酿酒酵母孢子壁结构及其合成相关基因研究进展

细胞壁是酵母细胞区别于哺乳动物细胞的重要特征结构。酵母细胞壁的结构组成、合成、再生等与酵母自身繁殖及环境胁迫压力密切相关。目前,酵母孢子壁的形成机理、调控过程机制及孢子壁合成相关基因的功能尚未研究清楚。本文以酿酒酵母为例,简要描述酵母孢子壁的形成过程,重点阐述孢子壁甘露糖层、葡聚糖层、壳聚糖层和二酪氨酸层的结构组成及其合成相关基因的国内外研究进展,以期为抗真菌药物的新靶点研究提供参考。     

The use of fluorescence enhancement to improve the microscopic diagnosis of falciparum malaria

Background

The Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) are enzymes of central importance in parasite metabolism. The dhfr and dhps gene mutations are known to be associated with sulphadoxine/pyrimethamine (SP) resistance.

Objective

To investigate the effects of dhfr/dhps mutations on parasite characteristics other than SP resistance.

Method

Parasite infections obtained from 153 Sudanese patients with uncomplicated falciparum malaria treated with SP or SP + chloroquine, were successfully genotyped at nine codons in the dhfr/dhps genes by PCR-ELISA.

Results & conclusion

Mutations were detected in dhfr at N51I, S108N and C59R, and in at dhps at A/S436F, A437G, K540E and A581G, the maximum number of mutations per infection were five. Based on number of mutant codons per infection (multiplicity of mutation, MOM), the infections were organized into six grades: wild-types (grade 0; frequency, 0.03) and infections with MOM grades of 1 to 5, with the following cumulative frequency; 0.97, 0.931, 0.866, 0.719, 0.121, respectively. There was no significant association between the MOM and SP response. Importantly, immunity, using age as a surrogate marker, contributed significantly to the clearance of parasites with multiple dhfr/dhps mutations. However, these mutations have a survival advantage as they were associated with increased gametocytogenesis. The above implications of dhfr/dhps mutations were associated with MOM 2 to 5, regardless of the gene/codon locus.     

Studies of genotoxicity and mutagenicity of nitroimidazoles: demystifying this critical relationship with the nitro group

Nitroimidazoles exhibit high microbicidal activity, but mutagenic, genotoxic and cytotoxic properties have been attributed to the presence of the nitro group. However, we synthesised nitroimidazoles with activity against the trypomastigotes of Trypanosoma cruzi, but that were not genotoxic. Herein, nitroimidazoles (11-19) bearing different substituent groups were investigated for their potential induction of genotoxicity (comet assay) and mutagenicity (Salmonella/Microsome assay) and the correlations of these effects with their trypanocidal effect and with megazol were investigated. The compounds were designed to analyse the role played by the position of the nitro group in the imidazole nucleus (C-4 or C-5) and the presence of oxidisable groups at N-1 as an anion receptor group and the role of a methyl group at C-2. Nitroimidazoles bearing NO2 at C-4 and CH3 at C-2 were not genotoxic compared to those bearing NO₂ at C-5. However, when there was a CH3 at C-2, the position of the NO2 group had no influence on the genotoxic activity. Fluorinated compounds exhibited higher genotoxicity regardless of the presence of CH3 at C-2 or NO2 at C-4 or C-5. However, in compounds 11 (2-CH3; 4-NO2; N-CH2OHCH2Cl) and 12 (2-CH3; 4-NO2; N-CH2OHCH2F), the fluorine atom had no influence on genotoxicity. This study contributes to the future search for new and safer prototypes and provide.     

Optimizing harvest of corn stover fractions based on overall sugar yields following ammonia fiber expansion pretreatment and enzymatic hydrolysis

Background  

Corn stover composition changes considerably throughout the growing season and also varies between the various fractions of the plant. These differences can impact optimal pretreatment conditions, enzymatic digestibility and maximum achievable sugar yields in the process of converting lignocellulosics to ethanol. The goal of this project was to determine which combination of corn stover fractions provides the most benefit to the biorefinery in terms of sugar yields and to determine the preferential order in which fractions should be harvested. Ammonia fiber expansion (AFEX) pretreatment, followed by enzymatic hydrolysis, was performed on early and late harvest corn stover fractions (stem, leaf, husk and cob). Sugar yields were used to optimize scenarios for the selective harvest of corn stover assuming 70% or 30% collection of the total available stover.     

