The role of constant region carbohydrate in the assembly and secretion of human IgD and IgA1

Immunoglobulins are glycoproteins, containing N- linked carbohydrates in the heavy chain constant regions of all isotypes and O-linked carbohydrates in the hinge regions of human IgA1 and IgD. A previous study showed that IgD synthesized in the presence of tunicamycin and lacking the three N-linked glycans on the heavy chain was not secreted (Shin, S. U., Wei, D. F., Amin, A. R., Thorbecke, G. J., and Morrison, S. L. (1992) Hum. Antibodies 3, 65-74). The contribution of each of the carbohydrates in the Fc of IgD to assembly and secretion was now analyzed by eliminating the carbohydrate addition sequence, Asn-X-Ser/Thr, through site-directed mutagenesis. Only the carbohydrate nearest the sole disulfide bond between heavy chains, which remained high mannose and appeared to be buried within the folded molecule, was found to be essential for secretion. When IgD lacked that glycan, assembly reached only the heavy/light chain half-molecule stage, and heavy chains were held inside the endoplasmic reticulum. Using benzyl 2-acetamido-2-deoxy-alpha-d-galactopyranoside (BADG) to inhibit complete O-linked glycosylation, we found that IgA1 and IgD with incomplete hinge carbohydrates were assembled and secreted from cells. Thus, one N-linked glycan plays a structural role in IgD and is required for proper assembly and secretion, but the O-linked carbohydrates in the hinge of IgD and IgA1 are not required for folding and export.     

Determination of enantiomeric compositions of pharmaceutical products by near-infrared spectrometry

A new method for the determination of enantiomeric compositions of a variety of drugs including propranolol, atenolol, and ibuprofen has been developed. The method is based on the use of the near-infrared technique to measure diastereomeric interactions between an added carbohydrate compound and both enantiomeric forms of a drug followed by evaluation of the data by partial least square analysis. The fact that the method works well with all three macrocyclic carbohydrates with different cavity sizes (i.e., alpha-, beta-, and gamma-cyclodextrin) and with sucrose, which is a linear carbohydrate, clearly demonstrates that it is not necessary to have inclusion complex formation to produce effective diastereomeric interactions. Rather a simple adsorption of the drug onto a carbohydrate is sufficient. Since inclusion complex formation is not a requisite, this method is not limited to the three drugs evaluated in this study but is rather universal as it can, in principle, be used for the sensitive and accurate determination of enantiomeric compositions of many different types of drugs with only about 1.5mg/mL concentration and enantiomeric excess as low as 0.80%, in water or in a mixture of water with organic solvent. Furthermore, it does not rely on the use of rather expensive carbohydrates such as cyclodextrins but is equally as effective even with a simple and inexpensive carbohydrate such as sucrose.     

Histone-DNA binding free energy cannot be measured in dilution-driven dissociation experiments

Despite decades of study on nucleosomes, there has been no experimental determination of the free energy of association between histones and DNA. Instead, only the relative free energy of association of the histone octamer for differing DNA sequences has been available. Recently, a method was developed based on quantitative analysis of nucleosome dissociation in dilution experiments that provides a simple practical measure of nucleosome stability. Solution conditions were found in which nucleosome dissociation driven by dilution fit well to a simple model involving a noncooperative nucleosome assembly/disassembly equilibrium, suggesting that this approach might allow absolute equilibrium affinity of the histone octamer for DNA to be measured. Here, we show that the nucleosome assembly/disassembly process is not strictly reversible in these solution conditions, implying that equilibrium affinities cannot be obtained from these measurements. Increases in [NaCl] or temperature, commonly employed to suppress kinetic bottlenecks in nucleosome assembly, lead to cooperative behavior that cannot be interpreted with the simple assembly/disassembly equilibrium model. We conclude that the dilution experiments provide useful measures of kinetic but not equilibrium stability. Kinetic stability is of practical importance: it may govern nucleosome function in vivo, and it may (but need not) parallel absolute thermodynamic stability.     

