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J E Casanova

Publications and source records attributed to J E Casanova.

At least 19 recordsLinked to original sources
PubMed1998

ARNO is a guanine nucleotide exchange factor for ADP-ribosylation factor 6.

ADP-ribosylation factors (ARFs) constitute a family of small monomeric GTPases. ARFs 1 and 3 function in the recruitment of coat proteins to membranes of the Golgi apparatus, whereas ARF6 is localized to the plasma membrane, where it appears to modulate both the assembly of the actin cytoskeleton and endocytosis. Like other GTPases, ARF activation is facilitated by specific guanine nucleotide exchange factors (GEFs). ARNO (ARF nucleotide-binding site opener) is a member of a growing family of ARF-GEFs that share a common, tripartite structure consisting of an N-terminal coiled-coil domain, a central domain with homology to the yeast protein Sec7p, and a C-terminal pleckstrin homology domain. Recently, ARNO and its close homologue cytohesin-1 were found to catalyze in vitro nucleotide exchange on ARF1 and ARF3, respectively, raising the possibility that these GEFs function in the Golgi. However, the actual function of these proteins may be determined in part by their ability to interact with specific ARFs and in part by their subcellular localization. We report here that in vitro ARNO can stimulate nucleotide exchange on both ARF1 and ARF6. Furthermore, based on subcellular fractionation and immunolocalization experiments, we find that ARNO is localized to the plasma membrane in mammalian cells rather than the Golgi. It is therefore likely that ARNO functions in plasma membrane events by modulating the activity of ARF6 in vivo. These findings are consistent with the previous observation that cytohesin-1 regulates the adhesiveness of alphaLbeta2 integrins at the plasma membrane of lymphocytes.

ADP-Ribosylation Factor 1↗
PubMed1998

US family physicians' experiences with practice guidelines.

BACKGROUND: Practice guidelines were developed to improve medical outcomes and cost-effectiveness. The experiences of family physicians, who may need to use multiple guidelines in their practices, are crucial for effective development and implementation of practice guidelines. We surveyed a national sample of US family physicians about factors that affect their adoption and use of practice guidelines. METHODS: We mailed a structured survey to a national random sample of 400 family physician members of the American Academy of Family Physicians. RESULTS: The response rate was 51%. Most respondents (69%) reported a positive attitude about practice guidelines, but only 44% reported using any guidelines. More younger physicians thought that guidelines could be useful tools. Most preferred guidelines that could be modified (87%) and that were no longer than two pages. Only 27% of respondents knew where to locate a guideline on a particular topic. Forty-three percent of respondents reported that it would be useful if guidelines were a component of an electronic medical record. CONCLUSIONS: If guidelines are to be used by practicing family physicians, a generalist perspective needs to be considered in future guideline development and implementation. Younger physicians had more positive attitudes toward guidelines.

Adult↗
PubMed1997

Cloning and characterization of human phosphomannomutase, a mammalian homologue of yeast SEC53.

Phosphomannomutase (PMM) catalyzes the conversion of mannose-6-phosphate to mannose-1-phosphate, which is a substrate for the synthesis of GDP-mannose. This nucleotide sugar is then used in the synthesis of dolichol-phosphate-mannose, which is essential for N-linked glycosylation and thus the secretion of several glycoproteins as well as for the synthesis of glycosyl-phosphatidyl-inositol (GPI) anchored proteins. In the yeast Saccharomyces cerevisiae, SEC53, a gene required for viability, encodes PMM. Given the importance of PMM in glycoprotein synthesis, it is surprising that very little is known about the enzyme in higher eukaryotes. Recently, an autosomal recessive human disease, Carbohydrate-deficient glycoprotein syndrome type I (CDGS-I) has been correlated with severely reduced PMM activity. Here we report the isolation of a cDNA encoding human PMM, a protein of 29 kDa that is 55% identical and 66% similar to yeast Sec53p. Northern blot analysis shows a single 1.4 kb transcript that is ubiquitously expressed, although levels vary markedly among tissues. Expression of the human cDNA in a temperature-sensitive mutant sec53 yeast strain confers growth at the restrictive temperature, strongly suggesting that this gene encodes a functional PMM. Finally, when expressed in BHK cells, PMM is localized exclusively to the cytosol corresponding to its localization in yeast.

Amino Acid Sequence↗
PubMed1995

Altered hepatic transport of immunoglobulin A in mice lacking the J chain.

