The value of clinical teachers for EMR implementations and conversions.

Effective physician training is an essential aspect of EMR implementation. However, it can be challenging to find instructors who can present the material in a clinically relevant manner. The authors describe a unique physician-training program, utilizing medical students as course instructors. This approach resulted in high learner satisfaction rates and provided significant cost-savings compared to alternative options.

Successful physician training program for large scale EMR implementation.

End-user training is an essential element of electronic medical record (EMR) implementation and frequently suffers from minimal institutional investment. In addition, discussion of successful EMR training programs for physicians is limited in the literature. The authors describe a successful physician-training program at Stanford Children's Health as part of a large scale EMR implementation. Evaluations of classroom training, obtained at the conclusion of each class, revealed high physician satisfaction with the program. Free-text comments from learners focused on duration and timing of training, the learning environment, quality of the instructors, and specificity of training to their role or department. Based upon participant feedback and institutional experience, best practice recommendations, including physician engagement, curricular design, and assessment of proficiency and recognition, are suggested for future provider EMR training programs. The authors strongly recommend the creation of coursework to group providers by common workflow.

Multi-screen electronic alerts to augment venous thromboembolism prophylaxis.

Venous thromboembolism (VTE) prophylaxis in high-risk patients is frequently underutilised. We previously devised a one-screen computer alert program that identified hospitalised patients at high risk for VTE who were not receiving prophylaxis and advised their physicians to prescribe prophylaxis. While this strategy reduced the 90-day incidence of symptomatic VTE by 41%, the majority of electronic alerts were ignored. We have now developed a serial three-screen alert computer program designed to educate physicians who initially declined to order prophylaxis after a single screen alert. Of a total cohort of 880, the responsible physicians for 425 patients received a single electronic alert, whereas 455 who declined prophylaxis after the first screen received the second and third screens of the novel three-screen alert. Our enhanced serial three-screen alert program generated VTE prophylaxis orders for 58.4% of the 455 patients whose physicians initially declined to order prophylaxis following the one-screen alert. There was no significant difference in symptomatic 90-day VTE rates between the two cohorts (2.8% for the one-screen vs. 2.2% for the three-screen, p=0.55). We conclude that our three-screen computer alert program can markedly increase prophylaxis among physicians who decline an initial single screen alert.

Muscarinic receptor function, density and G-protein coupling in the overactive diabetic rat bladder.

1 Bladder smooth muscle sensitivity to muscarinic agonists is increased in the overactive bladder. Treatment of rats with streptozotocin induces a diabetic state in which the bladder muscle is overactive and also supersensitive to muscarinic agonists. This study has examined bladder contraction, muscarinic receptor density and receptor/G-protein coupling in the streptozotocin-induced overactive bladder of the rat. 2 Diabetes was induced by a single intraperitoneal dose of streptozotocin. Seven days later contraction of isolated detrusor muscle strips was assessed in tissue bath experiments, while receptor density was assayed in saturation experiments with [3H]-QNB (quinuclidinyl benzilate, L-[benzilic-4,4'-3H]) and receptor/G-protein coupling was determined in agonist displacement experiments with this radioligand. 3 Isolated detrusor strips from diabetic animals displayed an enhanced degree of spontaneous activity (0.060 +/- 0.016 g mg(-1), compared with 0.015 +/- 0.004 g mg(-1), P < 0.05). Carbachol produced contractile responses in tissues from both control and diabetic rats, but the diabetic tissues were more sensitive to this agonist, the pEC50 being 6.52 +/- 0.17 compared with 5.93 +/- 0.06 in controls (P < 0.001). Maximum responses to carbachol were similar in both groups of animals. The increase in carbachol potency was accompanied by a 40% increase in receptor density from 158 +/- 5 to 221 +/- 22 fmol mg(-1) protein (P < 0.05), but this was not enough to fully account for the change in tissue sensitivity. 4 In the absence of GTP-gamma-S, carbachol displaced [3H]-QNB from two binding sites, the high-affinity site (pKi = 7.06 +/- 0.26) which represents the receptors coupled to G-proteins made up 43.1 +/- 5.9% of the total binding sites in control tissues and this value was similar (41.0 +/- 4.0%) in the diabetic tissues (pKi = 6.64 +/- 0.31). In the presence of GTP-gamma-S, carbachol displaced [3H]-QNB from a single binding site which had a low-affinity, similar to the low-affinity site observed in the absence of GTP-gamma-S. 5 These data demonstrate that detrusor supersensitivity is observed after only 1 week of untreated diabetes in the rat. The overactivity is associated with an enhanced sensitivity to carbachol, which is partly explained by an increase in receptor density, but there appears to be no change in receptor/G-protein coupling.

