Emergence of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> as human fungal pathogen and molecular assay for future medical diagnosis

Despite the great importance of Aureobasidium pullulans in biotechnology, the fungus had emerged as an opportunistic human pathogen, especially among immunocompromised patients. Clinical detection of this rare human fungal pathogen presently relies on morphology diagnosis which may be misleading. Thus, a sensitive and accurate quantitative molecular assay for A. pullulans remains lacking. In this study, we presented the microscopy observations of A. pullulans that reveals the phenotypic plasticity of the fungus. A. pullulans-specific primers and molecular beacon probes were designed based on the fungal 18S ribosomal RNA (rRNA) gene. Comparison of two probes with varied quencher chemistry, namely BHQ-1 and Tamra, revealed high amplification efficiency of 104% and 108%, respectively. The optimized quantitative real-time PCR (qPCR) assays could detect and quantify up to 1 pg concentration of A. pullulans DNA. Both assays displayed satisfactory performance parameters at fast thermal cycling mode. The molecular assay has great potential as a molecular diagnosis tool for early detection of fungal infection caused by A. pullulans, which merits future study in clinical diagnosis.     

Hexokinase isoenzymes in diabetes

Hexokinase is present in the tissues in four isoenzymic forms. Cerebral tissue contains predominantly Type I hexokinase which is believed to be insulin-insensitive. In cerebral tissue about 60 to 70% of the hexokinase is bound to the particulate fraction. The changes in the distribution of hexokinase Type I and Type II together with the bound and free hexokinase have been studied in control, diabetic and diabetic animals treated with insulin. The results indicate that the presence of insulin is essential for the normal binding of the hexokinase to the particulate fraction. In heart tissue, Type II hexokinase bound to the pellet shows a significant decrease in diabetes, which is reversed on insulin administration.     

Less label, more free: approaches in label-free quantitative mass spectrometry

In this review we examine techniques, software, and statistical analyses used in label-free quantitative proteomics studies for area under the curve and spectral counting approaches. Recent advances in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further experiments to validate results from label-free quantitation. Limitations of label-free approaches are considered, label-free approaches are compared with labelling techniques, and forward-looking applications for label-free quantitative data are presented. We conclude that label-free quantitative proteomics is a reliable, versatile, and cost-effective alternative to labelled quantitation.     

Genetic analysis of temperature-sensitive male sterilty in rice

The present study of genetic analysis is an attempt to precisely characterize diverse temperature-sensitive genic male-sterile (TGMS) lines so as to explore the possibilities of utilizing the most promising in large-scale hybrid seed production. Genetical studies revealed that the TGMS segregants derived from crosses involving TGMS lines ID24 and SA2 expressed differential fertility levels at low-temperature conditions. A majority of these progenies expressed transgressive segregation towards either sterility of fertility, causing instability of sterility and low reversibilty of fertility which may be due to large numbers of single-locus QTLs and their epistatic interactions. We identified two putative genes imparting temperature-sensitive male sterility after observing crosses involving diverse TGMS sources. To identify suitable molecular markers closely linked to the trait we used RAPD, AFLP and microsatellites which generated polymorphism through bulked segregant analysis. AFLP analysis using a smaller genome kit resulted in enormous polymorphism, out of which the combination EAA/MCAG amplified a 330-bp fragment, which closely segregated with the gene at a distance of 5.3 cM. This fragment was eluted for cloning and from the sequence a STS primer (TS200) was developed which produced a dominant polymorphism specific to TGMS. The microsatellite RM257, located earlier on chromosome 9, was linked with the TGMS trait in SA2 at a distance of 6.2 cM. RM257 produced a codominant polymorphism with 145-bp (sterile) and 132-bp (fertile) products. Both individually and collectively, the markers TS200 and RM257 located on either side of the TGMS locus are very useful for marker-assisted selection. Received: 10 April 1999 / Accepted: 29 July 1999     

Inhibition of Na+, K+-ATPase activates swelling-induced taurine efflux in a human neuroblastoma cell line

The Na⁺ pump (Na⁺, K⁺-ATPase) has been implicated in the regulation of many cellular functions, including cell volume regulation. The effects of inhibiting Na⁺ pump activity on cell volume and taurine efflux were evaluated in the human neuroblastoma cell line CHP-100. Cell volume changes monitored with the Coulter Multisizer technique and confocal microscopy showed that neuroblastoma cells exposed to ouabain swelled by 22 ± 4% (n = 5). The rapid cell swelling was followed by regulatory volume decrease (RVD). In cells treated with ouabain, ¹⁴C-taurine efflux increased by 183 ± 11% compared with controls. However, cells exposed simultaneously to ouabain and hypoosmotic solution resulted in a ¹⁴C-taurine efflux of 207 ± 18%. Western blot and immunofluorescence microscopy with specific monoclonal antibodies for the catalytic α isoforms of Na⁺, K⁺-ATPase demonstrated high levels of the ubiquitously expressed α1 and the neuronal-specific α3. Ouabain-binding data showed that CHP-100 cells express ∼3 × 10⁵ pump units/cell. The present data indicate that efflux of taurine may be involved during volume recovery subsequent to blockade of Na⁺, K⁺-ATPase in CHP-100 cells. J. Cell. Physiol. 174:145–153, 1998. © 1998 Wiley-Liss, Inc.     

Guanine deaminase inhibitor from rat liver. Isolation and characterization

1. An inhibitor of cytoplasmic guanine deaminase of rat liver was isolated from liver ;heavy mitochondrial' fraction after freezing and thawing and treatment with Triton X-100. 2. Submitochondrial fractionation revealed that the inhibitor was localized in the outer-membrane fraction. 3. The method of purification of inhibitor, involving precipitation with (NH(4))(2)SO(4) and chromatography on DEAE-cellulose, its precipitability by trichloroacetic acid and the pattern of absorption in the u.v. indicated that the inhibitor was a protein. In confirmation, tryptic digestion of the isolated material resulted in destruction of the inhibitor activity. The inhibitor was stable to acid, but labile to heat. 4. The isolated inhibitor required phosphatidylcholine (lecithin) for activity. Phosphatidylcholine also partially protected the inhibitor against heat inactivation. 5. When detergent treatment was omitted, the inhibitor activity of frozen mitochondria was precipitated by (NH(4))(2)SO(4) in a fully active form without supplementation with phosphatidylcholine, indicating that Triton X-100 ruptured the linkage between inhibitor and lipid. 6. A reconstituted sample of inhibitor-phosphatidylcholine complex was precipitated in a fully active form by dialysis against 2-mercaptoethanol, but treatment of the precipitate with NaCl yielded an extract which was inactive unless supplemented with fresh phosphatidylcholine. 7. We interpret the results as evidence that the inhibitor was present in vivo as a lipoprotein and that once the complex was dissociated by the action of detergent and the protein precipitated, there was an absolute need for exogenous phosphatidylcholine for its activity. The manner in which inhibitor associated with the outer membrane of rat liver mitochondria might regulate the activity of the enzyme in the supernatant has been suggested.