We have created J chain knockout mice to define the physiologic role of the J chain in immunoglobulin synthesis and transport. The J chain is covalently associated with pentameric immunoglobulin (Ig) M and dimeric IgA and is also expressed in most IgG-secreting cells. J chain-deficient mice have normal serum IgM and IgG levels but markedly elevated serum IgA. Although polymeric IgA was present in the mutant mice, a larger proportion of their serum IgA was monomeric than was found in wild-type mouse serum. Bile and fecal IgA levels were decreased in J chain-deficient mice compared with wild-type mice, suggesting inefficient transport of J chain-deficient IgA by hepatic polymeric immunoglobulin receptors (pIgR). The pIgR-mediated transport of serum-derived IgA from wild-type and mutant mice was assessed in Madin-Darby canine kidney (MDCK) cells transfected with the pIgR. These studies revealed selective transport by pIgR-expressing MDCK cells of wild-type IgA but not J chain-deficient IgA. We conclude that although the J chain is not required for IgA dimerization, it does affect the efficiency of polymerization or have a role in maintaining IgA dimer stability. Furthermore, the J chain is essential for efficient hepatic pIgR transport of IgA.

Animals↗
PubMed1995

Wortmannin, an inhibitor of phosphoinositide 3-kinase, inhibits transcytosis in polarized epithelial cells.

Wortmannin, an inhibitor of phosphoinositide 3-kinase, inhibits both basolateral to apical and apical to basolateral transcytosis of ricin in Fisher rat thyroid (FRT) cells by 50% at 100 nM in a continuous transcytosis assay. In MDCK cells, a similar effect of wortmannin on basolateral to apical transcytosis of ricin was found, whereas apical to basolateral transcytosis was inhibited to a lesser degree. Transcytosis of dimeric IgA in MDCK cells expressing the polymeric immunoglobulin receptor was also reduced to 50% of controls, suggesting that wortmannin inhibits membrane translocation rather than sorting of specific proteins in the transcytotic pathway. This effect of wortmannin is selective, however, in that endocytosis at the basolateral domain and recycling at both the basolateral and apical membrane domains are unaffected, and apical endocytosis and apical secretion are only moderately reduced. We have shown previously that cAMP stimulates a late stage in basolateral to apical transcytosis in MDCK cells through activation of protein kinase A (Hansen, S. H., and Casanova, J.E. (1994) J. Cell Biol. 126, 677-687). Elevation of cellular cAMP still induced a 100% increase in transcytosis in wortmannin-treated cells, but transcytosis was no longer increased when compared to cells which received no drugs. In contrast, in experiments using a 17 degrees C block to accumulate ricin internalized from the basolateral surface in the apical compartment of MDCK cells, wortmannin had little effect on the stimulation of transcytosis by activators of protein kinase A observed under these conditions. The data thus suggest the existence of a wortmannin-sensitive step in the transcytotic pathway, positioned after endocytosis but prior to translocation into the protein kinase A-sensitive apical compartment, implying a role for phosphoinositide 3-kinase in an intermediate step in transcytosis in polarized epithelial cells.

Androstadienes↗
PubMed1995

Evidence for a regulated interaction between heterotrimeric G proteins and caveolin.

Caveolae are flask-shaped plasma membrane specializations. A 22-kDa protein, caveolin, is a principal component of caveolar membranes in vivo. As recent evidence suggests that caveolae may participate in G protein-coupled signaling events, we have investigated the potential interaction of caveolin with heterotrimeric G proteins. Using cell fractionation techniques, we found that mutational or pharmacologic activation of Gs alpha prevents its cofractionation with caveolin. In a second independent approach, we directly examined the interaction of G proteins with caveolin. For this purpose, we recombinantly expressed caveolin as a glutathione S-transferase fusion protein. Using an in vitro binding assay, we found that caveolin interacts with G protein alpha subunits (Gs, Go, and Gi). Mutational or pharmacologic activation (with guanosine 5'-O-(thiotriphosphate)) of G alpha subunits prevents this interaction, indicating that the inactive GDP-bound form of G alpha subunits preferentially interacts with caveolin. This G protein binding activity is located within a 41-amino acid region of caveolin's cytoplasmic N-terminal domain (residues 61-101). Further functional analysis shows that a polypeptide derived from this region of caveolin (residues 82-101) effectively suppresses the basal activity of purified G proteins, apparently by inhibiting GDP/GTP exchange. This caveolin sequence is homologous to a region of the Rab GDP dissociation inhibitor, a known inhibitor of GDP/GTP exchange for Rab proteins. These data suggest that caveolin could function to negatively regulate the activation state of heterotrimeric G proteins.

