Changes of a high molecular mass proteinase activity during Bufo bufo development

1. A chymotrypsin-like proteolytic activity was found both in unfertilized eggs and in embryos of Bufo bufo. 2. A dramatical change of activity can be observed in the course of embryonic development. The activity rapidly increases after fertilization up to the stage 9 followed by a fall to a level close to unfertilized eggs. 3. Gel chromatography analysis reveals, in all stages of development, the presence of a single peak of proteinase activity characterized by a very high molecular mass. 4. Proteinase activity, found change during the development of Bufo bufo, was characterized by substrate specificity, protease inhibitor and pH effect. All results obtained suggest that the chymotrypsin-like activity can be assigned to the multicatalytic proteinase.     

Whole Grain Intakes in the Diets Of Malaysian Children and Adolescents – Findings from the MyBreakfast Study

Background

Diets rich in whole grain are associated with several health benefits. Little is known however, about whole grain consumption patterns in Malaysia. The aim of this study was to assess whole grain intakes and dietary source in Malaysian children and adolescents.

Methods

This analysis is from the MyBreakfast study, a national cross sectional study investigating eating habits among primary and secondary school children throughout Malaysia, conducted in 2013. Children (n = 5,165) and adolescents (n = 2,947) who completed two days of dietary assessment using a food record or recall respectively were included. The whole grain content of foods was estimated mainly through the use of quantitative ingredient declarations on food labels. All wholegrain foods were considered irrespective of the amount of whole grain they contained.

Results

Overall, only 25% of children and 19% of adolescents were wholegrain consumers. Mean daily intakes in the total sample were 2.3g/d (SD 5.8g/d) in children and 1.7g/d (SD 4.7g/d) in adolescents and in the consumer’s only sample, mean intakes reached 9.1g/d (SD 8.6) and 9.2g/d (SD 7.1g/d) respectively. Wheat was the main grain source of whole grain while ready to eat breakfast cereals and hot cereals were the main food contributors. Less than 3% of the children and adolescents reached the US quantitative whole grain recommendation of 48g/day.

Conclusion

Whole grain is consumed by only a minority of Malaysian children and adolescents and even among consumers, intakes are well below recommendations. Efforts are needed to firstly understand the barriers to whole grain consumption among Malaysian children in order to design effective health promotion initiatives to promote an increase in whole grain consumption.     

Investigation of neuro-inflammatory parameters in a cuprizone induced mouse model of multiple sclerosis

Cuprizone, copper chelator, treatment of mouse is a toxic model of multiple sclerosis (MS) in which oligodendrocyte death, demyelination and remyelination can be observed. Understanding T and B cell subset as well as their cytokines involved in MS pathogenesis still requires further scrutiny to better understand immune component of MS. The study presented here, aimed to evaluate relevant cytokines, lymphocytes, and gene expressions profiles during demyelination and remyelination in the cuprizone mouse model of MS. Eighty male C57BL/6J mice fed with 0.2% cuprizone for eight weeks. Cuprizone has been removed from the diet in the following eight weeks. Cuprizone treated and control mice sacrificed biweekly, and corpus callosum of the brain was investigated by staining. Lymphocyte cells of mice analyzed by flow cytometry with CD3e, CD11b, CD19, CD80, CD86, CD4, CD25 and FOXP3 antibodies. IFN-gamma, IL-1alpha, IL-2, IL-5, IL-6, IL-10, IL-17, TNF-alpha cytokines were analyzed in plasma samples. Neuregulin 1 (Nrg1), ciliary neurotrophic factor (Cntf) and C-X-C chemokine receptor type 4 (Cxcr4) gene expressions in corpus callosum sections of the mice brain were quantified. Histochemistry analysis showed that demyelination began at the fourth week of cuprizone administration and total demyelination occurred at the twelfth week in chronic model. Remyelination occurred at the fourth week of following withdrawal of cuprizone from diet. The level of mature and activated T cells, regulatory T cells, T helper cells and mature B cells increased during demyelination and decreased when cuprizone removed from diet. Further, both type 1 and type 2 cytokines together with the proinflammatory cytokines increased. The level of oligodendrocyte maturation and survival genes showed differential gene expression in parallel to that of demyelination and remyelination. In conclusion, for the first-time, involvement of both cellular immune response and antibody response as well as oligodendrocyte maturation and survival factors having role in demyelination and remyelination of cuprizone mouse model of MS have been shown.     

Purification and properties of cytoplasmic and mitochondrial hexokinase from the rabbit brain

The soluble Hexokinase from rabbit brain was purified 4,700-fold to near homogeneity by a combination of ion-exchange chromatography, dye-ligand chromatography and affinity chromatography. The purified enzyme showed a specific activity of 110 units/mg of protein and was obtained in 70% yield. The properties of the purified cytoplasmic hexokinase were compared with those of the solubilized mitochondrial enzyme. No significant differences were found in M.W., pI and electrophoretic mobility. However, the temperature dependence of activity and specificity for several hexose substrates were markedly different.     

