Oleosin expression and trafficking during oil body biogenesis in tobacco leaf cells

We have established a versatile method for studying the interaction of the oleosin gene product with oil bodies during oil body biogenesis in plants. Our approach has been to transiently express a green fluorescent protein (GFP)-tagged Arabidopsis oleosin gene fusion in tobacco leaf cells containing bona fide oil bodies and then to monitor oleosin-GFP expression using real-time confocal laser scanning microscopy. We show that normally non-oil-storing tobacco leaf cells are able to synthesize and then transport oleosin-GFP fusion protein to leaf oil bodies. Synthesis and transport of oleosin-GFP fusion protein to oil bodies occurred within the first 6 h posttransformation. Oleosin-GFP fusion protein exclusively associated with the endoplasmic reticulum and was trafficked in a Golgi-independent manner at speeds approaching 0.5 microm sec(-1) along highly dynamic endoplasmic reticulum positioned over essentially static polygonal cortical endoplasmic reticulum. Our data indicate that oil body biogenesis can occur outside of the embryo and that oleosin-GFP can be used to monitor early events in oil body biogenesis in real-time.     

Functional specialization of presynaptic Cav2.3 Ca2+ channels

Ca2+ influx into presynaptic terminals via voltage-dependent Ca2+ channels triggers fast neurotransmitter release as well as different forms of synaptic plasticity. Using electrophysiological and genetic techniques we demonstrate that presynaptic Ca2+ entry through Cav2.3 subunits contributes to the induction of mossy fiber LTP and posttetanic potentiation by brief trains of presynaptic action potentials while they do not play a role in fast synaptic transmission, paired-pulse facilitation, or frequency facilitation. This functional specialization is most likely achieved by a localization remote from the release machinery and by a Cav2.3 channel-dependent facilitation of presynaptic Ca2+ influx. Thus, the presence of Cav2.3 channels boosts the accumulation of presynaptic Ca2+ triggering presynaptic LTP and posttetanic potentiation without affecting the low release probability that is a prerequisite for the enormous plasticity displayed by mossy fiber synapses.     

The IIIb isoform of fibroblast growth factor receptor 2 is required for proper growth and branching of pancreatic ductal epithelium but not for differentiation of exocrine or endocrine cells

Fibroblast growth factors (Fgfs) and their receptors have been implicated in embryonic pancreas development. Recently it was shown that Fgf10, a major ligand for the IIIb isoform of fibroblast growth factor receptor 2 (Fgfr2b), has an important regulatory role in early pancreas development. The aim of our study was to define the role of Fgfr2b in pancreas development by analyzing the phenotype of Fgfr2b (-/-) mice. Pancreases of Fgfr2b (-/-) embryos were noticeably smaller than the wild type littermates during embryogenesis, and pancreatic ductal branching as well as duct cell proliferation was significantly reduced. However, both exocrine and endocrine pancreatic differentiation occurred relatively normally. Exogenous addition of Fgfr2b ligands (Fgf7 and Fgf10) stimulated duct cell proliferation and inhibited endocrine cell differentiation in the ex vivo embryonic organ cultures of wild type pancreas. Our results thus suggest that Fgfr2b-mediated signaling plays a major role in pancreatic ductal proliferation and branching morphogenesis, but has little effect on endocrine and exocrine differentiation.     

Leukocyte arrest during cytokine-dependent inflammation in vivo

Leukocyte rolling along the walls of inflamed venules precedes their adhesion during inflammation. Rolling leukocytes are thought to arrest by engaging beta2 integrins following cellular activation. In vitro studies suggest that chemoattractants may instantaneously activate and arrest rolling leukocytes. However, how leukocytes stop rolling and become adherent in inflamed venules in vivo has remained rather mysterious. In this paper we use a novel method of tracking individual leukocytes through the microcirculation to show that rolling neutrophils become progressively activated while rolling down the venular tree. On average, leukocytes in wild-type mice roll for 86 s (and cover 270 microm) before becoming adherent with an efficiency around 90%. These rolling leukocytes exhibit a gradual beta2 integrin-dependent decrease in rolling velocity that correlates with an increase in intracellular free calcium concentration before arrest. Similar tracking analyses in gene-targeted mice demonstrate that the arrest of rolling leukocytes is very rare when beta2 integrins are absent or blocked by a mAb. Arrest is approximately 50% less efficient in the absence of E-selectin. These data suggest a model of leukocyte recruitment in which beta2 integrins play a critical role in stabilizing leukocyte rolling during a protracted cellular activation period before arrest and firm adhesion.     