Kidney function estimating equations: where do we stand?

PURPOSE OF REVIEW: Estimation of the glomerular filtration rate (GFR) is central to the diagnosis, evaluation and management of chronic kidney disease. This review summarizes recent data on the performance of equations using serum creatinine to estimate the GFR, particularly the Modification of Diet in Renal Disease (MDRD) Study equation. RECENT FINDINGS: During 2005 GFR estimation has received substantial attention with a focus on comparing the MDRD Study equation with the Cockcroft-Gault equation. Several large studies (n>500) have appeared. Most studies discuss creatinine calibration but few were able to standardize measurements. Studies that did calibrate the creatinine had improved performance. Overall, the MDRD Study equation performed well in populations with a low range of GFR and often outperformed the Cockcroft-Gault equation. Both equations have lower precision in high GFR populations, and the MDRD equation under-estimated the GFR in a number of studies. Efforts are underway to develop improved prediction equations by pooling data across many study populations. SUMMARY: Equations for estimating the GFR from serum creatinine are useful for systematic staging of chronic kidney disease. The MDRD Study equation and systematic creatinine assay calibration improve the level of precision and accuracy in many settings. GFR estimates are less useful in the normal range of GFR, however, and are sensitive to the population under study.

Nicotinamide adenine dinucleotide (NAD) and its metabolites inhibit T lymphocyte proliferation: role of cell surface NAD glycohydrolase and pyrophosphatase activities.

The presence of NAD-metabolizing enzymes (e.g., ADP-ribosyltransferase (ART)2) on the surface of immune cells suggests a potential immunomodulatory activity for ecto-NAD or its metabolites at sites of inflammation and cell lysis where extracellular levels of NAD may be high. In vitro, NAD inhibits mitogen-stimulated rat T cell proliferation. To investigate the mechanism of inhibition, the effects of NAD and its metabolites on T cell proliferation were studied using ART2a+ and ART2b+ rat T cells. NAD and ADP-ribose, but not nicotinamide, inhibited proliferation of mitogen-activated T cells independent of ART2 allele-specific expression. Inhibition by P2 purinergic receptor agonists was comparable to that induced by NAD and ADP-ribose; these compounds were more potent than P1 agonists. Analysis of the NAD-metabolizing activity of intact rat T cells demonstrated that ADP-ribose was the predominant metabolite, consistent with the presence of cell surface NAD glycohydrolase (NADase) activities. Treatment of T cells with phosphatidylinositol-specific phospholipase C removed much of the NADase activity, consistent with at least one NADase having a GPI anchor; ART2- T cell subsets contained NADase activity that was not releasable by phosphatidylinositol-specific phospholipase C treatment. Formation of AMP from NAD and ADP-ribose also occurred, a result of cell surface pyrophosphatase activity. Because AMP and its metabolite, adenosine, were less inhibitory to rat T cell proliferation than was NAD or ADP-ribose, pyrophosphatases may serve a regulatory role in modifying the inhibitory effect of ecto-NAD on T cell activation. These data suggest that T cells express multiple NAD and adenine nucleotide-metabolizing activities that together modulate immune function.