Vacuolar reticulum in oomycete hyphal tips: An additional component of the Ca2+Regulatory system?

Cultures of Achlya sp., Phytophthora cinnamomi, Saprolegnia diclina, S. ferax, and S. parasitica, treated with 6-carboxyfluorescein diacetate solution, accumulate 6-carboxyfluorescein in a reticulate system of fine tubules. The network shows longitudinal polarity within the hyphae, tubules being finest toward the hyphal tips. In more mature subapical regions the network is connected with large vacuoles that also accumulate 6-carboxyfluorescein. A morphologically similar system has also been identified in freeze-substituted hyphae of S. ferax. The network is considered to be vacuolar, but differs from the tubular vacuole system of true fungi in that tubules are less motile, more frequently branched, and do not alternate with clusters of spherical vacuoles. The appearance of the network resembles patterns of calcium-sensitive dye staining and it is suggested that the vacuolar reticulum in the tip region of oomycete hyphae may act as a Ca2+ sink. The tubular reticulum in oomycetes is very fragile and can be shown with 6-carboxyfluorescein in only those hyphal tips with a motility and organelle distribution characteristic of growing hyphae with normal morphology. Diverse abnormal hyphae show a range of other fluorochrome localizations. These include large irregular compartments filled with fluorochrome, and fluorescent cytoplasm with organelles and vacuoles standing out in negative contrast. These localizations in abnormal hyphae are correlated with other structural changes indicative of damage. Special care is required in experiments with oomycetes to avoid such artefacts of localization. Copyright 1997 Academic Press. Copyright 1997 Academic Press     

Developmental and wound-, cold-, desiccation-, ultraviolet-B-stress-induced modulations in the expression of the petunia zinc finger transcription factor gene ZPT2-2

The ZPT2-2 gene belongs to the EPF gene family in petunia (Petunia hybrida), which encodes proteins with TFIIIA-type zinc-finger DNA-binding motifs. To elucidate a possible function for ZPT2-2, we analyzed its pattern of expression in relation to different developmental and physiological stress signals. The activity of the ZPT2-2 promoter was analyzed using a firefly luciferase (LUC) reporter gene, allowing for continuous measurements of transgene activity in planta. We show that ZPT2-2::LUC is active in all plant tissues, but is strongly modulated in cotyledons upon germination, in leaves in response to desiccation, cold treatment, wounding, or ultraviolet-B light, and in petal tissue in response to pollination of the stigma. Analysis of mRNA levels indicated that the modulations in ZPT2-2::LUC expression reflect modulations in endogenous ZPT2-2 gene expression. The change in ZPT2-2::LUC activity by cold treatment, wounding, desiccation, and ultraviolet-B light suggest that the phytohormones ethylene and jasmonic acid are involved in regulating the expression of ZPT2-2. Although up-regulation of expression of ZPT2-2 can be blocked by inhibitors of ethylene perception, expression in plants is not induced by exogenously applied ethylene. The application of jasmonic acid does result in an up-regulation of gene activity and, thus, ZPT2-2 may play a role in the realization of the jasmonic acid hormonal responses in petunia.     

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.     

<Emphasis Type="Italic">Candida albicans</Emphasis>genome sequence: a platform for genomics in the absence of genetics

Publication of the complete diploid genome sequence of the yeast Candida albicans will accelerate research into the pathogenesis of Candida infections. Comparative genomic analysis highlights genes that may contribute to C. albicans survival and its fitness as a human commensal and pathogen.     

Towards Harmonisation of Critical Laboratory Result Management - Review of the Literature and Survey of Australasian Practices

Timely release and communication of critical test results may have significant impact on medical decisions and subsequent patient outcomes. Laboratories therefore have an important responsibility and contribution to patient safety. Certification, accreditation and regulatory bodies also require that laboratories follow procedures to ensure patient safety, but there is limited guidance on best practices. In Australasia, no specific requirements exist in this area and critical result reporting practices have been demonstrated to be heterogeneous worldwide.Recognising the need for agreed standards and critical limits, the AACB started a quality initiative to harmonise critical result management throughout Australasia. The first step toward harmonisation is to understand current laboratory practices. Fifty eight Australasian laboratories responded to a survey and 36 laboratories shared their critical limits. Findings from this survey are compared to international practices reviewed in various surveys conducted elsewhere. For the successful operation of a critical result management system, critical tests and critical limits must be defined in collaboration with clinicians. Reporting procedures must include how critical results are identified; who can report and who can receive critical results; what is an acceptable timeframe within which results must be delivered or, if reporting fails, what escalation procedures should follow; what communication channels or systems should be used; what should be recorded and how; and how critical result procedures should be maintained and evaluated to assess impact on outcomes.In this paper we review the literature of current standards and recommendations for critical result management. Key elements of critical result reporting are discussed in view of the findings of various national surveys on existing laboratory practices, including data from our own survey in Australasia. Best practice recommendations are made that laboratories are expected to follow in order to provide high quality and safe service to patients.