Cryptococcus neoformans and its cell wall components induce similar cytokine profiles in human peripheral blood mononuclear cells despite differences in structure

We studied the cytokine profile of peripheral blood mononuclear cells after stimulation with various cryptococcal strains or its purified cell wall components. After 3 h of stimulation, tumor necrosis factor (TNF) alpha levels were strongly increased, whereas interferon (IFN) gamma and interleukin (IL) 10 levels were increased only slightly, or not at all (respectively). In contrast, after 18 h, TNF-alpha and IFN-gamma levels were (strongly) decreased, whereas the IL-10 levels were increased. The IL-1beta, IL-6 and IL-8 levels were equally high throughout the experiment. In order to establish which of the cryptococcal envelope components contributed most to the observed cytokine profile induced by whole cryptococci, glucuronoxylomannan, galactoxylomannan and mannoproteins were purified and partially characterized biochemically. All cryptococcal components elicited a similar cytokine pattern despite the differences in structure.     

Production of T-2 toxin by a Fusarium resembling Fusarium poae

A Fusarium species with a micro morphology similar to F. poae and a metabolite profile resembling that of F. sporotrichioides has been identified. Like typical F. poae, the microconidia have a globose to pyriform shape, but the powdery appearance, especially on Czapek-Dox Iprodione Dichloran agar (CZID), less aerial mycelium and the lack of fruity odour on Potato Sucrose Agar (PSA) make it different from F. poae. The lack of macroconidia, polyphialides and chlamydospores differentiates it from F. sporotrichioides. All 18 isolates investigated, 15 Norwegian, two Austrian and one Dutch, produced T-2 toxin (25–400 μg/g) on PSA or Yeast Extract Sucrose agar (YES). In addition, neosolaniol, iso-neosolaniol, HT-2 toxin, 4- and 15-acetyl T-2 tetraol, T-2 triol and T-2 tetraol and4,15-diacetoxyscirpenol were formed in variable amounts. Neither nivalenol, 4- or 15-acetylnivalenolor 4,15-diacetylnivalenol were detected in any of the cultures, while these toxins were produced at least in small amounts by all the 12 typical F. poae isolates studied. The question of whether this Fusarium should be classified as F. poae or F. sporotrichioides or a separate taxon should be addressed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Application of short-form oral health impact profile on elderly Japanese

Objectives: The purpose of this study was to use the oral health impact profile (OHIP‐14) to evaluate the impact of oral disease on the quality of life of a group of independently‐living elderly persons in an urban area of Japan. Subjects: A total of 1244 participants of the Senior Citizen's College, who attended the lectures once a week. They were community‐dwelling, independently‐living people over 60 years of age. Measurements: Japanese version of the short‐form OHIP‐14. Results: Internal reliability for the 14 items overall was very high (Cronbach's α = 0.95). Report of ‘painful aching’ and ‘uncomfortable to eat’ were the two most highly scored items using the mean sum OHIP‐14 score. A multiple logistic regression analysis indicated that the sum OHIP‐14 score had significant associations with self‐assessment of general health, dental status, and a perceived need for dental treatment. However, age, gender, dissatisfaction with financial status or education level was not significantly associated with the sum OHIP‐14. Compared with that of other countries, the items were ranked similarly, whereas the perceived magnitudes of the problems were quite different from other population. Conclusions: The OHIP‐14 in Japanese had a high internal reliability, was significantly associated with dental status and comparable ranking for items when compared with studies from other countries.     

