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A J Morris

Publications and source records attributed to A J Morris.

At least 145 records · Page 8Linked to original sources
PubMed1996

Activated phosphatidylinositol 3-kinase is sufficient to mediate actin rearrangement and GLUT4 translocation in 3T3-L1 adipocytes.

Insulin stimulation of 3T3-L1 adipocytes causes rapid translocation of actin and the GLUT4 glucose transporter to the plasma membrane. Both processes depend on the activity of phosphatidylinositol 3-kinase. Using single cell microinjection, we have transiently expressed a constitutively activated mutant of phosphatidylinositol 3-kinase, p110*, in 3T3-L1 adipocytes. Fluorescent detection of GLUT4 protein and actin within these cells demonstrates that expression of p110* is sufficient to cause translocation of GLUT4 to the plasma membrane and the formation of actin membrane ruffles. These effects are inhibited by wortmannin in the p110*-expressing cells, indicating that the phosphatidylinositol 3-kinase activity of the protein is required. Overexpression of an identical protein containing a point mutation in the kinase domain, p110*Deltakin, was incapable of mediating either action, confirming that neither the microinjection process nor a nonspecific effect of the protein was responsible for the observed effects. These data suggest that although insulin is capable of inducing numerous signaling pathways, the isolated activation of phosphatidylinositol 3-kinase can initiate the signaling cascade leading to both actin rearrangement and GLUT4 translocation in the absence of insulin stimulation.

3T3 Cells↗
PubMed1996

The molecular nature of the F-actin binding activity of aldolase revealed with site-directed mutants.

We used site-directed mutagenesis of rabbit muscle aldolase, falling ball viscometry, co-sedimentation binding assays, and negative stain electron microscopy, to identify specific residues involved in the aldolase-actin interaction. Three mutants, R42A (Arg --> Ala), K107A (Lys --> Ala), and R148A (Arg --> Ala), had minimal actin binding activity relative to wild type (wt) aldolase, and one mutant, K229A (Lys --> Ala), had intermediate actin binding activity. A mutant with approximately 4,000-fold reduced catalytic activity, D33S (Asp --> Ser), had normal actin binding activity. The aldolase substrates and product, fructose 1,6-bisphosphate, fructose 1-phosphate, and dihydroxyacetone phosphate, reversed the gelling of wt aldolase and F-actin, consistent with at least partial overlap of catalytic and actin-binding sites on aldolase. Molecular modeling reveals that the actin-binding residues we have identified are clustered in or around the catalytic pocket of the molecule. These data confirm that the aldolase-actin interaction is due to specific binding, and they suggest that electrostatic interactions between specific residues, rather than net charge, mediate this interaction. Low concentration of wt and D33S aldolase caused formation of high viscosity actin gel networks, while high concentrations of wt and D33S aldolase resulted in solation of the gel by bundling actin filaments, consistent with a potential role for this enzyme in the regulation of cytoplasmic structure.

Actins↗
PubMed1996

The changing face of candidemia: emergence of non-Candida albicans species and antifungal resistance.

OBJECTIVES: To assess the changing epidemiology of candidemia in the 1990s, to evaluate the clinical implications for the presence of non-Candida albicans in blood, and to evaluate the presence of antifungal resistance in relation to prior antifungal administration. DESIGN: Multicenter prospective observational study of patients with positive blood cultures for Candida species or Torulopsis glabrata. SETTING: Four tertiary care medical centers. RESULTS: Four hundred twenty-seven consecutive patients were enrolled. The frequency of candidemia due to non-C. albicans species significantly increased in each hospital throughout the 3.5-year study period (P = 0.01). Thirteen percent of candidemias occurred in patients who were already receiving systemic antifungal agents. Candidemias developing while receiving antifungal therapy were more likely caused by non-C. albicans species than by C. albicans species (P = 0.0005). C. parapsilosis and C. krusei were more commonly seen with prior fluconazole therapy, whereas T. glabrata was more commonly seen with prior amphotericin B therapy. Candida species isolated during episodes of breakthrough candidemia exhibited a significantly higher MIC to the antifungal agent being administered (P < 0.001). CONCLUSION: In this large scale study, the non-C. albicans species, especially T. glabrata, emerged as important and frequent pathogens causing fungemia. This finding has major clinical implications given the higher complication and mortality rate associated with the non-C. albicans species. The change in the pattern of candidemia might be partly attributed to the increase in number of immunocompromised hosts and the widespread use of prophylactic or empiric antifungal therapy. This is an ominous sign given the in vitro resistance of the non-C. albicans species to currently available antifungal agents.

