Ab initio molecular dynamics simulations of beta-D-glucose and beta-D-xylose degradation mechanisms in acidic aqueous solution

Ab initio molecular dynamics simulations were employed to investigate, with explicit solvent water molecules, beta-D-glucose and beta-D-xylose degradation mechanisms in acidic media. The rate-limiting step in sugar degradation was found to be protonation of the hydroxyl groups on the sugar ring. We found that the structure of water molecules plays a significant role in the acidic sugar degradation pathways. Firstly, a water molecule competes with the hydroxyl group on the sugar ring for protons. Secondly, water forms hydrogen bonds with the hydroxyl groups on the sugar rings, thus weakening the C-C and C-O bonds (each to a different degree). Note that the reaction pathways could be altered due to the change of relative stability of the C-C and C-O bonds. Thirdly, water molecules that are hydrogen-bonded to sugar hydroxyls could easily extract a proton from the reaction intermediate, terminating the reaction. Indeed, the sugar degradation pathway is complex due to multiple protonation probabilities and the surrounding water structure. Our experimental data support multiple sugar acidic degradation pathways.     

Quantitative assessment of FGF regulation by cell surface heparan sulfates

Heparin/heparan sulfate-like glycosaminoglycans (HSGAGs) modulate the activity of the fibroblast growth factor (FGF) family of proteins. Through interactions with both FGFs and FGF receptors (FGFRs), HSGAGs mediate FGF-FGFR binding and oligomerization leading to FGFR phosphorylation and initiation of intracellular signaling cascades. We describe a methodology to examine the impact of heparan sulfate fine structure and source on FGF-mediated signaling. Mitogenic assays using BaF3 cells transfected with specific FGFR isoforms allow for the quantification of FGF1 and FGF2 induced responses independent of conflicting influences. As such, this system enables a systematic investigation into the role of cell surface HSGAGs on FGF signaling. We demonstrate this approach using cell surface-derived HSGAGs and find that distinct HSGAGs elicit differential FGF response patterns through FGFR1c and FGFR3c. We conclude that this assay system can be used to probe the ability of distinct HSGAG species to regulate the activity of specific FGF-FGFR pairs.     

Evolutionary and biochemical differences between human and monkey acidic mammalian chitinases

Acidic mammalian chitinase (AMCase), an enzyme implicated in the pathology of asthma, is capable of chitin cleavage at a low pH optimum. The corresponding gene (CHIA) can be found in genome databases of a variety of mammals, but the enzyme properties of only the human and mouse proteins were extensively studied. We wanted to compare enzymes of closely related species, such as humans and macaques. In our attempt to study macaque AMCase, we searched for CHIA-like genes in human and macaque genomes. We found that both genomes contain several additional CHIA-like sequences. In humans, CHIA-L1 (hCHIA-L1) is an apparent pseudogene and has the highest homology to CHIA. To determine which of the two genes is functional in monkeys, we assessed their tissue expression levels. In our experiments, CHIA-L1 expression was not detected in human stomach tissue, while CHIA was expressed at high levels. However, in the cynomolgus macaque stomach tissue, the expression pattern of these two genes was reversed: CHIA-L1 was expressed at high levels and CHIA was undetectable. We hypothesized that in macaques CHIA-L1 (mCHIA-L1), and not CHIA, is a gene encoding an acidic chitinase, and cloned it, using the sequence of human CHIA-L1 as a guide for the primer design. We named the new enzyme MACase (Macaca Acidic Chitinase) to emphasize its differences from AMCase. MACase shares a similar tissue expression pattern and pH optimum with human AMCase, but is 50 times more active in our enzymatic activity assay. DNA sequence of the mCHIA-L1 has higher percentage identity to the human pseudogene hCHIA-L1 (91.7%) than to hCHIA (84%). Our results suggest alternate evolutionary paths for human and monkey acidic chitinases.     

