Serotypes and mating types of clinical strains ofCryptococcus neoformans isolated in Taiwan

Twenty-one strains ofCryptococcus neoformans isolated from patients in Taiwan were characterized for serotypes and mating types. Slide agglutination test was performed with 8 factor-specific sera (Iatron Company, Japan) to determine the serotypes. Wheat bran agar (WBA) and malt extract agar (MEA, Wickerham) media were used for the mating tests. Twenty of the isolates were of serotype A, and one was serotype B. Except for 2 strains of serotype A, all of the serotype A strains mated withFilobasidiella neoformans var.neoformans, mating type a. The only serotype B strain mated withF. neoformans var.bacillispora mating type a in MEA medium. These data revealed the low prevalence (1/21; 4.8%) ofC. neoformans var.gattii in Taiwan, a subtropically located island.     

In vitro and in vivo antifungal activities of liposomal amphotericin B,and amphotericin B lipid complex

The in vitro and in vivo antifungal activities of liposomal amphotericin B (L-AMPH) and amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, were compared with those of conventional amphotericin B (Fungizone®, AMPH). The acute intravenous toxicity was markedly lower in BALB/c mice; 50% lethal doses (LD_50s) were 2.75 mg/kg in AMPH, 32.9 mg/kg in L-AMPH and >75 mg/kg in ABLC. In vitro antifungal activities againstCandida albicans, C. parapsilosis, C. tropicalis, C. glabrata, andC. krusei were evaluated by the agar plate dilution method. The activities were unchanged againstC. albicans, but MICs increased more than four fold in 18 of the 20 strains other thanC. albicans in L-AMPH and in 9 of the 20 in ABLC. L-AMPH and ABLC were as efficacious as AMPH in the treatment of mice infected withC. albicans, and at a dose of 0.5 and 1.0 mg/kg of body weight, ABLC was more efficacious on survival. A ten-times larger dose (10 mg/kg) of L-AMPH and ABLC was administered to mice with 100% survival, suggesting improved tolerability as compared to amphotericin B.     

The action of structural analogues of ethidium bromide on the mitochondrial genome of yeast

We have studied the effects on the yeast mitochondrial genome of four analogues of ethidium bromide, in which the phenyl moiety has been replaced by linear alkyl chains of lengths varying from seven to fifteen carbon atoms. These analogues are more efficient than ethidium bromide in inducing petite mutants inSaccharomyces cerevisiae. The drugs also cause a loss of mtDNA from the cellsin vivo; however these analogues are in fact less effective inhibitors of mitochondrial DNA replicationper se, as shown by directin vitro studies. It is concluded that these analogues are more efficient than ethidium bromide in causing the fragmentation of mitochondrial DNA inS. cerevisiae.     

Quantitative analysis of collagen gel contractile forces generated by dermal fibroblasts and the relationship to cell morphology

The force generated in granulation tissue during wound contraction is thought to be cell mediated; however, it is unclear whether contractile forces are generated by fibroblast locomotion or contraction of myofibroblasts. To help clarify this question the force of this contraction can now be determined accurately in a human dermal fibroblast collagen lattice system using a novel instrument known as a Culture Force Monitor. Three distinct phases of contraction of such collagen gels could be identified over the first 24 hours. Most of the force generated by human dermal fibroblasts was produced during the first stage in parallel with cell attachment and associated changes in cell shape, and the appearance of cell processes. During this initial 24 hours no evidence could be found for the presence of myofibroblasts, but stereoscopic and electron microscopic analysis at a range of time points indicated that migratory fibroblasts were present in the system. Comparison of the contraction profiles of cells extracted from other tissues (tendon and articular cartilage), and extracted by different means from the same tissue specimen, indicated that different populations of fibroblasts can be distinguished on the basis of their pattern of contractions. It would seem that most of the force generated in this model is a result of fibroblast attachment and movement within the collagen lattice. Furthermore, different groups of fibroblasts, even within the same tissue, may vary in their contraction (hence locomotory) activity. © 1996 Wiley-Liss, Inc.     