Amphotericin B↗
PubMed1996

Push enteroscopy and heater probe therapy for small bowel bleeding.

BACKGROUND: Blood loss from the small bowel is a significant cause of obscure gastrointestinal bleeding. Small bowel angiodysplasia may be the source of bleeding in 30% to 40% of patients with this problem. In other areas of the bowel, angiodysplasia has been effectively treated by endoscopic methods. METHODS: We used a 1.7 meter push enteroscope and heater probe ablation to examine and treat 11 transfusion-dependent patients with significant bleeding from small bowel angiodysplasia. Patients had push enteroscopy to target all lesions identified and had follow-up hemoglobin and fecal occult blood tests for a minimum of 6 months after final enteroscopy. RESULTS: There were a median of 2 (range 1 to 7) small bowel lesions per patient. Patients required a median of 1 (range 1 to 5) examination to treat lesions identified at enteroscopy. Following therapy, hemoglobin levels rose significantly from a median of 8.5 (range 5.3 to 10.6) gm/dL) to a median of 13.5 (range 7.6 to 16.5) gm/dL (p < 0.01 Wilcoxon matched pair signed rank test). CONCLUSION: Push enteroscopy and heater probe ablation offer potential therapy for bleeding from small bowel angiodysplasia and result in reduction of blood loss and transfusion requirements along with a significant improvement in levels of hemoglobin.

Adult↗
PubMed1996

Candida infection of the arteriovenous fistula used for hemodialysis.

Arteriovenous fistula infection is an exceedingly rare manifestation of Candida infection, with only one case previously reported in the English literature. This report describes three cases of Candida arteriovenous fistula infection seen in a prospective study and reviews the previously reported case. All cases involved the polytetrafluoroethylene graft. The presentations can be either acute or subacute in onset, with drainage at the fistula site as the most common sign. The diagnosis of Candida arteriovenous fistula infection relies on the isolation of the organism from an intraoperative culture of the fistula. Candida was isolated from blood in three of four cases, and wound drainage in two of three cases. Antifungal therapy should be given, but complete removal of the arteriovenous fistula may be necessary for long-term cure.

Aged↗
PubMed1996

A lysine to arginine substitution at position 146 of rabbit aldolase A changes the rate-determining step to Schiff base formation.

Lys146 of rabbit aldolase A [D-fructose-1,6-bis(phosphate): D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13] was changed to arginine by site-directed mutagenesis. The kcat of the resulting mutant protein, K146R, was 500 times slower than wild-type in steady-state kinetic assays for both cleavage and condensation of fructose-1,6-bis(phosphate), while the K(m) for this substrate was unchanged. Analysis of the rate of formation of catalytic intermediates showed K146R was significantly different from the wild-type enzyme and other enzymes mutated at this site. Single-turnover experiments using acid precipitation to trap the Schiff base intermediate on the wild-type enzyme failed to show a build-up of this intermediate on K146R. However, K146R retained the ability to form the Schiff base intermediate as shown by the significant amounts of Schiff base intermediate trapped with NaBH4. In the single-turnover experiments it appeared that the Schiff base intermediate was converted to products more rapidly than it was produced. This suggested a maximal rate of Schiff base formation of 0.022 s-1, which was close to the value of kcat for this enzyme. This observation is strikingly different from the wild-type enzyme in which Schiff base formation is > 100 times faster than kcat. For K146R it appears that steps up to and including Schiff base formation are rate limiting for the catalytic reaction. The carbanion intermediate derived from either substrate or product, and the equilibrium concentrations of covalent enzyme-substrate intermediates, were much lower on K146R than on the wild-type enzyme. The greater bulk of the guanidino moiety may destabilize the covalent enzyme-substrate intermediates, thereby slowing the rate of Schiff base formation such that it becomes rate limiting. The K146R mutant enzyme is significantly more active than other enzymes mutated at this site, perhaps because it maintains a positively charged group at an essential position in the active site or perhaps the Arg functionally substitutes as a general acid/base catalyst in both Schiff base formation and in subsequent abstraction of the C4-hydroxyl proton.