Shift in the localization of sites of hydrogen peroxide production in brain mitochondria by mitochondrial stress

We have determined the underlying sites of H(2)O(2) generation by isolated rat brain mitochondria and how these can shift depending on the presence of respiratory substrates, electron transport chain modulators and exposure to stressors. H(2)O(2) production was determined using the fluorogenic Amplex red and peroxidase system. H(2)O(2) production was higher when succinate was used as a respiratory substrate than with another FAD-dependent substrate, alpha-glycerophosphate, or with the NAD-dependent substrates, glutamate/malate. Depolarization by the uncoupler p-trifluoromethoxyphenylhydrazone decreased H(2)O(2) production stimulated by all respiratory substrates. H(2)O(2) production supported by succinate during reverse transfer of electrons was decreased by inhibitors of complex I (rotenone and diphenyleneiodonium) whereas in glutamate/malate-oxidizing mitochondria diphenyleneiodonium decreased while rotenone increased H(2)O(2) generation. The complex III inhibitors antimycin and myxothiazol decreased succinate-induced H(2)O(2) production but stimulated H(2)O(2) production in glutamate/malate-oxidizing mitochondria. Antimycin and myxothiazol also increased H(2)O(2) production in mitochondria using alpha-glycerophosphate as a respiratory substrate. In substrate/inhibitor experiments maximal stimulation of H(2)O(2) production by complex I was observed with the alpha-glycerophosphate/antimycin combination. In addition, three forms of in vitro mitochondrial stress were studied: Ca(2+) overload, cold storage for more than 24 h and cytochrome c depletion. In each case we observed (i) a decrease in succinate-supported H(2)O(2) production by complex I and an increase in succinate-supported H(2)O(2) production by complex III, (ii) increased glutamate/malate-induced H(2)O(2) generation by complex I and (iii) increased alpha-glycerophosphate-supported H(2)O(2) generation by complex III. Our results suggest that all three forms of mitochondrial stress resulted in similar shifts in the localization of sites of H(2)O(2) generation and that, in both normal and stressed states, the level and location of H(2)O(2) production depend on the predominant energetic substrate.     

Mitogenic activity of pituitary hormones on cell cultures of normal and carcinogen-induced tumor epithelium from rat mammary glands

Cell suspension containing normal or tumor epithelium were readily obtained by enzymatically digesting rat mammary glands from perphenazine-treated (prolactin-hypersecreting) cycling, female virgin animals or hormone- responsive mammary tumors from animal treated with dimethylbenzanthracene. Cell suspensions were fractioned into predominantly epithelial and predominantly stromal cells by their differential rates of attachment to culture dishes. Both normal mammary and tumor epithelial cells were characterized by the presence of specific cell-junctional complexes, desmosome-like structures, surface microvilli, and their ability to synthesize casein. Serum-dependent protease activity was greater in cultures derived from tumors, and cells from such cultures grew in agarose whereas those from the non-neoplastic gland did not. The addition of prolactin to the culture medium stimulated DNA synthesis in primary or secondary epithelial cultures from tumors, whereas additional insulin and hydrocortisone with prolactin were required for similar levels of DNA synthesis in cultures from non-neoplastic glands. The fraction of cells synthesizing DNA was, however, smaller than that with 10 percent serum measured in the same time period. Both growth hormone and epidermal growth factor stimulated DNA synthesis but to a lesser extent than did prolactin. Prolactin with hydrocortisone and insulin were relatively inactive in promoting DNA synthesis of the nonepithelial cells whereas pituitary fibroblast growth factor was more active. These mitogenic effects were obtained when the hormones were added to the medium at near physiological concentrations, and paralleled the known activities of the hormones in control of mammary gland growth and development in the rat.     

Calcitonin gene-related peptide (CGRP) inhibits contractions of the prostatic stroma of the rat but not the guinea-pig

This study investigated the presence and effects of calcitonin gene-related peptide (CGRP) within the rat and guinea-pig prostate glands. Immunohistochemical studies demonstrated that CGRP immunoreactive nerve fibres are sparsely distributed throughout the prostatic fibromuscular stroma in both species. These CGRP immunopositive nerve fibres shared a similar distribution profile but were not colocalized with tyrosine hydroxylase immunopositive nerve fibres which also innervate the prostatic stroma of these species. Nerve terminals within rat and guinea-pig prostatic tissues were electrically field stimulated (60 V, 0.5 ms, 10 Hz, 20 pulses every 60 s). In guinea-pig preparations, application of human alpha-CGRP, rat adrenomedullin or rat amylin (0.1 nM-1 microM) had no effect on responses to field stimulation. In contrast, both rat and human alpha-CGRP (10 pM-300 nM), rat adrenomedullin (0.3 nM-1 microM) and rat amylin (3 nM-1 microM) concentration-dependently inhibited electrically evoked contractile responses in the rat prostate. The relative order of potency was rat alpha-CGRP=human alpha-CGRP>rat adrenomedullin>rat amylin. The inhibition by rat alpha-CGRP of field stimulation-induced contractions in the rat prostate was competitively antagonized by human CGRP((8-37)) (1, 3 and 10 microM) with a pA(2) of 6.20+/-0.13. Rat alpha-CGRP (10 nM) attenuated contractile responses of the rat prostate to exogenously added noradrenaline (1-100 microM). Inhibitory concentration-response curves to rat alpha-CGRP in rat prostates were unaffected by preincubation in either glibenclamide (10-100 microM), N-nitro-L-arginine methyl ester (L-NAME) (10 microM), bestatin (10 microM), captopril (10 microM) or phosphoramidon (3 microM). Our results indicate that CGRP-induced inhibition of electrically evoked contractions in the rat prostate occurs through activation of postjunctional CGRP(2) receptors which act independently of a K(ATP) channel or nitrergic mechanisms. Degradation of rat alpha-CGRP via peptidases does not appear to occur in the rat prostate.     