The RT6 (Art2) family of ADP-ribosyltransferases in rat and mouse.

Recent evidence suggests that a new member of the mono-ADP-ribosyltransferase/NAD glycohydrolase family, RT6, may be important in immune regulation. RT6 is expressed in two allelic forms and is present on post-thymic T cells in the rat. RT6-expressing T cells in the rat may have a regulatory role, a conclusion based on their ability to prevent autoimmune diabetes in the BB rat model of insulin-dependent diabetes mellitus. This observation led to investigation of RT6 at a molecular and biochemical level resulting in the determination that RT6 protein exists as both glycosylated and non-glycosylated glycosylphosphatidylinositol (GPI)-linked cell surface molecules. RT6, like many GPI-linked proteins, can mediate cell signal transduction events associated with T cell activation, and is also present in a soluble form in the circulation. The discovery that RT6 is an NAD glycohydrolase and auto-ADP-ribosyltransferase led to the ongoing investigations into the role that enzymatic activity may have in the immunoregulatory function of rat RT6+ T cells. A homologue of rat RT6, termed Rt6, has been identified in the mouse. Rt6 is predominately an ADP-ribosyltransferase enzyme as determined using simple guanidino compounds (e.g. arginine) as ribose acceptors. Abnormalities in mouse Rt6 mRNA are associated with the expression of autoimmunity. In the present manuscript, we review recent data on RT6/Rt6, and discuss the potential mechanisms by which RT6-expressing cells, and perhaps RT6 protein itself, may mediate immune regulation.

Characterization of NAD:arginine ADP-ribosyltransferases.

NAD:arginine mono-ADP-ribosyltransferases catalyze the transfer of ADP-ribose from NAD to the guanidino group of arginine on a target protein. Deduced amino acid sequences of one family (ART1) of mammalian ADP-ribosyltransferases, cloned from muscle and lymphocytes, show hydrophobic amino and carboxyl termini consistent with glycosylphosphatidylinositol (GPI)-anchored proteins. The proteins, overexpressed in mammalian cells transfected with the transferase cDNAs, are released from the cell surface with phosphatidylinositol-specific phospholipase C (PI-PLC), and display immunological and biochemical characteristics consistent with a cell surface, GPI-anchored protein. In contrast, the deduced amino acid sequence of a second family (ART5) of transferases, cloned from murine lymphoma cells and expressed in high abundance in testis, displays a hydrophobic amino terminus, consistent with a signal sequence, but lacks a hydrophobic signal sequence at its carboxyl terminus, suggesting that the protein is destined for export. Consistent with the surface localization of the GPI-linked transferases, multiple surface substrates have been identified in myotubes and activated lymphocytes, and, notably, include integrin alpha subunits. Similar to the bacterial toxin ADP-ribosyltransferases, the mammalian transferases contain the characteristic domains involved in NAD binding and ADP-ribose transfer, including a highly acidic region near the carboxy terminus, which, when disrupted by in vitro mutagenesis, results in a loss of enzymatic activity. The carboxyl half of the protein, synthesized as a fusion protein in E. coli, possessed NADase, but not ADP-ribosyltransferase activity. These findings are consistent with the existence at the carboxyl terminus of ART1 of a catalytically active domain, capable of hydrolyzing NAD, but not of transferring ADP-ribose to a guanidino acceptor.

Pharmacokinetics of glycosylated recombinant human granulocyte colony-stimulating factor (lenograstim) in healthy male volunteers.