Animals↗
PubMed1996

Push enteroscopy in the investigation of small-intestinal disease.

We report our experience with small-bowel push enteroscopy in 50 patients. The indications for push enteroscopy were: anaemia/occult gastrointestinal bleeding (22 patients); overt gastrointestinal bleeding (17 patients); abnormal small-bowel radiology (8 patients) and miscellaneous (3 patients). In those with undiagnosed gastrointestinal bleeding/anaemia, abnormalities were detected in 24/39 patients (62%): small bowel arteriovenous malformations (AVMs) were detected in 19 (49%), and five (13%) had lesions in the upper gastrointestinal tract. Seventeen patients had heater-probe ablation therapy of vascular lesions: nine patients had small-intestinal lesions, four patients gastric lesions, and four patients combined gastric and small-intestinal lesions. In those with abnormal small-bowel radiology, abnormalities were detected in 6/8 patients. We conclude that (i) push enteroscopy can establish a diagnosis in a high proportion of patients with gastrointestinal bleeding; (ii) heater-probe ablation therapy of vascular lesions can be performed routinely at the time of enteroscopy; (iii) a significant proportion of patients (9/50) referred for enteroscopy with undiagnosed gastrointestinal bleeding have lesions in the stomach/proximal duodenum missed at diagnostic endoscopy. Push enteroscopy is a valuable diagnostic and therapeutic endoscopic procedure.

Adult↗
PubMed1996

Cost and time savings following introduction of rejection criteria for clinical specimens.

We have evaluated the yield of several tests and have instituted specimen rejection criteria to reduce costs and save time. For a 12-month period, we recorded the reduction of these tests and calculated the resultant cost and time savings. Seven changes were analyzed: not performing fungal or mycobacterial (acid-fast bacillus) cultures on cerebrospinal fluid (CSF) specimens from patients without known immunosuppression when chemistry and cell count are normal; not performing routine stool culture or ovum and parasite examination on specimens from patients in the hospital for > 3 days; not culturing endotracheal suction aspirates when no organisms or > 10 squamous epithelial cells are present; discontinuing broth cultures on all specimens except for tissue, continuous ambulatory peritoneal dialysis fluid, and CSF from patients with shunts; and eliminating bacterial antigen tests. For each test, the number not performed (n), reagent savings, and technologist time saved, respectively, were as follows: CSF fungal culture, 267, $999, and 67 h; CSF acid-fast bacillus culture, 275, $1,662, and 124 h; stool cultures, 320, $2,991, and 98 h; ovum and parasite examinations, 216, $525, and 108 h; endotracheal suction aspirate cultures, 1,505, $4,447, and 306 h; broth cultures, 5,218, $4,931, and 80 h; and bacterial antigen tests, 2,598, $2,293, and 299 h. Overall, 5,181 tests were rejected and 5,218 broth cultures were omitted. Achievable savings were $28,000 in reagent costs and 1,082 h of technologist time. In conclusion, rejecting specimens of proven low yield saves reagent costs and, more importantly, saves technologist time. This time can be spent on specimens having greater diagnostic utility.

Antigens, Bacterial↗
PubMed1996

Duration of incubation of fungal cultures.

To determine the optimum duration of incubation for recovery of fungi, the results of 2,173 consecutive clinical cultures were reviewed. Overall, 94% of fungal isolates were detected by day 7 and 98% were detected by day 14. Yeasts were usually (98%) detected within the first week of incubation. Recovery of molds required more time, but 81% were detected by day 7 and more than 96% were detected by day 14.

Cross Infection↗
PubMed1996

Contemporary testing for enteric pathogens: the potential for cost, time, and health care savings.

We sent a questionnaire to 79 clinical microbiology laboratories seeking information on contemporary practices when investigating for bacterial and protozoan enteric pathogens. Data from the 67 respondents (response rate of 85%) showed that a minority of laboratories (40% for stool culture and 45% for ova and parasite [O&P] examinations) had restrictions for testing in place and that fewer laboratories (24% for stool culture and 19% for O&P examinations) rejected specimens from patients who had been in the hospital for > 3 days. Using two estimates, 15 and 40%, for the proportion of all specimens received from patients in the hospital for > 3 days, we calculated savings for the average hospital in this survey. Reagent savings of $4,000 to $10,000 and time savings of 274 to 731 h per year might have been realized. Moreover, between $26,000 and $71,000 in patient charges could have been prevented. On the basis of this survey, wider application of rejection criteria when testing for enteric pathogens appears possible. If implemented, savings to the nation's health care system could be between $27 and $73 million a year.