Analytical description of the activation of multi-state receptors by continuous neurotransmitter signals at brain synapses.

Chemical synaptic transmission is a fundamental component of interneuronal communications in the central nervous system (CNS). Discharge of a presynaptic vesicle containing a few thousand molecules (a quantum) of neurotransmitter into the synaptic cleft generates a transmitter concentration signal that drives postsynaptic ion-channel receptors. These receptors exhibit multiple states, with state transition kinetics dependent on neurotransmitter concentration. Here, a novel and simple analytical approach for describing gating of multi-state receptors by signals with complex continuous time courses is used to describe the generation of glutamate-mediated quantal postsynaptic responses at brain synapses. The neurotransmitter signal, experienced by multi-state N-methyl-D-aspartate (NMDA)- and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors at specific points in a synaptic cleft, is approximated by a series of step functions of different intensity and duration and used to drive a Markovian, multi-state kinetic scheme that describes receptor gating. Occupancy vectors at any point in time can be computed interatively from the occupancy vectors at the times of steps in transmitter concentration. Multi-state kinetic schemes for both the low-affinity AMPA subtype of glutamate receptor and for the high-affinity NMDA subtype are considered, and expected NMDA and AMPA components of synaptic currents are calculated. The amplitude of quantal responses mediated by postsynaptic receptor clusters having specific spatial distributions relative to foci of quantal neurotransmitter release is then calculated and related to the displacement between the center of the postsynaptic receptor cluster and the focus of synaptic vesicle discharge. Using this approach we show that the spatial relation between the focus of release and the center of the postsynaptic receptor cluster affects synaptic efficacy. We also show how variation in this relation contributes to variation in synaptic current amplitudes.     

Contour detection threshold: repeatability and learning with 'contour cards'.

Human observers are able to locate contours that are defined solely on the basis of long-range, orientation-domain correlations. The integrity of the mechanisms responsible for second-order contour detection is disrupted by amblyopia (Kovacs et al., 1996; Hess et al., 1997) and it is therefore of interest to develop methods for assessing pediatric patients undergoing treatment for amblyopia. In this study, we have determined the inter-observer and test-retest reliability of a card-based test of second-order contour integration. The magnitude of practice effects was also assessed in both adult and pediatric patient groups. Contour detection thresholds were measured for a closed contour, defined by Gabor patches, embedded in a randomly oriented Gabor-patch background. The visibility of the contour was controlled by varying the density of the background elements. Thresholds, defined in terms of the ratio of contour element spacing to average background spacing were measured with a clinical staircase procedure. Thresholds measured by two observers differed on average by 0.023 +/- 0.075 or about one half the increment between cards. Children and adults showed only small practice effects (0.022 +/- 0.051 vs 0.053 +/- 0.077, respectively) and average unsigned differences between repeated measures were equivalent to approximately 1 card across groups. A card-based test of second-order contour integration produces reliable estimates of contour integration performance in normal and amblyopic observers, including children.     

Involvement of the Reck tumor suppressor protein in maternal and embryonic vascular remodeling in mice

Background

The accessibility of the developing zebrafish pharyngeal dentition makes it an advantageous system in which to study many aspects of tooth development from early initiation to late morphogenesis. In mammals, hedgehog signaling is known to be essential for multiple stages of odontogenesis; however, potential roles for the pathway during initiation of tooth development or in later morphogenesis are incompletely understood.

Results

We have identified mRNA expression of the hedgehog ligands shha and the receptors ptc1 and ptc2 during zebrafish pharyngeal tooth development. We looked for, but did not detect, tooth germ expression of the other known zebrafish hedgehog ligands shhb, dhh, ihha, or ihhb, suggesting that as in mammals, only Shh participates in zebrafish tooth development. Supporting this idea, we found that morphological and gene expression evidence of tooth initiation is eliminated in shha mutant embryos, and that morpholino antisense oligonucleotide knockdown of shha, but not shhb, function prevents mature tooth formation. Hedgehog pathway inhibition with the antagonist compound cyclopamine affected tooth formation at each stage in which we applied it: arresting development at early stages and disrupting mature tooth morphology when applied later. These results suggest that hedgehog signaling is required continuously during odontogenesis. In contrast, over-expression of shha had no effect on the developing dentition, possibly because shha is normally extensively expressed in the zebrafish pharyngeal region.

Conclusion

We have identified previously unknown requirements for hedgehog signaling for early tooth initiation and later morphogenesis. The similarity of our results with data from mouse and other vertebrates suggests that despite gene duplication and changes in the location of where teeth form, the roles of hedgehog signaling in tooth development have been largely conserved during evolution.