AIMS: The aim of this open, randomised, crossover, parallel-group study was to compare the pharmacokinetics and neutrophil responses of lenograstim when administered subcutaneously (s.c.) and intravenously (i.v.). METHODS: A total of 27 healthy male volunteers was recruited. Lenograstim doses (0.5, 2, 5, or 10 microg kg(-1)) were administered s.c. or i.v. once-daily for 5 days, and then, after a 10-day washout period, vice versa for a further 5 days. Lenograstim concentrations and absolute neutrophil counts (ANCs) were measured predosing and postdosing on days 1 and 5. RESULTS: Maximum serum concentrations of lenograstim were higher following i.v. dosing (mean 5.2-185.5 vs 0.7-30.0 ng ml(-1) after s.c. dosing on day 1) and attained sooner (median 0.5-0.8 vs 4.7-8.7 h on day 1). However, apparent elimination half-lives of lenograstim were longer following s.c. dosing (mean 2.3-3.3 vs 0.8-1.2 h after i.v. dosing on days 1 and 5). ANCs increased in a dose-dependent manner with both routes of lenograstim, but more prolonged rises and higher ANC peaks were attained following s.c. doses. ANCs peaked on day 6 following 5 microg kg(-1) s.c. doses (mean peak=26.3x10(9) cells l(-1)), but on day 2 after 5 microg kg(-1) i.v. doses (mean peak = 12.4 x 10(9) cells l(-1)). Irrespective of route, the most common adverse events were headaches and back/spine pain; at doses of up to 5 microg kg(-1) these were mild and generally well tolerated. CONCLUSIONS: While supporting the use of both s.c. and i.v. administered lenograstim to treat neutropenia, these results demonstrate that neutrophil responses are more sustained and prolonged with the s.c. route.

Effects of site-directed mutagenesis of Escherichia coli heat-labile enterotoxin on ADP-ribosyltransferase activity and interaction with ADP-ribosylation factors.

Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsalpha, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction.

Expression in BALB/c and C57BL/6 mice of Rt6-1 and Rt6-2 ADP-ribosyltransferases that differ in enzymatic activity: C57BL/6 Rt6-1 is a natural transferase knockout.

Several proteins with NAD+:arginine ADP-ribosyltransferase (ART) activity are expressed in T cells and affect their function. Rat T cells that express the ART designated RT6 are determinants of the expression of autoimmune diabetes. In the mouse, a 35-kDa ecto-ART modulates the proliferation and functional activity of CTL. Here we report on mouse ARTs designated Rt6-1 and Rt6-2 in BALB/c and C57BL/6 mice. mRNAs for Rt6-1 and Rt6-2 were found in spleen, thymus, and intestinal tissue of both strains, but Rt6-1 mRNA in C57BL/6 mice was detected only at low levels. Rt6-1 and Rt6-2 cDNAs from both strains were cloned and sequenced. Predicted amino acid sequences of Rt6-2 were identical in both strains, but there was an in-frame stop codon in the sequence of Rt6-1 in C57BL/6 mice not present in BALB/c mice. Recombinant C57BL/6 Rt6-2 and BALB/c Rt6-1 proteins expressed in COS1 cells exhibited ART activity and were documented to be glycosylphosphatidylinositol-linked membrane proteins. COS-1 cells transfected with a C57BL/6 Rt6-1 cDNA construct expressed a truncated protein consistent in size with that predicted by the presence of the stop codon. This approximately 21-kDa protein appeared not to be glycosylphosphatidylinositol linked to the cell surface and lacked ART activity. C57BL/6 Rt6-1 therefore appears to be a naturally occurring ART knockout. The expression of Rt6-1 and Rt6-2 mRNAs in lymphoid tissues suggests that these ARTs may regulate immune system functions. Expression of Rt6-2 or another redundant ART may compensate for the lack of enzymatically active Rt6-1 in C57BL/6 mice.

Characterization of mouse Rt6.1 NAD:arginine ADP-ribosyltransferase.