Mutants for photosystem I subunit D of Arabidopsis thaliana: effects on photosynthesis, photosystem I stability and expression of nuclear genes for chloroplast functions

In Arabidopsis thaliana, the D-subunit of photosystem I (PSI-D) is encoded by two functional genes, PsaD1 and PsaD2, which are highly homologous. Knock-out alleles for each of the loci have been identified by a combination of forward and reverse genetics. The double mutant psad1-1 psad2-1 is seedling-lethal, high-chlorophyll-fluorescent and deficient for all tested PSI subunits, indicating that PSI-D is essential for photosynthesis. In addition, psad1-1 psad2-1 plants show a defect in the accumulation of thylakoid multiprotein complexes other than PSI. Of the single-gene mutations, psad2 plants behave like wild-type (WT) plants, whereas psad1-1 markedly affects the accumulation of PsaD mRNA and protein, and photosynthetic electron flow. Additional effects of the psad1-1 mutation include a decrease in growth rate under greenhouse conditions and downregulation of the mRNA expression of most genes involved in the light phase of photosynthesis. In the same mutant, a marked decrease in the levels of PSI and PSII polypeptides is evident, as well as a light-green leaf coloration and increased photosensitivity. Increased dosage of PsaD2 in the psad1-1 background restores the WT phenotype, indicating that PSI-D1 and PSI-D2 have redundant functions.     

Changes in nitrogen content and protein profiles following <Emphasis Type="Italic">in vitro</Emphasis> selection of NaCl resistant mung bean and tomato

Mung bean and tomato were in vitro selected on media containing 0, 25, 50, 100 and 150 mM NaCl. Two types of media (hormone supplemented media, CB and hormone free media, MS) were used for mung bean using cotyledon explants whereas two types of explants (cotyledons and shoot apices) were used for tomato on MS media. Total-N, protein content, nitrite reductase (NiR) activity and protein protein profiles were checked in selected plants and compared to original non selected ones. NaCl at low concentrations slightly increased total-N in shoots and roots of in vitro selected mung bean and tomato whereas higher concentrations induced significant reductions. Similar increases in protein content were detected at lower concentrations with no significant effects thereover. On the contrary, NaCl gradually inhibited NiR activity. Similar responses of total-N, protein and NiR activity, but with greater magnitudes, were detected in original plants. In addition, NaCl significantly reduced dry weights of shoots and roots of either in vitro selected or, in particular, original intact plants. Moreover, electrophoresis (SDS-PAGE) of protein from shoots of either in vitro selected or intact plants showed that NaCl induced new protein bands while some others were concomitantly disappeared. The induction of one or more of the 86.4, 79, 77.6, 77 and 71.5 kDa bands following in vitro selection and/or the disappearance of the 86 kDa band from intact plants seemed necessary for mung bean resistance. Also, the presence of 86.2 kDa band and/or the loss of the 85.8 and 57.5 kDa bands might be included in tomato resistance. Of these induced bands in mung bean selected on CB media, only two bands were detected in plants selected on MS media. In tomato, two bands lost following selection from cotyledons but only one band lost following selection from shoot apices. These changes in protein pattern therefore might serve as adaptive regulators for resistance to NaCl.     

Non-methylated Genomic Sites Coincidence Cloning (NGSCC): an approach to large scale analysis of hypomethylated CpG patterns at predetermined genomic loci

We have developed a new approach to the analysis of hypomethylated CpG patterns within predetermined, megabase long, genome regions. The approach, which we term Non-methylated Genomic Sites Coincidence Cloning (NGSCC), includes three main steps. First, total genomic DNA is digested with a methylation sensitive restriction endonuclease, such as Hpa II or Hha I. Then the fragments corresponding to the genomic area of interest are selected. To this end the fragmented genome DNA is hybridized with a mixture of clones (BACs, cosmids etc.) representing a given region and digested with the same restriction enzyme(s). A special version of the coincidence cloning procedure was developed to make this hybridization selection highly efficient and specific. Finally, fragments of the locus under study are mapped and sequenced. The technique proved to be efficient and specific. As a test, it was applied to the analysis of hypomethylated CpG patterns along the 1-Mb D19S208-COX7A1 (Chr 19q13.12) locus, on human chromosome 19, in normal testis and in seminoma tissues. Some differences in the distribution of hypomethylated CpGs between the two tissues were demonstrated. The methylation profiles in both tissues revealed a clear trend to clustering of non-methylated sites. We also analyzed the expression of genes located within hypomethylated clusters in both tissues. It was shown that, whereas the expression of some of the genes investigated was correlated with hypomethylation of the region, other genes were expressed regardless of their methylation status. NGSCC thus promises to be a useful approach for the analysis of the role of dynamic epigenetic factors in genome function.Communicated by G. P. Georgiev     

DNA chip-based expression profile analysis indicates involvement of the phosphatidylinositol signaling pathway in multiple plant responses to hormone and abiotic treatments

The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors, and previous studies have indicated the involvement of multiple PI-related gene families during cellular responses.Through a detailed analysis of the Arabidopsis thaliana genome, 82 polypeptides were identified as being involved in PI signaling. These could be grouped into different families including PI synthases (PIS), PI-phosphate kinases (PIPK),phospholipases (PL), inositol polyphosphate phosphatases (IPPase), inositol polyphosphate kinases (IPK), PI transfer proteins and putative inositol polyphosphate receptors. The presence of more than 10 isoforms of PIPK, PLC, PLD and IPPase suggested that these genes might be differentially expressed during plant cellular responses or growth and development. Accordingly, DNA chip technology was employed to study the expression patterns of various isoforms.In total, 79 mRNA clones were amplified and used for DNA chip generation. Expression profile analysis was performed using samples that represented multiple tissues or cellular responses. Tested samples included normal leaf, stem and flower tissues, and leaves from plants treated with various hormones (auxin, cytokinin, gibberellin, abscisic acid and brassinosteroid) or environmental factors (temperature, calcium, sodium, drought, salicylic acid and jasmonic acid).Results showed that many PI pathway-related genes were differentially expressed under these experimental conditions.In particular, the different isoforms of each family were specifically expressed in many cases, suggesting their involvement in tissue specificity and cellular responses to environmental conditions. This work provides a starting point for functional studies of the relevant PI-related proteins and may help shed light onto the role of PI pathways in development and cellular responses.     

肿瘤相关基因表达检测寡核苷酸芯片的制备及其初步应用

为研制肿瘤相关寡核苷酸芯片,并实现其在抗肿瘤反义核酸“癌泰得”作用机理研究方面的初步应用,制备了包含近450种肿瘤相关基因特异寡核苷酸探针的寡核苷酸芯片,建立了相应的质控标准.“癌泰得”用脂质体转染HepG2肿瘤细胞,提取细胞总RNA反转录并荧光标记cDNA,用制备的寡核苷酸芯片检测肝癌细胞HepG2的肿瘤相关基因表达水平,用软件分析获得其差异基因表达谱.0.4 μmol/L的反义核酸“癌泰得”作用于HepG2细胞15 h后,MDNCF、DHS等基因mRNA表达下调,MUC2、MPP11、LAT、HRIF-B、JNK3A1等mRNA基因表达上调,初步检测到了“癌泰得”的抗肿瘤作用可能的相关基因,为进一步的分子作用机理的探讨奠定基础.结果表明,制备的肿瘤相关芯片敏感度高、特异性高、重复性均较好,可用于检测肿瘤相关基因的表达谱,为临床诊断和基础研究提供了技术平台.     

db/db小鼠糖尿病肾病相关基因的分析和克隆

用GM-U74A基因芯片分别检测了正常对照组（db/m小鼠）、糖尿病肾病组（db/db小鼠）、大黄酸治疗组（大黄酸150 mg/kg治疗12周）肾脏基因表达谱.发现在12 437个基因（包括表达序列标签）中,与正常对照组相比,糖尿病肾病组有1 085个基因表达下调,37个基因表达上调,其中变化幅度大于2倍,表达下调的有166个和表达上调的有29个.与糖尿病肾病组相比,大黄酸治疗组有384个基因表达下调,155个表达上调,其中变化幅度大于2倍,表达下调的有47个和表达上调的有30个.在此基础上,对其中的一个差异表达的表达序列标签(EST)进行了详细的生物信息学分析,发现它是一个未知功能基因——“REKEN cDNA 0610006H10”基因的一部分.在用RT-PCR进一步验证了其与糖尿病肾病的相关性后,对“REKEN cDNA 0610006H10”基因进行了克隆.