Expression and contribution of endogenous IL-13 in an experimental model of sepsis

IL-13 has been shown to exert potent anti-inflammatory properties. In this study, we elucidated the functional role of endogenous IL-13 in a murine model of septic peritonitis induced by cecal ligation and puncture (CLP). Initial studies demonstrated that the level of IL-13 increased in tissues including liver, lung, and kidney, whereas no considerable increase was found in either peritoneal fluid or serum after CLP. Immunohistochemically, IL-13-positive cells were Kupffer cells in liver, alveolar macrophages in lung, and epithelial cells of urinary tubules in kidney. IL-13 blockade with anti-IL-13 Abs significantly decreased the survival rate of mice after CLP from 53% to 14% on day 7 compared with control. To determine the potential mechanisms whereby IL-13 exerted a protective role in this model, the effects of anti-IL-13 Abs on both local and systemic inflammation were investigated. Administration of anti-IL-13 Abs did not alter the leukocyte infiltration and bacterial load in the peritoneum after CLP but dramatically increased the neutrophil influx in tissues after CLP, an effect that was accompanied by significant increases in the serum levels of aspartate transaminase, alanine transaminase, blood urea nitrogen, and creatinine. Tissue injury caused by IL-13 blockade was associated with increases in mRNA and the protein levels of CXC chemokines macrophage inflammatory protein-2 and KC as well as the CC chemokine macrophage inflammatory protein-1alpha and the proinflammatory cytokine TNF-alpha. Collectively, these results suggest that endogenous IL-13 protected mice from CLP-induced lethality by modulating inflammatory responses via suppression of overzealous production of inflammatory cytokines/chemokines in tissues.     

Multi-allelic origin of congenital disorder of glycosylation (CDG)-Ic

Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndrome, represent a family of genetic diseases with variable clinical presentations. Common to all types of CDG characterized to date is a defective Asn-linked glycosylation caused by enzymatic defects of N-glycan synthesis. Previously, we have identified a mutation in the ALG6 alpha1,3 glucosyltransferase gene as the cause of CDG-Ic in four related patients. Here, we present the identification of seven additional cases of CDG-Ic among a group of 35 untyped CDG patients. Analysis of lipid-linked oligosaccharides in fibroblasts confirmed the accumulation of dolichyl pyrophosphate-Man9GlcNAc2 in the CDG-Ic patients. The genomic organization of the human ALG6 gene was determined, revealing 14 exons spread over 55 kb. By polymerase chain reaction amplification and sequencing of ALG6 exons, three mutations, in addition to the previously described A333 V substitution, were detected in CDG-Ic patients. The detrimental effect of these mutations on ALG6 activity was confirmed by complementation of alg6 yeast mutants. Haplotype analysis of CDG-Ic patients revealed a founder effect for the ALG6 allele bearing the A333 V mutation. Although more than 80% of CDG are type Ia, CDG-Ic may be the second most common form of the disease.     

Macrophage infiltration in human non-small-cell lung cancer: the role of CC chemokines

 Bronchogenic carcinoma is the leading cause of malignancy-related mortality in the United States, with an overall 5-year survival rate of less than 15%. This aggressive behavior reflects, among other traits, the capacity of the tumor to evade normal host immune defenses, and to induce a pro-angiogenic environment. A central feature of any immune response toward tumors is the recruitment of specific immune cell populations. In the present study we investigated the infiltration of monocytes in human specimens of non-small-cell lung cancer (NSCLC). The presence of macrophages in NSCLC tumors was documented by immunohistochemistry. In vitro chemotaxis assays demonstrated higher monocyte chemotactic activity in NSCLC tumor homogenates than in normal lung tissue. We next investigated the expression of CC chemokines within specimens of NSCLC tumors. Levels of the CC chemokines were higher in NSCLC tumor tissue than in normal lung tissue. Immunolocalization showed that the cells associated with antigenic CC chemokines were the malignant tumor cells, as well as occasional stromal cells. Maximal inhibition of monocyte chemotaxis induced by NSCLC in vitro occurred in the presence of neutralizing antibodies to MCP-1 and MIP-1β. On follow-up of 15 patients in whom we quantified macrophage infiltration, we found that those with recurrence of disease had higher levels of macrophage infiltration in their initial tumors. However, the functional significance of CC-chemokine-mediated macrophage infiltration into NSCLC remains to be determined. Received: 12 November 1999 / Accepted: 10 December 1999     

Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein

Mutations in genes encoding for the sarcoglycans, a subset of proteins within the dystrophin-glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known. To understand the role of the sarcoglycan complex, we looked for sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin-glycoprotein complex. Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a gamma- and delta-sarcoglycan interacting protein. In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals. Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival. Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application. The finding that FLN2 interacts with the sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy.