石耳科(子囊菌亚门)的间断分布和替代现象

本文报道了石耳科的两个亚洲的,也是美洲以外的新记录种,即角石耳与深色石耳。进一步证实了单果石耳在南大西洋的,即南美洲和南部非洲的间断分布。对于上述种类以及本科其它一些种类的间断分布和替代现象进行了分析讨论。     

Independent regulation of matrix metalloproteinases and plasminogen activators in human fibrosarcoma cells

Serine proteases and matrix metalloproteinases have been shown to often cooperate in multiple physiological and pathological processes associated with changes in the extracellular matrix (ECM). We have examined the interaction between the plasminogen activator (PA)-plasmin system and matrix metalloproteinases (MMPs) in HT1080 human fibrosarcoma cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). While TPA treatment evoked a temporary increased expression of urokinase type PA (uPA), the production of both types of plasminogen activator inhibitors (PAI) was induced and sustained over 12 h by TPA treatment shifting the protease-protease inhibitors balance in favor of the inhibitors. TPA treatment of HT1080 cells induced the expression of interstitial collagenase (MMP-1) and increased the expression of gelatinase B (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1), and MT-MMP, a membrane-bound activator of progelatinase A (proMMP-2), while MMP-2 and TIMP-2 expression were decreased. Increased MT-MMP expression by TPA treatment was associated with increased activation of proMMP-2. These data show that the regulation of PA-plasmin and metalloproteinase and their specific inhibitors is uncoordinated. In addition, inhibition of the PA-plasmin system by PAI-2 or aprotinin did not prevent the activation of proMMP-2 by TPA, suggesting that plasmin is not involved in MT-MMP-mediated activation of proMMP-2. © 1996 Wiley-Liss, Inc.     

Effects of high temperature shocks on the development and biology ofTrichogramma brassicae [Hym.: Trichogrammatidae]

Trichogramma brassicae Bezdenko is used throughout Europe as an effective biological control agent against the European corn borer (Ostrinia nubilalis Hübner). However, in certain climatic regions, the biological control exerted by this parasitoid is less effective. High temperatures inside the released capsules are suspected to have a negative influence on the parasitoids. To simulate these adverse conditions, we applied heat shocks (35°C and 44°C) for 6 hours to pupae ofTrichogramma brassicae at two periods: white pupae and melanized pupae. The results showed the susceptibility of both the white pupae and melanized pupae, especially at 44°C. At this temperature, the adults (G₀ generation) derived from pupae treated at any age showed reduced longevity and fecundity. Moreover, those descended from treated melanized pupae showed an emergence rate lower than that of the control. These effects reduced to half and more the parasitic efficiency of the G₀ generation. For the progeny (G₁ generation), we observed a decrease of the female ratio: 38% for the progeny of adults derived from treated melanized pupae; 88% for the progeny of those derived from treated white pupae. In that condition, the G₁ generation, almost totally male, could not have any parasitic activity.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

CLNR1, the AREA/NIT2-like global nitrogen regulator of the plant fungal pathogen Colletotrichum lindemuthianum is required for the infection cycle

Nitrogen starvation is generally assumed to be encountered by biotrophic and hemibiotrophic plant fungal pathogens at the beginning of their infection cycle. We tested whether nitrogen starvation constitutes a cue regulating genes that are required for pathogenicity of Colletotrichum lindemuthianum, a fungal pathogen of common bean. The clnr1 (C. lindemuthianumnitrogen regulator 1) gene, the areA/nit-2 orthologue of C. lindemuthianum, was isolated. The predicted CLNR1 protein exhibits high amino acid sequence similarities with the AREA and NIT2 global fungal nitrogen regulators. Targeted clnr1- mutants are unable to use a wide array of nitrogen sources, indicating that clnr1 is the C. lindemuthianum major nitrogen regulatory gene. The clnr1- mutants are non-pathogenic, although few anthracnose lesions seldom occur on whole plantlets. Surprisingly, cytological analysis reveals that the clnr1- mutants are not disturbed from the penetration stage until the end of the biotrophic phase, but that they are impaired during the setting up of the necrotrophic phase. Thus, through CLNR1, nitrogen starvation constitutes a cue for the regulation of genes that are compulsory for this stage of the C. lindemuthianum infection process. Additionally, clnr1- mutants complemented with the Aspergillus nidulans areA gene are fully pathogenic, indicating that areA is able to activate the C. lindemuthianum suited genes, normally under the control of clnr1.     

