol for 30 s contained long (1-2 μm) serpentine-like membrane structures adjacent to the plasma membrane. Three-dimensional electron microscopy confirmed the existence of fused multigranular aggregates within the beta cell, the frequency of which increased about fourfold in response to stimulation with carbachol. CONCLUSIONS/INTERPRETATION: Although contributing marginally to glucose-induced insulin secretion, compound exocytosis becomes quantitatively significant under conditions associated with global elevation of cytoplasmic calcium. These findings suggest that compound exocytosis is a major contributor to the augmentation of glucose-induced insulin secretion by muscarinic receptor activation

Effects of genetic variation in H3K79 methylation regulatory genes on clinical blood pressure and blood pressure response to hydrochlorothiazide

<p>Abstract</p> <p>Background</p> <p>Nearly one-third of the United States adult population suffers from hypertension. Hydrochlorothiazide (HCTZ), one of the most commonly used medications to treat hypertension, has variable efficacy. The renal epithelial sodium channel (ENaC) provides a mechanism for fine-tuning sodium excretion, and is a major regulator of blood pressure homeostasis. <it>DOT1L, MLLT3, SIRT1</it>, and <it>SGK1 </it>encode genes in a pathway that controls methylation of the histone H3 globular domain at lysine 79 (H3K79), thereby modulating expression of the ENaCα subunit. This study aimed to determine the role of variation in these regulatory genes on blood pressure response to HCTZ, and secondarily, untreated blood pressure.</p> <p>Methods</p> <p>We investigated associations between genetic variations in this candidate pathway and HCTZ blood pressure response in two separate hypertensive cohorts (clinicaltrials.gov NCT00246519 and NCT00005520). In a secondary, exploratory analysis, we measured associations between these same genetic variations and untreated blood pressure. Associations were measured by linear regression, with only associations with <it>P </it>≤ 0.01 in one cohort and replication by <it>P </it>≤ 0.05 in the other cohort considered significant.</p> <p>Results</p> <p>In one cohort, a polymorphism in <it>DOT1L </it>(rs2269879) was strongly associated with greater systolic (<it>P </it>= 0.0002) and diastolic (<it>P </it>= 0.0016) blood pressure response to hydrochlorothiazide in Caucasians. However, this association was not replicated in the other cohort. When untreated blood pressure levels were analyzed, we found directionally similar associations between a polymorphism in <it>MLLT3 </it>(rs12350051) and greater untreated systolic (<it>P </it>< 0.01 in both cohorts) and diastolic (<it>P </it>< 0.05 in both cohorts) blood pressure levels in both cohorts. However, when further replication was attempted in a third hypertensive cohort and in smaller, normotensive samples, significant associations were not observed.</p> <p>Conclusions</p> <p>Our data suggest polymorphisms in <it>DOT1L, MLLT3, SIRT1</it>, and <it>SGK1 </it>are not likely associated with blood pressure response to HCTZ. However, a possibility exists that rs2269879 in <it>DOT1L </it>could be associated with HCTZ response in Caucasians. Additionally, exploratory analyses suggest rs12350051 in <it>MLLT3 </it>may be associated with untreated blood pressure in African-Americans. Replication efforts are needed to verify roles for these polymorphisms in human blood pressure regulation.</p

Microarray gene expression profiling and analysis in renal cell carcinoma

BACKGROUND: Renal cell carcinoma (RCC) is the most common cancer in adult kidney. The accuracy of current diagnosis and prognosis of the disease and the effectiveness of the treatment for the disease are limited by the poor understanding of the disease at the molecular level. To better understand the genetics and biology of RCC, we profiled the expression of 7,129 genes in both clear cell RCC tissue and cell lines using oligonucleotide arrays. METHODS: Total RNAs isolated from renal cell tumors, adjacent normal tissue and metastatic RCC cell lines were hybridized to affymatrix HuFL oligonucleotide arrays. Genes were categorized into different functional groups based on the description of the Gene Ontology Consortium and analyzed based on the gene expression levels. Gene expression profiles of the tissue and cell line samples were visualized and classified by singular value decomposition. Reverse transcription polymerase chain reaction was performed to confirm the expression alterations of selected genes in RCC. RESULTS: Selected genes were annotated based on biological processes and clustered into functional groups. The expression levels of genes in each group were also analyzed. Seventy-four commonly differentially expressed genes with more than five-fold changes in RCC tissues were identified. The expression alterations of selected genes from these seventy-four genes were further verified using reverse transcription polymerase chain reaction (RT-PCR). Detailed comparison of gene expression patterns in RCC tissue and RCC cell lines shows significant differences between the two types of samples, but many important expression patterns were preserved. CONCLUSIONS: This is one of the initial studies that examine the functional ontology of a large number of genes in RCC. Extensive annotation, clustering and analysis of a large number of genes based on the gene functional ontology revealed many interesting gene expression patterns in RCC. Most notably, genes involved in cell adhesion were dominantly up-regulated whereas genes involved in transport were dominantly down-regulated. This study reveals significant gene expression alterations in key biological pathways and provides potential insights into understanding the molecular mechanism of renal cell carcinogenesis

Catecholamine Storage Vesicles: Role of Core Protein Genetic Polymorphisms in Hypertension

Hypertension is a complex trait with deranged autonomic control of the circulation. The sympathoadrenal system exerts minute-to-minute control over cardiac output and vascular tone. Catecholamine storage vesicles (or chromaffin granules) of the adrenal medulla contain remarkably high concentrations of chromogranins/secretogranins (or “granins”), catecholamines, neuropeptide Y, adenosine triphosphate (ATP), and Ca2+. Within secretory granules, granins are co-stored with catecholamine neurotransmitters and co-released upon stimulation of the regulated secretory pathway. The principal granin family members, chromogranin A (CHGA), chromogranin B (CHGB), and secretogranin II (SCG2), may have evolved from shared ancestral exons by gene duplication. This article reviews human genetic variation at loci encoding the major granins and probes the effects of such polymorphisms on blood pressure, using twin pairs to probe heritability and individuals with the most extreme blood pressure values in the population to study hypertension

Effects of dietary carotenoids on mouse lung genomic profiles and their modulatory effects on short-term cigarette smoke exposures

Male C57BL/6 mice were fed diets supplemented with either β-carotene (BC) or lycopene (LY) that were formulated for human consumption. Four weeks of dietary supplementations results in plasma and lung carotenoid (CAR) concentrations that approximated the levels detected in humans. Bioactivity of the CARs was determined by assaying their effects on the activity of the lung transcriptome (~8,500 mRNAs). Both CARs activated the cytochrome P450 1A1 gene but only BC induced the retinol dehydrogenase gene. The contrasting effects of the two CARs on the lung transcriptome were further uncovered in mice exposed to cigarette smoke (CS) for 3 days; only LY activated ~50 genes detected in the lungs of CS-exposed mice. These genes encoded inflammatory-immune proteins. Our data suggest that mice offer a viable in vivo model for studying bioactivities of dietary CARs and their modulatory effects on lung genomic expression in both health and after exposure to CS toxicants