Lipid production in batch and fed-batch cultures of Rhodosporidium toruloides from 5 and 6 carbon carbohydrates

ABSTRACT: BACKGROUND: Microbial lipids are a potential source of bio- or renewable diesel and the red yeast Rhodosporidium toruloides is interesting not only because it can accumulate over 50% of its dry biomass as lipid, but also because it utilises both five and six carbon carbohydrates, which are present in plant biomass hydrolysates. METHODS: R. toruloides was grown in batch and fed-batch cultures in 0.5 l bioreactors at pH 4 in chemically defined, nitrogen restricted (C/N 40 to 100) media containing glucose, xylose, arabinose, or all three carbohydrates as carbon source. Lipid was extracted from the biomass using chloroform-methanol, measured gravimetrically and analysed by GC. RESULTS: R. toruloides was grown on glucose, xylose, arabinose or mixtures of these carbohydrates in batch and fed-batch, nitrogen restricted conditions. Lipid production was most efficient with glucose (up to 25 g lipid L1, 48 to 75% lipid in the biomass, at up to 0.21 g lipid L1h1) as the sole carbon source, but high lipid concentrations were also produced from xylose (36 to 45% lipid in biomass). Lipid production was low (15-19% lipid in biomass) with arabinose as sole carbon source and was lower than expected (30% lipid in biomass) when glucose, xylose and arabinose were provided simultaneously. The presence of arabinose and/or xylose in the medium increased the proportion of palmitic and linoleic acid and reduced the proportion of oleic acid in the fatty acids, compared to glucose-grown cells. High cell densities were obtained in both batch (37 g L1, with 49% lipid in the biomass) and fed-batch (35 to 47 g L1, with 50 to 75% lipid in the biomass) cultures. The highest proportion of lipid in the biomass was observed in cultures given nitrogen during the batch phase but none with the feed. However, carbohydrate consumption was incomplete when the feed did not contain nitrogen and the highest total lipid and best substrate consumption were observed in cultures which received a constant low nitrogen supply. CONCLUSIONS: Lipid production in R. toruloides was lower from arabinose and mixed carbohydrates than from glucose or xylose. Although high biomass and lipid production were achieved in both batch and fed-batch cultures with glucose as carbon source, for lipid production from mixtures of carbohydrates fed-batch cultivation was preferable. Constant feeding was better than intermittent feeding. The feeding strategy did not affect the relative proportion of different fatty acids in the lipid, but the presence of C5 sugars did.     

1,3-beta-D glucan binding protein (BGBP) from the white shrimp,Penaeus vannamei,is also a heparin binding protein

Shrimp BGBP was purified as a 100 kDa glycoprotein by affinity chromatography using immobilised heparin. BGBP bound simple carbohydrates, glycosaminoglycans like heparin sulphate and glycoproteins, but it was unable to agglutinate erythrocytes. Using an ELISA-based microplate assay, it was shown that simple carbohydrates such as n-glucose and D-mannose are competitive inhibitors of heparin sulphate binding to BGBP. Based on these properties BGBP is considered as a new type of heparin binding protein.     

Dynamics of extractable carbohydrates in Pisum sativum. III. The effect of IAA and temperature on content and translocation of carbohydrates in pea cuttings during rooting

The number of roots formed in cuttings of pea ( Pisum sativum L. cv. Alaska) was regulated both by the temperature and by IAA, whereas the time to the appearance of the first roots was regulated only by the temperature. Cuttings treated with 10^-3 M IAA had a smaller content of extractable carbohydrates than the control ones irrespective of the temperature. In the bases of cuttings rooted at 25°C the content of extractable carbohydrates was lower than in those rooted at 15°C. Cuttings treated with IAA showed up to elevenfold increase of extractable carbohydrates in the bases at day 3. This increase of soluble sugars was not correlated with the number of roots formed or the speed of rooting. It is concluded that IAA affects the accumulation of carbohydrates, and this is not connected with the rooting ability of the cuttings.     

Carbohydrate sources and sinks in woody plants

Each perennial woody plant is a highly integrated system of competing carbohydrate sinks (utilization sites). Internal competition for carbohydrates is shown by changes in rates of carbohydrate movement from sources to sinks and reversals in direction of carbohydrate transport as the relative sink strengths of various organs change. Most carbohydrates are produced in foliage leaves but some are synthesized in cotyledons, hypocotyls, buds, twigs, stems, flowers, fruits, and strobili. Although the bulk of the carbohydrate pool moves to sinks through the phloem, some carbohydrates are obtained by sinks from the xylem sap. Sugars are actively accumulated in the phloem and move passively to sinks along a concentration gradient. The dry weight of a mature woody plant represents only a small proportion of the photosynthate it produced. This discrepancy results not only from consumption of plant tissues by herbivores and shedding of plant parts, but also from depletion of carbohydrates by respiration, leaching, exudation, secretion, translocation to other plants through root grafts and mycorrhizae and losses to parasites. Large spatial and temporal variations occur in the use of reserve- and currently produced carbohydrates in metabolism and growth of shoots, stems, roots, and reproductive structures. A portion of the carbohydrate pool is diverted for production of chemicals involved in defense against fungi, herbivores, and competing plants. Woody plants accumulate carbohydrates during periods of excess production and deplete carbohydrates when the rate of utilization exceeds the rate of production. Stored carbohydrates play an important role in metabolism, growth, defense, cold hardiness, and postponement or prevention of plant mortality.     

