Characterization and optimization of in vitro assay conditions for (1,3)beta-glucan synthase activity from Aspergillus fumigatus and Candida albicans for enzyme inhibition screening.

(1,3)Beta-D-glucan synthase (E.C.2.4.1.34. UDP-glucose: 1,3-beta-D-glucan 3-beta-glucosyl transferase) catalyzes the polymerization of glucose ([1-3]-beta-linkages) using UDP-glucose as substrate. We have determined optimal in vitro conditions for the assay of (1,3)beta-glucan synthase activity from Aspergillus fumigatus and Candida albicans. These included lysis of cells in the following for C. albicans, 100 mM HEPES, pH 8.0, 10 microM guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), 2 mM ethylenediaminetetraacetic acid (EDTA), disodium salt, 5 mM NaF, 250 mM sucrose, and 10 mM NaH2PO4; and for A. fumigatus, 50 mM HEPES, 10mM EDTA, 750 mM sucrose, 10 mM NaH2PO4, 100 mM cellobiose and 50 microM GTPgammaS. Resulting low-speed supernatants were used as enzyme sources to determine the optimal in vitro assay conditions. We have characterized the resulting enzyme activities and tested the optimized assays with known (1,3)beta-glucan synthase inhibitors including cilofungin, papulacandin, aculeacin A, and echinocandin B. We have used both optimized assays to screen > 1000 extracts of marine macroorganisms and, using bioassay-guided purification, have identified (1,3)beta-glucan synthase inhibitors.

Expression of a plant protein by Neurospora crassa.

Heterologous expression of plant genes may serve as an important alternative for producing plant proteins. We have investigated the ability of the fungus Neurospora crassa to secrete zeamatin, a protein produced by Zea mays. Zeamatin was induced after being fused to glucoamylase, an extracellular hydrolase produced by N. crassa. Glucoamylase induction and other culture parameters were monitored in untransformed N. crassa grown in shaken liquid culture. A DNA plasmid, pGEZ, was constructed by inserting zeamatin-encoding cDNA into an expression cassette containing the promoter, a truncated open reading frame, and the terminator sequence of the N. crassa glucoamylase gene. Zeamatin-encoding cDNA was modified at the N terminus to include a kex-2 protease site, allowing cleavage of the chimeric product in the secretory pathway. Strains containing the chimeric gene construct were grown in liquid culture and induced for glucoamylase and zeamatin production. Zeamatin antibody detected a protein in a Western blot of concentrated culture supernatants that comigrated with authentic zeamatin. Secreted zeamatin was active in inhibiting the growth of Candida albicans in an agar diffusion assay, indicating that zeamatin had been correctly synthesized, processed, and secreted by N. crassa.

Basal nitric oxide expresses endogenous cardioprotection during reperfusion by inhibition of neutrophil-mediated damage after surgical revascularization.

Ischemia-reperfusion damages endothelium and impairs basal production of nitric oxide. Basally released nitric oxide is cardioprotective by its inhibition of neutrophil activities. Loss of endogenous nitric oxide with endothelial injury may occur during two phases: cardioplegic ischemia and reperfusion (aortic declamping). This study tested the hypothesis that inhibition of endogenously released nitric oxide in hearts subjected to regional ischemia, cardioplegic arrest, and reperfusion (1) restricts endogenous cardioprotection and permits neutrophil-mediated damage and (2) expresses damage during the reperfusion phase. L-Nitro-arginine was used to block basal nitric oxide production. In 22 anesthetized dogs, the left anterior descending artery was ligated for 90 minutes followed by 1 hour of arrest with cold multidose (every 20 minutes) blood cardioplegia. Dogs were divided into three groups: the first group received standard unsupplemented blood cardioplegia (group 1, n = 8), in the second group L-nitro-arginine was administered as an additive to blood cardioplegic solution (1 mmol) and as an infusion during reperfusion (34 mg/kg) (group 2, n = 7), and in the third group L-nitro-arginine was administered only at reperfusion (group 3, n = 7). The ligature was released during the second infusion of cardioplegic solution. Infarct size (triphenyltetrazolium chloride) was increased in group 3 (L-nitro-arginine only at reperfusion) compared with that in group 1 (standard blood cardioplegia) (49% +/- 6% vs 34% +/- 2%, respectively), but was not further extended in group 2 (L-nitro-arginine as an additive to blood cardioplegic solution and at reperfusion) (56% +/- 3%, p > 0.05 vs group 3), which suggests primarily a reperfusion process. Polymorphonuclear neutrophil-specific myeloperoxidase activity in the area at risk was elevated comparably in groups 2 and 3 (group 2: 2.9 +/- 0.5 units/gm tissue, p = 0.06 vs group 1; group 3: 3.9 +/- 1.0 units/gm tissue, p < 0.05 vs group 1) compared with that in the standard blood cardioplegia group (1.7 +/- 0.3 units/gm tissue), suggesting polymorphonuclear neutrophil accumulation occurs primarily during reperfusion. Polymorphonuclear neutrophil adherence in ischemic-reperfused left anterior descending artery segments was comparably greater in group 2 (L-nitro-arginine as an additive to blood cardioplegic solution and at reperfusion: 195 +/- 21 polymorphonuclear neutrophils/mm2 of artery, p < 0.05 vs group 1) and group 3 (L-nitro-arginine only at reperfusion: 224 +/- 20 polymorphonuclear neutrophils/mm2 of artery, p < 0.05 vs group 1) relative to that in group 1 (108 +/- 19 polymorphonuclear neutrophils/mm2 of artery). There was no significant adherence to nonischemic circumflex arteries. We conclude that blockade of endogenous nitric oxide augments postischemic injury mediated by polymorphonuclear neutrophils, and this damage is expressed primarily during the reperfusion phase.