Rat RT6 proteins, and perhaps mouse Rt6, identify a set of immunoregulatory T lymphocytes. Rat RT6.1 (RT6.1) and rat RT6.2 (RT6. 2) are NAD glycohydrolases, which catalyze auto-ADP-ribosylation, but not ADP-ribosylation of exogenous proteins. Mouse Rt6.1 (mRt6.1) also catalyzes auto-ADP-ribosylation. The activity of mouse cytotoxic T lymphocytes is reportedly inhibited by ADP-ribosylation of surface proteins, raising the possibility that mRt6 may participate in this process. The reactions catalyzed by mRt6, would, however, need to be more diverse than those of the rat homologues and include the ADP-ribosylation of acceptors other than itself. To test this hypothesis, mRt6.1 and rat RT6.2 were synthesized in Sf9 insect cells and rat mammary adenocarcinoma (NMU) cells. mRt6.1, but not rat RT6.2, catalyzed the ADP-ribosylation of guanidino-containing compounds (e.g. agmatine). Unlike RT6.2, mRt6.1 was a weak NAD glycohydrolase. In the presence of agmatine, however, the ratio of [adenine-14C]ADP-ribosylagmatine formation from [adenine-14C]NAD to [carbonyl-14C]nicotinamide formation from [carbonyl-14C]NAD was approximately 1.0, demonstrating that mRt6.1 is primarily a transferase. ADP-ribosylarginine hydrolase, which preferentially hydrolyzes the alpha-anomer of ADP-ribosylarginine, released [U-14C]arginine from ADP-ribosyl[U-14C]arginine synthesized by mRT6.1, consistent with the conclusion that mRt6.1 catalyzes a stereospecific Sn2-like reaction. Thus, mRt6.1 is an NAD:arginine ADP-ribosyltransferase capable of catalyzing a multiple turnover, stereospecific Sn2-like reaction.

Expression in human endothelial cells of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding proteins involved in the initiation of vesicular transport.

ADP-ribosylation factors (ARFs) are approximately 20-kDa, guanine nucleotide-binding proteins, initially discovered as stimulators of cholera toxin ADP-ribosyltransferase activity and subsequently shown to participate in vesicular trafficking. Five of the six mammalian ARFs have been identified in human tissues by molecular cloning. They fall into three classes (class I: ARFs 1-3; class II: ARFs 4, 5; class III: ARF 6) based on deduced amino acid sequence, size, phylogenetic analysis, and gene structure. Similar to the rab family of approximately 20 kDa guanine nucleotide-binding proteins, the ARFs appear to function in specific trafficking pathways. The presence of a specific ARF might serve as a marker for that pathway. To verify expression of ARF mRNA and protein in human umbilical vein endothelial cells, immunoreactivity using antibodies specific for each ARF class, quantitative polymerase chain reaction (PCR) using ARF-specific, internal cRNA standards containing unique restriction enzyme cleavage sites introduced by point mutations, and Northern analysis with probes specific for ARFs 1, and 3-6, were utilized. PCR and Northern analysis were in agreement in showing that amounts of mRNA for ARF 1 and ARF 4 were similar and higher than those of ARF 3 and ARF 5 which were greater than ARF 6. Primarily, Class 1 ARF proteins were detected by immunoreactivity, with the majority in the supernatant fraction. The relative expression of ARFs in endothelial cells thus differs from that in neuronal tissues where it had been found that ARF3 is the predominant species.

Rat RT6.2 and mouse Rt6 locus 1 are NAD+: arginine ADP ribosyltransferases with auto-ADP ribosylation activity.

RT6 is a glycosylphosphatidylinositol-linked protein found on the surface of mature rat T lymphocytes. Cells that express RT6 have an immunoregulatory function and modulate the expression of autoimmune diabetes mellitus in the BioBreeding rat. A homologue of the rat RT6 gene, designated Rt6, has been identified in the mouse, but expression of mouse Rt6 protein has not been documented. Rat RT6 is known to be a nicotinamide adenine dinucleotide (NAD+) glycohydrolase. We now report that rat RT6.2 and recombinant mouse Rt6 locus 1 proteins possess auto-ADP ribosylation activity. In addition, mouse Rt6 but not rat RT6, catalyzes the ADP ribosylation of exogenous acceptors such as histones. The ADP-ribosyl-protein bonds in auto-ADP-ribosylated rat RT6.2, auto-ADP-ribosylated mouse Rt6, and ADP-ribosylhistone synthesized by Rt6 were stable to HgCl2 and HCl, but labile to NH2OH, consistent with ADP ribosylarginine linkages. To determine if these enzymatic activities could affect the function of rat T cells, the effect of substrate availability on lymphocyte proliferation was examined. An inverse correlation was observed between NAD+ concentration in the medium and the ability of rat T cells to respond to anti-CD3, ConA, and PMA plus ionomycin. The data suggest that lymphocyte surface ADP ribosyltransferases could be involved in signaling and immunoregulatory processes.