Sugar for my honey: carbohydrate partitioning in ectomycorrhizal symbiosis

Simple, readily utilizable carbohydrates, necessary for growth and maintenance of large numbers of microbes are rare in forest soils. Among other types of mutualistic interactions, the formation of ectomycorrhizas, a symbiosis between tree roots and certain soil fungi, is a way to overcome nutrient and carbohydrate limitations typical for many forest ecosystems. Ectomycorrhiza formation is typical for trees in boreal and temperate forests of the northern hemisphere and alpine regions world-wide. The main function of this symbiosis is the exchange of fungus-derived nutrients for plant-derived carbohydrates, enabling the colonization of mineral nutrient-poor environments. In ectomycorrhizal symbiosis up to 1/3 of plant photoassimilates could be transferred toward the fungal partner. The creation of such a strong sink is directly related to the efficiency of fungal hexose uptake at the plant/fungus interface, a modulated fungal carbohydrate metabolism in the ectomycorrhiza, and the export of carbohydrates towards soil growing hyphae. However, not only the fungus but also the plant partner increase its expression of hexose importer genes at the plant/fungus interface. This increase in hexose uptake capacity of plant roots in combination with an increase in photosynthesis may explain how the plant deals with the growing fungal carbohydrate demand in symbiosis and how it can restrict this loss of carbohydrates under certain conditions to avoid fungal parasitism.     

Nectar formation and floral nectary anatomy of Anigozanthos flavidus: a combined magnetic resonance imaging and spectroscopy study

Metabolic processes underlying the formation of floral nectar carbohydrates, especially the generation of the proportions of fructose, glucose, and sucrose, are important for understanding ecological plant-pollinator interactions. The ratio of sucrose-derived hexoses, fructose and glucose, in the floral nectar of Anigozanthos flavidus (Haemodoraceae) was observed to be different from 1:1, which cannot be explained by the simple action of invertases. Various NMR techniques were used to investigate how such an unbalanced ratio of the two nectar hexoses can be formed. High-resolution (13)C NMR spectroscopy in solution was used to determine the proportion of carbohydrates in vascular bundles of excised inflorescences fed with (13)C-labelled carbohydrates. These experiments verified that feeding did not affect the metabolic processes involved in nectar formation. In vivo magnetic resonance imaging (e.g. cyclic J cross-polarization) was used to detect carbohydrates in vascular bundles and (1)H spin echo imaging non-invasively displayed the architecture of tepal nectaries and showed how they are connected to the vascular bundles. A model of the carbohydrate metabolism involved in forming A. flavidus floral nectar was established. Sucrose from the vascular bundles is not directly secreted into the lumen of the nectary but, either before or after invertase-catalysed hydrolyses, taken up by nectary cells and cycled at least partly through glycolysis, gluconeogenesis, and the pentose phosphate pathway. Secretion of the two hexoses in the cytosolic proportion could elegantly explain the observed fructose:glucose ratio of the nectar.     

Sugar sensing and α-amylase gene repression in rice embryos

We used a transient expression system to study the mechanism by which carbohydrates repress a rice (Oryza sativa L.) α-amylase (EC 3.2.1.1) gene. Exogenously fed metabolizable carbohydrates are able to elicit repression of the α-amylase gene RAmy3D in the rice embryo, and our results indicate that repression is also triggered efficiently by endogenous carbohydrates. Glucose analogs that are taken up by plant cells but not phosphorylated by hexokinase are unable to repress the α-amylase gene studied, while 2-deoxyglucose, which is phosphorylable but not further metabolized, down-regulates RAmy3D promoter activity, indicating a role for hexokinase in the sugar-sensing mechanism triggering repression of the RAmy3D gene. We tested two different hexokinase inhibitors, mannoheptulose and glucosamine, but only the latter was able to relieve RAmy3D promoter activity from repression by endogenous carbohydrates. This correlates with the higher ability of glucosamine to inhibit the activity of rice hexokinases in vitro. The glucosamine-mediated relief of RAmy3D promoter activity from repression by endogenous carbohydrates does not correlate with a reduced rate of carbohydrate utilization. Received: 22 April 1997 / Accepted: 9 September 1997     