Overexpression of myotonic dystrophy kinase in BC3H1 cells induces the skeletal muscle phenotype.

Myotonic muscular dystrophy is an autosomal dominant defect that produces muscle wasting, myotonia, and cardiac conduction abnormalities. The myotonic dystrophy locus codes for a putative serine-threonine protein kinase of unknown function. We report that overexpression of human myotonic dystrophy protein kinase induces the expression of skeletal muscle-specific genes in undifferentiated BC3H1 muscle cells. BC3H1 clones expressing myotonic dystrophy kinase appear equivalent to differentiated cells with respect to expression of myogenin, retinoblastoma tumor supressor gene, M creatine kinase, beta-tropomyosin, and vimentin. In addition, differential display analysis demonstrates that the pattern of gene expression exhibited by myotonic dystrophy kinase-expressing cells is essentially identical to that of differentiated BC3H1 muscle cells. These observations suggest that myotonic dystrophy kinase may function in the myogenic pathway.

Identification, tissue-specific expression, and subcellular localization of the 80- and 71-kDa forms of myotonic dystrophy kinase protein.

The protein product of the myotonic dystrophy (DM) gene is a putative serine-threonine protein kinase (DM kinase). Previous reports have characterized the DM gene product as various 50-62-kDa proteins. The predicted protein size from DM cDNA sequence is 69 kDa. We therefore expressed a full-length recombinant human DM kinase protein and compared its size and expression to heart, cardiac Purkinje fibers, and skeletal muscle from normal and DM subjects. Recombinantly expressed DM kinase and endogenous DM kinase in human heart, displayed two immunoreactive DM kinase proteins with apparent molecular sizes of 71 and 80 kDa, suggesting that these prior reports are incorrect. In cardiac Purkinje fibers the 71-kDa protein was the major form, and in skeletal muscle the 80-kDa protein was the major form. Immunostaining showed DM kinase localized to neuromuscular junctions in skeletal muscle and intercalated discs in heart and Purkinje fibers. DM subjects showed low abundance of DM kinase in heart and skeletal muscle, suggesting haplotype insufficiency as a potential mechanism for disease expression. These studies describe differential expression of two protein forms of DM kinase, which are localized to specialized cellular structures associated with impulse transmission.

Endogenous nitric oxide (NO) protects against ischaemia-reperfusion injury in the rabbit.

OBJECTIVES: Recent studies suggest that nitric oxide (NO) is deleterious in models of shock and hypoxia-reoxygenation However, the role of endogenous NO in ischaemia-reperfusion injury in vivo remains controversial. We tested the hypothesis that blockade of endogenous NO produced during myocardial ischaemia-reperfusion or during reperfusion alone in vivo increases infarct size after coronary occlusion in the rabbit, and conversely, supplementation with L-arginine would reduce infarct size. METHODS: Ketamine-xylazine anaesthetised New Zealand white rabbits were subjected to left coronary artery occlusion for 30 min and reperfusion for 120 min. The rabbits were divided into five groups: (1) saline (VEH); (2) L-nitro arginine (L-NA), a NO-synthase inhibitor, was infused intravenously (15 mg/kg bolus followed by 7.5 mg/kg h-1) before coronary occlusion to block NO synthase activity during ischaemia and reperfusion (IR); (3) L-NA was administered during reperfusion only (R) at the same dose as in the IR group; (4) D-arginine (D-ARG) (25 mg/kg bolus followed by 4 mg/kg min-1), the non-metabolised enantiomer of L-arginine was infused intravenously during reperfusion only; (5) L-arginine (L-ARG) (25 mg/kg bolus followed by 4 mg/kg min-1), the physiological precursor of NO, was infused intravenously during reperfusion only. RESULTS: L-NA infusion in the IR and R groups caused an increase in mean arterial pressure and a decrease in heart rate; however, no significant change in pressure rate product (PRP) occurred immediately after drug infusion. PRP did not change significantly during the experiment across groups except at the end of reperfusion. The area at risk was comparable in all groups, averaging 29(1)%. The infarct size (triphenyltetrazolium chloride) expressed as a percent of area at risk was 27(2)% for the untreated vehicle group. In contrast, L-NA significantly (P < 0.05) increased infarct size in the IR group, 51(2)%; this augmented infarct size persisted when NO synthase activity was blocked during reperfusion only in the R group, 50(2)%. There was no significant (P < 0.05) difference in infarct size between the IR and the R groups. D-ARG-treated group showed a comparable increase in infarct size 48(2)% versus the IR and R groups. However, supplementation of NO with L-arginine (L-ARG) showed no reduction in infarct size, 24(3)%, over vehicle group (VEH). CONCLUSIONS: We conclude that (1) blockade of NO synthase activity with L-NA increases infarct size, (2) this effect was expressed primarily during reperfusion, (3) D-arginine mimicked the infarct augmentation of L-NA, while (4) L-arginine supplementation did not reduce infarct size. These data imply that endogenous NO production exerts a tonic cardioprotective effect on myocardial infarct following coronary reperfusion.