Tracheal colonization factor: a Bordetella pertussis secreted virulence determinant.

We report here the identification of a virulence-associated factor, Tcf, (tracheal colonization factor), produced by strains of Bordetella pertussis but not Bordetella parapertussis or Bordetella bronchiseptica. This protein is encoded by the tcfA gene. When a strain of B. pertussis 18323 lacking this protein is used to infect mice with an aerosol challenge, the number of bacteria isolated from the tracheas is decreased 10-fold when compared with the parent 18323. The derived amino acid sequence of tcfA predicts a 68 kDa RGD-containing, proline-rich protein, which after cleavage of a typical prokaryotic signal sequence would be 64 kDa. Amino acid sequence analysis demonstrates that the C-terminal 30 kDa of this protein shows 50% identity to the 30 kDa C-terminus of another Bordetella protein, the pertactin precursor. The N-terminal 34 kDa region contains the three amino-acid motif RGD and is 16.5% proline. Coupled in vitro transcription and translation analysis indicates that the tcfA gene product migrates as two bands of approximately 90 kDa. A fusion protein of the N-terminal, 34 kDa portion of Tcf to maltose-binding protein migrates, on SDS-PAGE, 30 kDa higher than expected from the combined molecular weights. Polyclonal antisera raised against the unique N-terminal portion of Tcf recognizes 90 kDa and 60 kDa bands in immunoblots of whole-cell lysates of strains of B. pertussis; it does not recognize any protein in whole-cell lysates of B. bronchiseptica or B. parapertussis. Supernatants of cultures of B. pertussis 18323 contain the 60 kDa form of the protein. Southern blot analysis of chromosomal DNA from strains of B. bronchiseptica and B. parapertussis, using a probe derived from tcfA, shows strong hybridization only to B. pertussis DNA. Thus, Tcf appears to be a unique virulence factor of B. pertussis.

Involvement of N-myristoylation in monoclonal antibody recognition sites on chimeric G protein alpha subunits.

Monoclonal antibody, LAS-2, directed against the alpha subunit of transducin (Gt alpha), inhibited Gt beta gamma-dependent, pertussis toxin-catalyzed ADP ribosylation of Gt alpha and was specific for Gt alpha. Immunoblotting studies on proteolytic fragments of Gt alpha were consistent with an amino-terminal epitope. To define the antibody recognition site, recombinant Gt alpha was synthesized in Escherichia coli cotransfected with or without yeast N-myristoyl-transferase. Amino-terminal fatty acylation of Gt alpha, verified by use of radiolabeled fatty acid, was required for immunoreactivity. LAS-2 did not react with a chimeric protein consisting of residues 1-9 of Gt alpha and the remainder Go alpha, regardless of its myristoylation. Immunoreactivity was observed when amino acids 1-17 of Gt alpha were present in a Go alpha chimera and the protein was amino-terminally myristoylated; there was no reactivity without myristoylation. It appears that the LAS-2 epitope requires both Gt alpha-specific sequence in amino acids 10-17 and a fatty acyl group in proximity to these residues. These results are consistent with the hypothesis that the myristoyl group is essential for protein structure; conceivably it "folds back" on and stabilizes the amino-terminal structure of Gt alpha as opposed to protruding from an amino-terminal alpha-helix and serving as an amino-terminal membrane anchor.