Amino Acid Sequence↗
PubMed1994

Mutational analysis of polymeric immunoglobulin receptor/ligand interactions. Evidence for the involvement of multiple complementarity determining region (CDR)-like loops in receptor domain I.

The polymeric Ig receptor (pIgR) mediates the transport of IgA and IgM across a variety of mucosal epithelia. The ectodomain of this receptor consists of five immunoglobulin-like domains (I-V), the first four being structurally similar to immunoglobulin variable regions, and the fifth to Ig constant regions. This study examines the structural features of the pIgR that participate in binding of the ligand, dimeric IgA (dIgA). Recent evidence suggests that a highly conserved region of the first Ig-like domain (domain I) may be important in this process (Bakos, M.A., Kurosky, A., and Goldblum, R. M. (1991) J. Immunol. 147, 3419-3426). In support of this hypothesis, molecular modeling of domain I places this conserved region in an exposed loop analogous to the CDR1 loop of Ig, suggesting that interactions between dIgA and the pIgR may be similar to those between antibodies and their cognate antigens. To test this hypothesis directly, we performed a mutagenic analysis of all three CDR-like loops in domain I of the pIgR. We found that point mutations in multiple residues of CDR1 produced effects on IgA binding ranging from minimal (90% of control) to profound (7%). In addition, we replaced regions corresponding to the CDR2 and CDR3 loops of domain I with their counterparts from domain II (which does not bind IgA), which in both cases resulted in complete abrogation of IgA binding. Taken together, these data suggest that each of the three CDR-like loops of domain I of the rabbit pIgR participates in the binding of dimeric IgA.

Amino Acid Sequence↗
PubMed1994

Gs alpha stimulates transcytosis and apical secretion in MDCK cells through cAMP and protein kinase A.

Recent evidence suggests a role for heterotrimeric G proteins in vesicular transport. Cholera toxin, which activates Gs alpha by ADP-ribosylation, has been reported to stimulate both apical secretion (Pimplikar, S.W., and K. Simons. 1993. Nature (Lond.). 352:456-458) and apically directed transcytosis (Bomsel, M., and K.E. Mostov. 1993. J. Biol. Chem. 268:25824-25835) in MDCK cells, via a cAMP-independent mechanism. Here, we demonstrate that apical secretion and apically directed transcytosis are significantly stimulated by agents that elevate cellular cAMP. Forskolin, which activates adenylyl cyclase directly, and 8BrcAMP augment both transport processes in MDCK cells. The increase is not limited to receptor-mediated transport (polymeric Ig receptor), since transcytosis of ricin, a galactose-binding lectin, is similarly stimulated. The effects of elevated cellular cAMP on apical secretion and transcytosis are apparently mediated via protein kinase A (PKA), as they are inhibited by H-89, a selective PKA inhibitor. Experiments employing a 17 degrees C temperature block indicate that cAMP/PKA acts at a late, possibly rate-limiting stage in the transcytotic pathway, after translocation of internalized markers into the apical cytoplasm. However, no significant stimulus of apical recycling was observed in the presence of FSK, suggesting that cAMP/PKA either affects transcytosis at a level proximal to apical early endosomes and/or specifically increases the efficiency by which transcytosing molecules are delivered to the apical plasma membrane. Finally, we overexpressed wild-type Gs alpha and a mutant, Q227L, which constitutively activates adenylyl cyclase, in MDCK cells. Although Q227L increased transcytosis more than wild-type Gs alpha, neither construct was as effective as FSK in stimulating transcytosis, arguing against a significant role of Gs alpha in transcytosis independent of cAMP and PKA.

Adenylyl Cyclases↗
PubMed1994

In vitro phosphorylation of caveolin-rich membrane domains: identification of an associated serine kinase activity as a casein kinase II-like enzyme.