A cost-effective competency program for a rehabilitation unit.

As competency evaluations become the standard for assessing proficient nursing practice, the need for validated, cost-effective, and efficient programs will increase. This article describes a competency assessment program that was implemented for a hospital-based, 15-bed acute rehabilitation unit in Chehalis, WA. The program, initiated and executed by the hospital's staff development department, was efficient in terms of financial costs to the unit and the time required for implementation for all participating staff. Appropriately selected psychomotor and cognitive skills were verified through return demonstration and written examination. The 14 members of the licensed staff successfully completed all aspects of the program.

A high throughput in vitro assay for fungal (1,3)beta-glucan synthase inhibitors.

An in vitro assay for (1,3)beta-glucan synthase activity from the filamentous ascomycete Neurospora crassa, suitable for use as a high throughput screen for enzyme inhibitors is described. Samples were added to 25 microliters reaction mixtures in 96-well V-bottom microtiter plates and plates incubated at 22 degrees C. Reactions were terminated by the addition of TCA and the contents transferred to a Milliblot D apparatus containing Inotech 201-A glass fiber filters. Filters were washed to remove unincorporated substrate and the amount of 14C-labeled (1,3)beta-glucan formed by each reaction was quantitated using a Phosphorlmager. As little as 50 ng of an inhibitor with a Ki of 4 microM was detected by this assay. This assay is rapid and cost effective, permitting its use as a screen to detect compounds that inhibit (1,3)beta-glucan synthase activity.

Angiotensin-converting enzyme DD genotype in patients with ischaemic or idiopathic dilated cardiomyopathy.

Polymorphism in the angiotensin-converting enzyme (ACE) gene has been shown to correlate with circulating ACE concentrations, and also to be an independent risk factor for the development of myocardial infarction, particularly in men thought to be at low risk by standard criteria. We determined the genotypes of individuals with end-stage heart failure due to either ischaemic dilated cardiomyopathy (102) or idiopathic dilated cardiomyopathy (112) and compared these to organ donors with normally functioning hearts (79). Genotypes were determined by the polymerase chain reaction with oligonucleotide primers flanking the polymorphic region in intron 16 of the ACE gene to amplify template DNA isolated from patients. Compared with the DD frequency in the control population, the frequency of the ACE DD genotype was 48% higher in individuals with idiopathic dilated cardiomyopathy (p = 0.008) and 63% higher in subjects with ischaemic cardiomyopathy (p = 0.008), suggesting that an ACE gene variant may contribute to the pathogenesis of both types of cardiomyopathy.

In vitro inhibition of stable 1,3-beta-D-glucan synthase activity from Neurospora crassa.

Glucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 microM GTP, 1 mM DTT, 10 mM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4 degrees C, and a Km(app) of 1.66 +/- 0.28 mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-beta-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki(app) of each inhibitor determined. It seems likely that this stable preparation of glucan synthase activity may be useful for in vitro enzyme screens for new glucan synthase inhibitors.

Cilofungin (LY121019) inhibits Candida albicans (1-3)-beta-D-glucan synthase activity.

Cilofungin (LY121019) inhibited Candida albicans growth and activity of (1-3)-beta-glucan synthase, for which it was a noncompetitive inhibitor with a Ki-app of 2.5 microM. Cilofungin had no effect on chitin synthase activity. Based on these and other data, it seems likely that cilofungin inhibits fungal growth by inhibiting (1-3)-beta-glucan synthase activity.