Genetic evidence for functional interaction of the Escherichia coli signal recognition particle receptor with acidic lipids in vivo

The mechanism underlying the interaction of the Escherichia coli signal recognition particle receptor FtsY with the cytoplasmic membrane has been studied in detail. Recently, we proposed that FtsY requires functional interaction with inner membrane lipids at a late stage of the signal recognition particle pathway. In addition, an essential lipid-binding α-helix was identified in FtsY of various origins. Theoretical considerations and in vitro studies have suggested that it interacts with acidic lipids, but this notion is not yet fully supported by in vivo experimental evidence. Here, we present an unbiased genetic clue, obtained by serendipity, supporting the involvement of acidic lipids. Utilizing a dominant negative mutant of FtsY (termed NG), which is defective in its functional interaction with lipids, we screened for E. coli genes that suppress the negative dominant phenotype. In addition to several unrelated phenotype-suppressor genes, we identified pgsA, which encodes the enzyme phosphatidylglycerophosphate synthase (PgsA). PgsA is an integral membrane protein that catalyzes the committed step to acidic phospholipid synthesis, and we show that its overexpression increases the contents of cardiolipin and phosphatidylglycerol. Remarkably, expression of PgsA also stabilizes NG and restores its biological function. Collectively, our results strongly support the notion that FtsY functionally interacts with acidic lipids.     

Contrasting response of seedlings of two tropical species Clusia minor and Clusia multiflora to mycorrhizal inoculation in two soils with different pH

Differences in mycotrophic growth and response to phosphorus (P) fertilization were studied in seedlings of two woody native species: Clusia minor L. and Clusia multiflora H.B.K. from a cloud montane forest of tropical America. Greenhouse investigation was undertaken to determine the relationships between mycorrhizal dependency of host species associated with P utilization and growth in two different soils contrasting in pH (acidic and neutral) and nutrient content. Four treatments were performed: sterilized soil; sterilized soil plus 375 mg/kg of triple superphosphate (TSP); sterilized soil inoculated with Scutellospora fulgida (20 g/pot); and sterilized soil plus S. fulgida and TSP, with 10 replications per treatment for the two species. Results showed that both Clusia species presented high growth response to increasing P availability, which indicates that the root morphology (magnolioid roots) of these species is not a limiting factor for the incorporation of P from soils. Plants inoculated with arbuscular mycorrhizal fungi (AMF) in acidic soil had significantly increased shoot and root biomass, leaf area and height, in comparison to the biomass of P-fertilized plants and nonmycorrhizal plants. In neutral soil, seedlings of C. minor and C. multiflora were negatively affected by inoculation with AMF. In contrast, a significant decrease in growth was observed when inoculated plants were compared with noninoculated plants on neutral soil. Results indicate that an increase in the availability of a limiting nutrient (P) can turn a balanced mutualistic relationship into a less balanced nonmutualistic one.     

Comparative study of alkali- and acidic organic solvent-soluble hemicellulosic polysaccharides from sugarcane bagasse

Two-stage treatments of sugarcane bagasse with mild alkali and acidic 1,4-dioxane were performed. Pretreatment with 1M NaOH aqueous solution at 20, 25, 30, 35, and 40 degrees C for 18 h released 55.5%, 57.3%, 59.1%, 60.9%, and 62.1% of the original hemicelluloses, respectively. Post-treatment of the corresponding alkali-treated residue with 1,4-dioxane-2M HCl (9:1, v/v) at 87 degrees C for 2h, respectively, degraded 11.6%, 11.9%, 11.4%, 10.9%, and 10.6% of hemicelluloses (% dry starting material). It was found that the five alkali-soluble hemicellulosic preparations contained a much higher amounts of xylose (78.0-82.2%) and slightly higher uronic acids (4.8-5.8%), mainly 4-O-methyl-alpha-d-glucopyranosyluronic acid, but were lower in arabinose (9.3-11.7%) and glucose (2.2-4.1%) than those of the corresponding five acidic dioxane-degraded hemicellulosic fractions in which xylose (44.9-46.8%), arabinose (35.9-38.1%), and glucose (13.0-13.7%) were the major sugar constituents. The studies revealed that the five alkali-soluble hemicellulosic preparations were more linear and acidic, and had a large molecular weight (35,200-37,430 g mol(-1)) than those of the hemicellulosic fractions (12,080-13,320 g mol(-1)) degraded during the acidic dioxane post-treatment. This demonstrated that the post-treatment with acidic dioxane under the condition used resulted in substantial degradation of the hemicellulosic polymers. The 10 hemicellulosic samples were further characterized by FT-IR and 1H and 13C NMR spectroscopy, GPC and thermal analysis, and the results are reported.     