Caveolae are flask-shaped micro-invaginations associated with the plasma membrane of a wide variety of cell types. Caveolin, an integral membrane component of caveolae, was first identified as the major phosphoprotein whose phosphorylation was elevated in v-Src transformed cells. As both v-Src transformation and elevated caveolin phosphorylation were dependent on membrane attachment of v-Src, it has been suggested that caveolin is a critical target in v-Src transformation. Although an increase in tyrosine phosphorylation of caveolin was evident, the increase in caveolin phosphorylation was predominantly on serine residues. In accordance with these in vivo observations, isolated caveolin-rich membrane domains undergo phosphorylation in vitro predominantly on serine and contain an unidentified serine kinase activity. Here, we have identified this serine kinase activity as a casein kinase II-like enzyme, since the phosphorylation of caveolin-rich membrane domains is stimulated and inhibited by known effectors of casein kinase II (poly-L-lysine, endogenous polyamines, and a casein kinase II inhibitor peptide), but is unaffected by modulators of other known kinases. In support of these observations, caveolin contains a consensus sequence for casein kinase II phosphorylation in its cytoplasmic N-terminal domain (Ser-88). A peptide containing this sequence inhibits the in vitro phosphorylation of caveolin-rich membrane domains, while many other peptides derived from the N-terminal domain of caveolin do not affect phosphorylation. Caveolin-rich membrane domains were also a substrate for exogenously added purified casein kinase II, but not casein kinase I. Finally, immunoblotting of these domains with an antibody directed against the alpha and alpha' subunits of casein kinase II reveals two bands with apparent molecular weights consistent with the known molecular weights of the alpha and alpha' subunits of casein kinase II. As casein kinase II appears to play a role in mitogenic signalling events and casein kinase II activators (endogenous polyamines) are required for v-Src transformation, our results may have implications for understanding the mechanism of v-Src oncogenesis.

Amino Acid Sequence↗
PubMed1993

Utility of pulmonary function testing in the management of chronic obstructive pulmonary disease.

This study sought to measure the effect of pulmonary function testing (PFT) data on the decisions made by generalist physicians in the management of chronic obstructive pulmonary disease (COPD). 148 physicians were randomly assigned to two groups, both of which were asked to manage two identical fictitious but representative cases of COPD, which included history, physical, x-ray, and laboratory results. The experimental group received PFT results in addition. No significant difference was noted between the two groups in management based on availability of PFT data. The optimum utility of PFT data in the management of COPD may be exaggerated and has yet to be determined.

Decision Support Techniques↗
PubMed1993

Hand dexterity in hospital personnel with multiple needlestick injuries.

OBJECTIVE: To test the hypothesis that multiple needlestick injuries in hospital employees may be due to intrinsic deficits in hand dexterity. DESIGN: A case-control study comparing employees with multiple reported needlestick injuries to those with none. Hand dexterity was tested using the Purdue Pegboard Test, a standardized validated test of hand dexterity. SETTING: A 300-bed, acute care teaching hospital. PARTICIPANTS: Fifteen hospital employees who sustained four or more injuries were compared to 19 controls. RESULTS: No differences were detected in hand dexterity between the case and control groups. CONCLUSIONS: While employees with multiple needlestick injuries accounted for 85% of reported injuries, underlying factors responsible for injuries in this high-risk subgroup do not include measurable deficits in hand dexterity.

Case-Control Studies↗
PubMed1991

Direct apical sorting of rat liver dipeptidylpeptidase IV expressed in Madin-Darby canine kidney cells.

In Madin-Darby canine kidney (MDCK) cells, apical and basolateral membrane proteins are segregated from each other in the trans-Golgi network (TGN) and are transported to the appropriate membrane domain via separate vesicle populations. In hepatocytes, however, all plasma membrane proteins are delivered basolaterally. Apical proteins are then selectively retrieved and reach the apical surface by transcytosis. The sorting of apical proteins in different cell types may be the result of differences in the cellular sorting machinery, or alternatively, due to expression of cell-specific sorting signals on the proteins themselves. To test this directly, we have stably expressed cDNA encoding an apical protein from rat liver, dipeptidylpeptidase IV (DPPIV), in MDCK cells. We found that approximately 90% of the exogenous DPPIV is expressed on the apical cell surface at steady state. Furthermore, we demonstrate that this distribution is primarily due to vectorial transport from the TGN to the apical plasma membrane. The small pool of mis-sorted DPPIV that appears basolaterally is slowly endocytosed (t1/2 approximately 60 min) and is subsequently transcytosed. These data are consistent with the notion that both hepatocytes and MDCK cells are capable of correctly sorting rat liver DPPIV, but that this sorting occurs at different sites in the two cell types.

Animals↗