Determining the optimal method for proteinuria detection in chronic spinal cord injury.

STUDY DESIGN: A retrospective analysis. OBJECTIVES: The objective of this study is to determine whether dipstick protein analysis (DSP) or random urine protein:creatinine ratios (UPC) are accurate in predicting clinical proteinuria in the chronic spinal cord injury (SCI) population. METHODS: A retrospective analysis was performed in 219 veterans with SCI, comparing DSP and 24-h urine protein excretion. Sensitivity, specificity, predictive values (PV) and receiver-operator characteristic (ROC) curves of DSP in predicting clinical proteinuria were calculated with and without correction for specific gravity (SG). A prospective study was also performed in 62 SCI patients, comparing the UPC and 24-h urines. Sensitivity, specificity, PV and ROC curves of UPC in predicting clinical proteinuria were calculated. RESULTS: Any level of positive DSP had high specificity, but low sensitivity, for detecting the presence of clinical proteinuria. ROC curves of DSP for identifying clinical proteinuria yielded area under the curve of 0.749 (95% confidence interval 0.699-0.794), and adjustment for SG did not significantly improve accuracy. A UPC of <0.3 was sensitive with a high negative PV for ruling out clinical proteinuria, whereas a ratio >0.8 was specific with a high positive PV. A UPC between 0.3-0.8 had an intermediate sensitivity and specificity. CONCLUSION: Urine collections of 24-h are still needed in the chronic SCI population for accurate detection of clinically significant proteinuria. DSP may not reliably detect low-grade clinical proteinuria, whereas a UPC below 0.3 may be used to rule out clinical range proteinuria.

Regulation of cyclic peptide biosynthesis in a plant pathogenic fungus by a novel transcription factor.

Strains of the filamentous fungus Cochliobolus carbonum that produce the host-selective compound HC-toxin, a cyclic tetrapeptide, are highly virulent on certain genotypes of maize (Zea mays L.). Production of HC-toxin is under the control of a complex locus, TOX2, which is composed of at least seven linked and duplicated genes that are present only in toxin-producing strains of C. carbonum. One of these genes, TOXE, was earlier shown to be required for the expression of the other TOX2 genes. TOXE has four ankyrin repeats and a basic region similar to those found in basic leucine zipper (bZIP) proteins, but lacks any apparent leucine zipper. Here we show that TOXE is a DNA-binding protein that recognizes a ten-base motif (the "tox-box") without dyad symmetry that is present in the promoters of all of the known TOX2 genes. Both the basic region and the ankyrin repeats are involved in DNA binding. A region of TOXE that includes the first ankyrin repeat is necessary and sufficient for transcriptional activation in yeast. The data indicate that TOXE is the prototype of a new family of transcription factor, so far found only in plant-pathogenic fungi. TOXE plays a specific regulatory role in HC-toxin production and, therefore, pathogenicity by C. carbonum.

An inhibitor-resistant histone deacetylase in the plant pathogenic fungus Cochliobolus carbonum.

We have partially purified and characterized histone deacetylases of the plant pathogenic fungus Cochliobolus carbonum. Depending on growth conditions, this fungus produces HC-toxin, a specific histone deacetylase inhibitor. Purified enzymes were analyzed by immunoblotting, by immunoprecipitation, and for toxin sensitivity. The results demonstrate the existence of at least two distinct histone deacetylase activities. A high molecular weight complex (430,000) is sensitive to HC-toxin and trichostatin A and shows immunoreactivity with an antibody against Cochliobolus HDC2, an enzyme homologous to yeast RPD3. The second activity, a 60,000 molecular weight protein, which is resistant even to high concentrations of well-known deacetylase inhibitors, such as HC-toxin and trichostatin A, is not recognized by antibodies against Cochliobolus HDC1 (homologous to yeast HOS2) or HDC2 and represents a different and/or modified histone deacetylase which is enzymatically active in its monomeric form. This enzyme activity is not present in the related filamentous fungus Aspergillus nidulans. Furthermore, in vivo treatment of Cochliobolus mycelia with trichostatin A and analysis of HDACs during the transition from non-toxin-producing to toxin-producing stages support an HC-toxin-dependent enzyme activity profile.

A gene related to yeast HOS2 histone deacetylase affects extracellular depolymerase expression and virulence in a plant pathogenic fungus.

A gene, HDC1, related to the Saccharomyces cerevisiae histone deacetylase (HDAC) gene HOS2, was isolated from the filamentous fungus Cochliobolus carbonum, a pathogen of maize that makes the HDAC inhibitor HC-toxin. Engineered mutants of HDC1 had smaller and less septate conidia and exhibited an approximately 50% reduction in total HDAC activity. Mutants were strongly reduced in virulence as a result of reduced penetration efficiency. Growth of hdc1 mutants in vitro was normal on glucose, slightly decreased on sucrose, and reduced by 30 to 73% on other simple and complex carbohydrates. Extracellular depolymerase activities and expression of the corresponding genes were downregulated in hdc1 mutant strains. Except for altered conidial morphology, the phenotypes of hdc1 mutants were similar to those of C. carbonum strains mutated in ccSNF1 encoding a protein kinase necessary for expression of glucose-repressed genes. These results show that HDC1 has multiple functions in a filamentous fungus and is required for full virulence of C. carbonum on maize.

Molecular cloning and characterization of cel2 from the fungus Cochlibolus carbonum.