Charge-based interaction conserved within histone H3 lysine 4 (H3K4) methyltransferase complexes is needed for protein stability, histone methylation, and gene expression

Histone H3 lysine 4 (H3K4) methyltransferases are conserved from yeast to humans, assemble in multisubunit complexes, and are needed to regulate gene expression. The yeast H3K4 methyltransferase complex, Set1 complex or complex of proteins associated with Set1 (COMPASS), consists of Set1 and conserved Set1-associated proteins: Swd1, Swd2, Swd3, Spp1, Bre2, Sdc1, and Shg1. The removal of the WD40 domain-containing subunits Swd1 and Swd3 leads to a loss of Set1 protein and consequently a complete loss of H3K4 methylation. However, until now, how these WD40 domain-containing proteins interact with Set1 and contribute to the stability of Set1 and H3K4 methylation has not been determined. In this study, we identified small basic and acidic patches that mediate protein interactions between the C terminus of Swd1 and the nSET domain of Set1. Absence of either the basic or acidic patches of Set1 and Swd1, respectively, disrupts the interaction between Set1 and Swd1, diminishes Set1 protein levels, and abolishes H3K4 methylation. Moreover, these basic and acidic patches are also important for cell growth, telomere silencing, and gene expression. We also show that the basic and acidic patches of Set1 and Swd1 are conserved in their human counterparts SET1A/B and RBBP5, respectively, and are needed for the protein interaction between SET1A and RBBP5. Therefore, this charge-based interaction is likely important for maintaining the protein stability of the human SET1A/B methyltransferase complexes so that proper H3K4 methylation, cell growth, and gene expression can also occur in mammals.     

八肋游仆虫酸性核糖体蛋白基因克隆与特征分析

原核和真核生物的核糖体大亚基上都存在着一类酸性核糖体蛋白,在大肠杆菌中,分别为L10和L7/L12蛋白,而在真核生物中是P0、P1和P2(简称P蛋白)。这些酸性核糖体蛋白共同组成五聚体复合物P0-(P1/P2)2,在大亚基上形成一个向外侧凸出的柄状结构(Ribosomalstalk)。酸性核糖体蛋白位于核糖体的活性部位,一方面     

妊娠晚期腹腔注射酸性磷酸缓冲液对两个近交系小鼠繁育生理影响的对比分析

目的对比分析妊娠晚期腹腔注射缓冲液对近交系SPF级C57BL/6J（B6）和BALB/c（B/c）小鼠繁育生理的影响。方法 B6和B/c小鼠随机、全同胞兄妹2∶1/1∶1（♀/♂）过夜同居交配,观察交配方式、阴栓与受孕率的关系;受孕鼠在妊娠的第17.5天（妊娠0d=交配后当天）接受腹腔注射酸性磷酸缓冲液,观察注射对母鼠及胚胎的作用及子鼠从离乳到8周的早期生长发育情况。结果 B6较B/c雌鼠的受孕率高（29.4%vs.21.1%[2∶1],33.2%vs.29.7%[1∶1]）;交配后10 d～14 d,根据雌鼠增大的腹部、结合体重来判断受孕较观察阴栓更为准确;比较而言,妊娠晚期腹腔注射对B6母鼠及胚胎的影响较大,表现在离乳子鼠数量减少（4.7±3.1 vs.6.1±2.1,P=0.231）,离乳时两性别的子鼠体重（g,雌性：11.7±1.1 vs.12.7±1.5;雄性：12.8±1.3 vs.13.6±1.5）显著降低（P均〈0.05）及两品系子鼠早期生长发育的方式显著不同（P=0.000）。结论近交系小鼠繁殖生理存在品系差异;两品系受孕鼠对妊娠晚期腹腔注射的耐受不同,并可能影响实验动物产后的哺乳过程及子鼠的早期生长发育。