Construction and characterization of recombinant Vibrio cholerae strains producing inactive cholera toxin analogs.

The catalytic A subunit of cholera toxin (CT-A) is capable of ADP-ribosylating the guanine nucleotide-binding protein, which regulates cell adenylyl cyclase, leading to the life-threatening diarrhea of cholera. Amino acids involved in the enzymatic activity of CT-A have previously been identified. By means of site-directed mutagenesis, an analog of the CT-A subunit gene was created with codon substitutions for both Arg-7 and Glu-112, each of which has been shown to produce subunits lacking ADP-ribosyltransferase activity. The mutated gene fragment was exchanged for the wild-type copy in the previously cloned ctxAB operon from El Tor biotype, Ogawa serotype Vibrio cholerae strain 3083, which produces CT-2. Further, the zonula occludens toxin gene, zot, was inactivated by an insertional mutation to create the new plasmid construct pCT-2*. Additionally, a DNA fragment encoding the B subunit of CT-1 (CT produced by classical biotype, Inaba serotype V. cholerae strain 569B) was exchanged for the homologous part in pCT-2*, resulting in the creation of pCT-1*. These plasmid constructs were introduced into the CT-negative V. cholerae mutant strain JBK70 (E1 Tor biotype, Inaba serotype); CT-A-B+ derivatives CVD101 and CVD103 of classical biotype Ogawa and Inaba serotype strains 395 and 569B, respectively; El Tor biotype Inaba and Ogawa serotype strains C6706 and C7258, respectively, recently isolated in Peru; and O139 (synonym Bengal) strain SG25-1 from the current epidemic in India. Recombinant toxins (CT-1* and CT-2*), partially purified from culture supernatants of transformed JBK70, were shown to be inactive on mouse Y1 adrenal tumor cells and in an in vitro ADP-ribosyltransferase assay. CT-1* and CT-2* reacted with polyclonal and monoclonal antibodies against both A and B subunits of CT. The toxin analogs reacted with antibodies against CT-A and CT-B on cellulose acetate strips and in a GM1 enzyme-linked immunosorbent assay; they reacted appropriately with B-subunit epitype-specific monoclonal antibodies in checkerboard immunoblots, and they formed precipitin bands with GM1-ganglioside in Ouchterlony tests. However, the reactions of the modified proteins with anti-A-subunit monoclonal antibodies were weaker than the reactions with wild-type holotoxins. V, cholerae strains carrying ctxA*, with either ctxB-1 or ctxB-2, and inactivated zot genes were created by homologous recombination. The recombinant strains and the purified toxin analogs were inactive in the infant rabbit animal model.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a glucose-repressible ADP-ribosylation factor 3 (ARF3) from Saccharomyces cerevisiae.

ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin, and are believed to participate in vesicular transport in both exocytic and endocytic pathways. Based on size, phylogenetic analysis, amino acid sequence, and gene structure, mammalian ARFs fall into three classes (class I, ARFs 1, 2, 3; class II, ARFs 4, 5; class III, ARF6). Two ARF genes (yARF1, yARF2) are known in Saccharomyces cerevisiae and believed to participate in vesicular trafficking in the Golgi system; the double deletion mutant is not viable. A third yeast ARF (yARF3) cDNA has been cloned by polymerase chain reaction-based procedures. It contains an open reading frame of 549 bases encoding a protein of 183 amino acids, with a deduced amino acid sequence more identical (60%) to that of the class III mammalian ARF than to those of the other two classes (52-56%). The yARF3 protein, however, reacted poorly with antibodies against any of the three classes of mammalian ARFs. In the presence of GTP, recombinant yARF3 protein stimulated cholera toxin-catalyzed auto-ADP-ribosylation. yARF3 gene transcription, similar to that of yARF2, was repressed by glucose. As yARF3 was not essential for cell viability and was not required for endoplasmic reticulum to Golgi protein transport, it may provide an opportunity to define an ARF function in another kind of vesicular trafficking.