A new cellulase gene, cel2, from the filamentous fungus Cochliobolus carbonum was cloned by using egl-1 of Trichoderma reesei as a heterologous probe. DNA blot analysis of cel2 showed that this gene is present as a single copy. The gene contains one 49-bp- intron. cel2 encodes a predicted protein (Cel2p) of 423 amino acids with a molecular mass of 45.8 kDa. The predicted pI is 4.96. It shows similarity to other endoglucanases from various fungi. From the comparison with other cellulase genes, cel2 belongs to family 7 of glucohydrolases. cel2 is located on a 2.5-Mb chromosome in C. carbonum and its expression is repressed by sucrose. A cel2 mutant of C. carbonum was created by transformation-mediated gene disruption. The pathogenicity of the mutant was indistinguishable from the wild type, indicating that cel2 by itself is not important for pathogenicity.

Provision of platelet support for fetuses and neonates affected by severe fetomaternal alloimmune thrombocytopenia.

Severe fetomaternal alloimmune thrombocytopenia requires urgent treatment with compatible platelet concentrates. As prompt treatment is sometimes delayed owing to the unavailability of compatible platelets, we established an accredited platelet donor panel to provide effective and timely transfusion support for fetal and neonatal therapy. After a mass screening programme of over 60,000 blood donations, 45 HPA-1a-negative donors with no antibodies to HPA, HLA, red cell antigens and granulocytes/lymphocytes, and with low titre anti-A and/or -B were accredited. All accredited donors were fully genotyped for HPA-1, -2, -3 and -5 by PCR-SSP. Ninety-one per cent of the accredited donors were also negative for HPA-5b.

Mutational analysis of beta-glucanase genes from the plant-pathogenic fungus Cochliobolus carbonum.

Two new beta-glucanase-encoding genes, EXG2 and MLG2, were isolated from the plant-pathogenic fungus Cochliobolus carbonum using polymerase chain reaction based on amino acid sequences from the purified proteins. EXG2 encodes a 46.6-kDa exo-beta1,3-glucanase and is located on the same 3.5-Mb chromosome that contains the genes of HC-toxin biosynthesis. MLG2 encodes a 26.8-kDa mixed-linked (beta1,3-beta1,4) glucanase with low activity against beta1,4-glucan and no activity against beta1,3-glucan. Specific mutants of EXG2 and MLG2 were constructed by targeted gene replacement. Strains with multiple mutations (genotypes exg1/mlg1, exg2/mlg1, mlg1/mlg2, and exg1/exg2/mlg1/mlg2) were also constructed by sequential disruption and by crossing. Total mixed-linked glucanase activity in culture filtrates of mlg1/mlg2 and exg1/exg2/mlg1/mlg2 mutants was reduced by approximately 73%. Total beta1,3-glucanase activity was reduced by 10, 54, and 96% in exg2, mlg1, and exg1/exg2/mlg1/mlg2 mutants, respectively. The quadruple mutant showed only a modest decrease in growth on beta1,3-glucan or mixed-linked glucan. None of the mutants showed any decrease in virulence.

Characterization of two putative histone deacetylase genes from Aspergillus nidulans.

In eukaryotic organisms, acetylation of core histones plays a key role in the regulation of transcription. Multiple histone acetyltransferases (HATs) and histone deacetylases (HDACs) maintain a dynamic equilibrium of histone acetylation. The latter form a highly conserved protein family in many eukaryotic species. In this paper, we report the cloning and sequencing of two putative histone deacetylase genes (rpdA, hosA) of Aspergillus nidulans, which are the first to be analyzed from filamentous fungi. Hybridization with a chromosome-specific cosmid library of A. nidulans allowed the localization of rpdA to chromosome III and hosA to chromosome II, respectively. PCR analyses and Southern hybridization experiments revealed that no further members of the RPD3 family are present in the genome of the fungus. Although sequence alignment displays significant amino acid similarity to other eukaryotic RPD3-type deacetylases, the deduced RPDA sequence reveals an unusual 200-amino acid extension at the C-terminus. Expression of both genes was determined by RNA blot analysis. Treatment of the cells with trichostatin A (TSA), a potent inhibitor of HDACs, was found to stimulate expression of rpdA of A. nidulans.

RPD3-type histone deacetylases in maize embryos.

Posttranslational core histone acetylation is established and maintained by histone acetyltransferases and deacetylases. Both have been identified as important transcriptional regulators in various eukaryotic systems. In contrast to nonplant systems where only RPD3-related histone deacetylases (HD) have been characterized so far, maize embryos contain three unrelated families of deacetylases (HD1A, HD1B, and HD2). Purification, cDNA cloning, and immunological studies identified the two maize histone deacetylase HD1B forms as close homologues of the RPD3-type deacetylase HDAC1. Unlike the other maize deacetylases, HD1A and nucleolar HD2, HD1B copurified as a complex with a protein related to the retinoblastoma-associated protein, Rbap46. Two HD1B mRNA species could be detected on RNA blots, encoding proteins of 58 kDa (HD1B-I) and 51 kDa (HD1B-II). HD1B-I (zmRpd3) represents the major enzyme form as judged from RNA and immunoblots. Levels of expression of HD1B-I and -II mRNA differ during early embryo germination; HD1B-I mRNA and protein are present during the entire germination pathway, even in the quiescent embryo, whereas HD1B-II expression starts when meristematic cells enter S-phase of the cell cycle. In line with previous results, HD1B exists as soluble and chromatin-bound enzyme forms. In vivo treatment of meristematic tissue with the deacetylase inhibitor HC toxin does not affect the expression of the three maize histone deacetylases, whereas it causes downregulation of histone acetyltransferase B.