Animals↗
PubMed1995

Phospholipase D signaling is essential for meiosis.

Phospholipid metabolism plays an important role in cellular regulation by generating second messengers for signal transduction. Many stimuli activate a phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine, producing phosphatidic acid and choline. Here we report that the yeast SP014 gene, which is essential for meiosis [Honigberg, S. M., Conicella, C. & Esposito, R. E. (1992) Genetics 130, 703-716], encodes a phospholipase D. SP014 RNA and protein activity are induced during late meiotic prophase, and the enzyme has properties similar to mammalian phosphatidylinositol 4,5-bisphosphate-regulated phospholipase D. Characterization of an unusual allele of SP014 defines regions of the protein important for enzyme catalysis and regulation. These results implicate phospholipase D signaling in regulating cellular differentiation.

Alleles↗
PubMed1995

Human ADP-ribosylation factor-activated phosphatidylcholine-specific phospholipase D defines a new and highly conserved gene family.

Activation of phosphatidylcholine-specific phospholipase D (PLD) has been implicated as a critical step in numerous cellular pathways, including signal transduction, membrane trafficking, and the regulation of mitosis. We report here the identification of the first human PLD cDNA, which defines a new and highly conserved gene family. Characterization of recombinant human PLD1 reveals that it is membrane-associated, selective for phosphatidylcholine, stimulated by phosphatidylinositol 4,5-bisphosphate, activated by the monomeric G-protein ADP-ribosylation factor-1, and inhibited by oleate. PLD1 likely encodes the gene product responsible for the most widely studied endogenous PLD activity.

ADP-Ribosylation Factors↗
PubMed1995

The pleckstrin homology domain of phospholipase C-delta 1 binds with high affinity to phosphatidylinositol 4,5-bisphosphate in bilayer membranes.

The pleckstrin homology (PH) domain of phospholipase C-delta 1 (PLC-delta 1) binds to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in phospholipid membranes with an affinity (Ka approximately 10(6) M-1) and specificity comparable to those of the native enzyme. PLC-delta 1 and its PH domain also bind inositol 1,4,5-trisphosphate, the polar head group of PI(4,5)P2, with comparable affinity and approximately 1:1 stoichiometry. A peptide corresponding to amino acids 30-43 of the PLC-delta 1 PH domain contains several basic residues predicted to bind PI(4,5)P2, but binds weakly and with little specificity for PI(4,5)P2; hence the tertiary structure of the isolated PH domain is required for high affinity PI(4,5)P2 binding. Our PI-(4,5)P2 binding results support the hypothesis that the intact PH domain, serving as a specific tether, directs PLC-delta 1 to membranes enriched in PI(4,5)P2 and permits the active site, located elsewhere in the protein, to hydrolyze multiple substrate molecules before this enzyme dissociates from the membrane surface.

Amino Acid Sequence↗
PubMed1995

Insulin-stimulated GLUT4 translocation is mediated by a divergent intracellular signaling pathway.

Insulin stimulates glucose transport largely by mediating translocation of the insulin-sensitive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane. Using single cell microinjection of 3T3-L1 adipocytes, coupled with immunofluorescence detection of GLUT4 proteins, we have determined that inhibition of endogenous p21ras or injection of oncogenic p21ras has no effect on insulin-stimulated GLUT4 translocation. On the other hand, microinjection of anti-phosphotyrosine antibodies or inhibition of endogenous phosphatidylinositol 3-kinase by microinjection of a GST-p85 SH2 fusion protein markedly inhibits this biologic effect of insulin. These data suggest that the p21ras/mitogen-activated protein kinase pathway is not involved in this metabolic effect of insulin, whereas tyrosine phosphorylation and stimulation of phosphatidylinositol 3-kinase activity are critical components of this signaling pathway.

1-Methyl-3-isobutylxanthine↗