Multiplex PCR/liquid chromatography assay for detection of gene rearrangements: application to RB1 gene

Screening for large gene rearrangements is established as an important part of molecular medicine but is also challenging. A variety of robust methods can detect whole-gene deletions, but will fail to detect more subtle rearrangements that may involve a single exon. In this paper, we describe a new, versatile and robust method to assess exon copy number, called multiplex PCR/liquid chromatography assay (MP/LC). Multiple exons are amplified using unlabeled primers, then separated by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and quantitated by fluorescent detection using a post-column intercalation dye. The relative peak intensities for each target directly reflect exon copy number. This novel technique was used to screen a panel of 121 unrelated retinoblastoma patients who were tested previously using a reference strategy. MP/LC correctly scored all deletions and demonstrated a previously undetected RB1 duplication, the first to be described. MP/LC appears to be an easy, versatile, and cost-effective method, which is particularly relevant to denaturing HPLC (DHPLC) users since it broadens the spectrum of available applications on a DHPLC system.     

Identification of a Disulfide Bridge Essential for Transport Function of the Human Proton-coupled Amino Acid Transporter hPAT1

The proton-coupled amino acid transporter 1 (PAT1, SLC36A1) mediates the uptake of small neutral amino acids at the apical membrane of intestinal epithelial cells after protein digestion. The transporter is currently under intense investigation, because it is a possible vehicle for oral drug delivery. Structural features of the protein such as the number of transmembrane domains, the substrate binding site, or essential amino acids are still unknown. In the present study we use mutagenesis experiments and biochemical approaches to determine the role of the three putative extracellular cysteine residues on transport function and their possible involvement in the formation of a disulfide bridge. As treatment with the reducing reagent dithiothreitol impaired transport function of hPAT1 wild type protein, substitution of putative extracellular cysteine residues Cys-180, Cys-329, and Cys-473 by alanine or serine was performed. Replacement of the two highly conserved cysteine residues Cys-180 and Cys-329 abolished the transport function of hPAT1 in Xenopus laevis oocytes. Studies of wild type and mutant transporters expressed in human retinal pigment epithelial (HRPE) cells suggested that the binding of the substrate was inhibited in these mutants. Substitution of the third putative extracellular nonconserved cysteine residue Cys-473 did not affect transport function. All mutants were expressed at the plasma membrane. Biotinylation of free sulfhydryl groups using maleimide-PEG₁₁-biotin and SDS-PAGE analysis under reducing and nonreducing conditions provided direct evidence for the existence of an essential disulfide bond between Cys-180 and Cys-329. This disulfide bridge is very likely involved in forming or stabilizing the substrate binding site.The solute carrier (SLC)² superfamily represents the second largest group of membrane proteins after the G-protein-coupled receptor (GPCR) superfamily in the human genome. Comprising 384 members, the 46 SLC families include transporters for inorganic ions, amino acids, neurotransmitters, sugars, purines, fatty acids, and other substances (1). Ten SLC families contain 47 known transporters for amino acids and 48 related orphan transporters. Phylogenetic analysis revealed four main clusters (α, β, γ, and δ). Together with members of the SLC32 and SLC38 families, the proton-coupled amino acid transporter 1 (PAT1) was placed into group β. PAT1 is a member of the SLC36 family (SLC36A1). It was originally identified as the lysosomal amino acid transporter (LYAAT1) in rat brain (2). Subsequently, mouse and human homologs were cloned from mouse intestine (3) and from Caco-2 cells (4), respectively. PAT1 is identical to the H⁺/amino acid cotransporter that has been functionally described in Caco-2 cells (5). It is localized mainly to the apical membrane of intestine epithelial cells and is also found in lysosomes in brain neurons (4) facilitating the transport of amino acids from luminal protein digestion or lysosomal proteolysis, respectively. The transport of substrates via PAT1 is driven by an inwardly directed H⁺ gradient. Recently we could identify the conserved His-55 as being responsible for binding and translocation of the proton (6).Prototypic substrates for PAT1 are small neutral amino acids (e.g. l-proline, glycine, β-alanine) and amino acid derivatives (e.g. γ-aminobutyric acid (GABA), α-(methylamino)-isobutyric acid) (3–5, 7–10). Recently, PAT1 gained much interest because it transports pharmaceutically relevant compounds such as d-cycloserine, l-azetidine-2-carboxylic acid, 3-amino-1-propanesulfonic acid, 3,4-dehydro-l-proline, vigabatrin, and other GABA analogs (8, 10, 11) rendering it an interesting target for the pharmaceutical industry. PAT1 seems to be one of the most important drug transporters in the intestine allowing oral availability of GABA-related and other drugs and prodrugs. Furthermore, a recent report shows involvement of this transporter family, namely the PAT2 subtype, in the autosomal dominant inherited disorder iminoglycinuria (12).Unfortunately, up to now the exact three-dimensional structure of PAT1, the transmembrane domain topology, and the substrate binding site are unknown. More structural information of PAT1 would allow a better understanding of the molecular mechanisms of its function and drug interaction, which is so far being investigated only in classic transport studies. Mutational analysis of putative extracellular regions is a suitable tool to get the first clue into transmembrane organization and relevant amino acid residues (6). This approach should also elucidate the spatial organization of the extracellular loops. The present study was performed to identify functionally important extracellular cysteine residues and their involvement in disulfide bridges. The relevance of disulfide bonds for membrane protein function is mainly based on the stabilization of a proper three-dimensional structure. The correct conformation in turn is essential for trafficking, surface expression, stability, and transport function. So far, intramolecular disulfide bonds have been identified for only very few SLCs, e.g. the serotonin transporter SERT and the dopamine transporter DAT (13–15). Native disulfide bonds are probably required for transporter function of the Na⁺/glucose cotransporter SGLT1 (16, 17). For the type IIa sodium/phosphate cotransporter, it was shown that cleavage of disulfide bonds results in conformational changes that lead to internalization and subsequent lysosomal degradation of the transport protein (18). A similar stabilizing effect of an intramolecular disulfide bridge was also reported for the human ATP-binding cassette (ABC) transporter ABCG2 (19).Linkage via cysteine residues can also be necessary for transporter oligomerization. For the rat serotonin transporter SERT (20) and for the human ABC transporter ABCG2 (21), intermolecular disulfide bridges could be identified. For the hexose transporter GLUT1, an intramolecular disulfide bond promotes tetramerization of the transporter (22, 23). On the other hand, removal of cysteine residues can also lead to an impaired trafficking and mislocalization of the transporter protein without a disulfide bridge being involved (13, 24, 25). In those cases, the cysteine residues themselves are assumed to play an important role for the trafficking and targeting of the transporter to the cell surface. Similarly, for several transporters, cysteine residues located in a transmembrane domain play a key role in substrate recognition. Single cysteines have been found to be essential for substrate binding of the rat organic cation transporters rOCT1 and rOCT2 (26) and the multidrug and toxin extrusion transporter MATE1 (27). The relevance of conserved cysteines for the integrity of a membrane protein has therefore to be investigated very thoroughly. Several earlier studies reported loss of function in cysteine mutants without testing membrane localization.After assessing a negative influence of the reducing reagent DTT on hPAT1 function, we performed systematic mutagenesis in this study. The three putative extracellular cysteine residues Cys-180, Cys-329, and Cys-473 were individually exchanged to either alanine or serine residues. The resulting mutants were analyzed for substrate binding and transport in human retinal pigment epithelial (HRPE) cells and electrogenic transport in Xenopus laevis oocytes. Biochemical approaches provided direct evidence for an essential disulfide bond between Cys-180 and Cys-329. A triple mutant was constructed and examined to exclude other juxtamembrane cysteine residues as potential partners for disulfide bridges. The data suggest that this disulfide bridge is involved in forming or stabilizing the putative substrate-binding pocket. In addition, our results strongly support the eleven transmembrane domain topology model of hPAT1. This is consistent with our recently published data on glycosylation of hPAT1 (28).     