Plasticizers increase adhesion of the deteriogenic fungus Aureobasidium pullulans to polyvinyl chloride.

Initial adhesion of fungi to plasticized polyvinyl chloride (pPVC) may determine subsequent colonization and biodeterioration processes. The deteriogenic fungus Aureobasidium pullulans was used to investigate the physicochemical nature of adhesion to both unplasticized PVC (uPVC) and pPVC containing the plasticizers dioctyl phthalate (DOP) and dioctyl adipate (DOA). A quantitative adhesion assay using image analysis identified fundamental differences in the mechanism of adhesion of A. pullulans blastospores to these substrata. Adhesion to pPVC was greater than that to uPVC by a maximum of 280% after a 4-h incubation with 10(8) blastospores ml(-1). That plasticizers enhance adhesion to PVC was confirmed by incorporating a dispersion of both DOA and DOP into the blastospore suspension. Adhesion to uPVC was increased by up to 308% in the presence of the dispersed plasticizers. Hydrophobic interactions were found to dominate adhesion to uPVC because (i) a strong positive correlation was observed between substratum hydrophobicity (measured by using a dynamic contact angle analyzer) and adhesion to a range of unplasticized polymers including uPVC, and (ii) neither the pH nor the electrolyte concentration of the suspension buffer, both of which influence electrostatic interactions, affected adhesion to uPVC. In contrast, adhesion to pPVC is principally controlled by electrostatic interactions. Enhanced adhesion to pPVC occurred despite a relative reduction of 13 degrees in the water contact angle of pPVC compared to that of uPVC. Furthermore, adhesion to pPVC was strongly dependent on both the pH and electrolyte concentration of the suspension medium, reaching maximum levels at pH 8 and with an electrolyte concentration of 10 mM NaCl. Plasticization with DOP and DOA therefore increases adhesion of A. pullulans blastospores to pPVC through an interaction mediated by electrostatic forces.     

Analysis of Human Proteome Organization Plasma Proteome Project (HUPO PPP) reference specimens using surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry: multi-institution correlation of spectra and identification of biomarkers

We report on a multicenter analysis of HUPO reference specimens using SELDI-TOF MS. Eight sites submitted data obtained from serum and plasma reference specimen analysis. Spectra from five sites passed preliminary quality assurance tests and were subjected to further analysis. Intralaboratory CVs varied from 15 to 43%. A correlation coefficient matrix generated using data from these five sites demonstrated high level of correlation, with values >0.7 on 37 of 42 spectra. More than 50 peaks were differentially present among the various sample types, as observed on three chip surfaces. Additionally, peaks at approximately 9200 and approximately 15,950 m/z were present only in select reference specimens. Chromatographic fractionation using anion-exchange, membrane cutoff, and reverse phase chromatography, was employed for protein purification of the approximately 9200 m/z peak. It was identified as the haptoglobin alpha subunit after peptide mass fingerprinting and high-resolution MS/MS analysis. The differential expression of this protein was confirmed by Western blot analysis. These pilot studies demonstrate the potential of the SELDI platform for reproducible and consistent analysis of serum/plasma across multiple sites and also for targeted biomarker discovery and protein identification. This approach could be exploited for population-based studies in all phases of the HUPO PPP.     

Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/phosphoinositide 3-kinase/Akt-dependent pathway and is essential for the enhanced contractile phenotype of fibrotic fibroblasts

The endothelins are a family of endothelium-derived peptides that possess a variety of functions, including vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and promotes myofibroblast contraction and migration, hence contributing to matrix remodeling during tissue repair. Here, we show that addition of ET-1 to normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including alpha-smooth muscle actin (alpha-SMA), ezrin, moesin, and paxillin. We confirm that ET-1 enhances the ability of lung fibroblasts to contract extracellular matrix, a function essential for tissue repair, through induction of de novo protein synthesis. Blockade of the Akt/phosphoinositide 3-kinase (PI3-kinase) pathway with LY294002 and wortmannin prevents the ability of ET-1 to induce alpha-SMA, ezrin, paxillin, and moesin and to promote matrix contraction. Dominant negative rac and Akt blocked the ability of ET-1 to promote formation of alpha-SMA stress fibers. Using specific ET-1 receptor inhibitors, we show that ET-1 induces collagen matrix contraction through the ETA, but not the ETB, receptor. Relative to normal pulmonary fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease systemic sclerosis (scleroderma) show enhanced ET-1 expression and binding. Systemic sclerosis lung fibroblasts show increased ability to contract a collagen matrix and elevated expression of the procontractile proteins alpha-SMA, ezrin, paxillin, and moesin, which are greatly reduced by antagonizing endogenous ET-1 signaling. Thus, blocking ET-1 or the PI3-kinase/Akt cascades might be beneficial in reducing scar formation in pulmonary fibrosis.     

Reduction of myocardial infarct size by doxycycline: A role for plasmin inhibition

Myocardial ischemia-reperfusion (I/R) is associated with the activation of matrix metalloproteinases (MMPs) and serine proteases. We hypothesized that activation of MMPs and the serine protease plasmin contribute to early cardiac myocyte death following I/R and that broad-spectrum protease inhibition with doxycycline (DOX) preserves myocyte viability. Rats treated daily with or without DOX beginning 48 h prior to experimentation were subjected to 30 min of coronary occlusion and 2 days of reperfusion. DOX pre-treatment reduced infarct size by 37%. DOX attenuated increases in MMP-9 and plasmin levels as determined by gelatin zymography and immunoblot, respectively. Neutrophil extravasation was unaltered by DOX as assessed by myeloperoxidase (MPO) activity. To examine the contribution of MMP-9 and plasmin to myocyte injury, cultures of neonatal rat ventricular myocytes (NRVMs) were treated for 48 h with 83 kDa MMP-9 or plasminogen in the presence or absence of DOX. MMP-9 treatment did not affect myocyte viability. Plasminogen treatment led to increased plasmin activity, resulting in loss of ₁-integrin, NRVM detachment and apoptosis. DOX co-treatment inhibited plasmin activity and preserved NRVM attachment, whereas co-treatment with the broad-spectrum MMP inhibitor GM6001 had no effect. These results indicate that plasmin causes disruption of myocyte attachment and viability independently of MMP activation in vitro and that inhibition of plasmin by DOX may reduce I/R-induced myocyte death in vivo through the inhibition of plasmin. (Mol Cell Biochem 270: 1–11, 2005)     

Cell proliferation rates in an artificial tissue-engineered environment

Worldwide, and particularly in Europe, Japan and the USA, cardiovascular disease is a major killer. It can be treated using tissue or organ transplant surgery, but donor organs may be scarce. Tissue engineering is the integration of engineering principles and biology to produce satisfactory synthetic replacement body parts, using viable cells in a suitable matrix, for regenerative medicine. The aim of this study was to measure and compare cell proliferation kinetics after different time intervals of myofibroblasts in a synthetic matrix, thus to be able to deduce the period that a transplanted-cell population can be expected to survive in a tissue-engineered environment. Porcine aortic wall cells were grown in a porous sponge scaffold, that later could be fashioned into aortic or heart valve substitutes. Freshly acquired cells were seeded on identical sponges and were grown under normal culture conditions for a period of 4 weeks. Seeding concentration was a million cells per sponge. Cells progressively populated the sponges, both covering the surface and infiltrating the depth of the matrix, via sponge pores. Samples were taken at 1 week and at 4 weeks, and the rate of cell proliferation was determined by the metaphase arrest technique. Specimens were also taken for light and electron microscopy to determine whether these transplanted cells were capable of synthesizing their own extracellular matrix.