Clinical patterns in asthma based on proximal and distal airway nitric oxide categories

Background

The exhaled nitric oxide (eNO) signal is a marker of inflammation, and can be partitioned into proximal [J''aw_NO (nl/s), maximum airway flux] and distal contributions [CA_NO (ppb), distal airway/alveolar NO concentration]. We hypothesized that J''aw_NO and CA_NO are selectively elevated in asthmatics, permitting identification of four inflammatory categories with distinct clinical features.

Methods

In 200 consecutive children with asthma, and 21 non-asthmatic, non-atopic controls, we measured baseline spirometry, bronchodilator response, asthma control and morbidity, atopic status, use of inhaled corticosteroids, and eNO at multiple flows (50, 100, and 200 ml/s) in a cross-sectional study design. A trumpet-shaped axial diffusion model of NO exchange was used to characterize J''aw_NO and CA_NO.

Results

J''aw_NO was not correlated with CA_NO, and thus asthmatic subjects were grouped into four eNO categories based on upper limit thresholds of non-asthmatics for J''aw_NO (≥ 1.5 nl/s) and CA_NO (≥ 2.3 ppb): Type I (normal J''aw_NO and CA_NO), Type II (elevated J''aw_NO and normal CA_NO), Type III (elevated J''aw_NO and CA_NO) and Type IV (normal J''aw_NO and elevated CA_NO). The rate of inhaled corticosteroid use (lowest in Type III) and atopy (highest in Type II) varied significantly amongst the categories influencing J''aw_NO, but was not related to CA_NO, asthma control or morbidity. All categories demonstrated normal to near-normal baseline spirometry; however, only eNO categories with increased CA_NO (III and IV) had significantly worse asthma control and morbidity when compared to categories I and II.

Conclusions

J''aw_NO and CA_NO reveal inflammatory categories in children with asthma that have distinct clinical features including sensitivity to inhaled corticosteroids and atopy. Only categories with increase CA_NO were related to poor asthma control and morbidity independent of baseline spirometry, bronchodilator response, atopic status, or use of inhaled corticosteroids.