Carbohydrates Facilitate Correct Disulfide Bond Formation and Folding of Rotavirus VP7

It is well established that glycosylation is essential for assembly of enveloped viruses, but no information is yet available as to the function of carbohydrates on the nonenveloped but glycosylated rotavirus. We show that tunicamycin and, more pronouncedly, a combination of tunicamycin and brefeldin A treatment caused misfolding of the luminal VP7 protein, leading to interdisulfide bond aggregation. While formation of VP7 aggregates could be prevented under reducing conditions, they reoccurred in less than 30 min after a shift to an oxidizing milieu. Furthermore, while glycosylated VP7 interacted during maturation with protein disulfide isomerase, nonglycosylated VP7 did not, suggesting that glycosylation is a prerequisite for protein disulfide isomerase interaction. While native NSP4, which does not possess S-S bonds, was not dependent on N-linked glycosylation or on protein disulfide isomerase assistance for maturation, nonglycosylated NSP4 was surprisingly found to interact with protein disulfide isomerase, further suggesting that protein disulfide isomerase can act both as an enzyme and as a chaperone. In conclusion, our data suggest that the major function of carbohydrates on VP7 is to facilitate correct disulfide bond formation and protein folding.     

The characterization of the uptake of avidin-biotin complex by HeLa cells

HeLa cells have been shown to internalize the avidin-biotin complex. Adsorptive pinocytosis seems to be the mechanism of this uptake as seen by the requirements of energy and the integrity of the microtubular assembly. Pretreatment of HeLa cells with cycloheximide inhibits uptake and binding of the avidin-biotin complex. Scatchard plot of specific binding of avidin indicates a single type of binding with a K_d of approx. 55 pM with about 21 500 receptors/cell. The lack of inhibition of binding by simple carbohydrates indicates that binding is not through the oligosaccharide chain of avidin.     

Role for fucose-sulfate-rich carbohydrates in the penetration of zona-pellucida-free hamster eggs by hamster spermatozoa

The interaction of acrosome-reacted hamster spermatozoa and zona-pellucida-free hamster eggs was investigated by incubating gametes in the presence of a variety of simple and complex carbohydrates. Significant inhibition of gamete fusion was achieved only in the presence of fucoidan and ascophyllin, two algal polysaccharides containing fucose sulfate. These compounds did not interfere with sperm motility, capacitation, or acrosome reactions. It is concluded that these two compounds share common structural features with putative cell-surface carbohydrates involved in sperm-oolemmal interaction.     

In vitro screening for molecules that affect virus capsid assembly (and other protein association reactions)

Protein self-assembly is critical for numerous biological processes. Yet, assembly is rarely targeted by therapeutic agents, in part because it is hard to identify molecules that interfere with protein-protein interactions. Here, we describe a simple fluorescence-based screen for self-association and its application to the assembly of hepatitis B virus capsids. These data are analyzed to identify kinetic and thermodynamic effects--both of which are critical for the viral lifecycle and for understanding the mechanism of assembly effectors. Suggestions are made for modification of this protocol so that it can be applied to other self-assembling systems. With manual pipetting, setting up a plate takes about 2 h, the initial reading takes 1 h and the end point reading the following day takes about 5 min.     

De novo kinetochore assembly requires the centromeric histone H3 variant

Kinetochores mediate chromosome attachment to the mitotic spindle to ensure accurate chromosome segregation. Budding yeast is an excellent organism for kinetochore assembly studies because it has a simple defined centromere sequence responsible for the localization of >65 proteins. In addition, yeast is the only organism where a conditional centromere is available to allow studies of de novo kinetochore assembly. Using a conditional centromere, we found that yeast kinetochore assembly is not temporally restricted and can occur in both G1 phase and prometaphase. We performed the first investigation of kinetochore assembly in the absence of the centromeric histone H3 variant Cse4 and found that all proteins tested depend on Cse4 to localize. Consistent with this observation, Cse4-depleted cells had severe chromosome segregation defects. We therefore propose that yeast kinetochore assembly requires both centromeric DNA specificity and centromeric chromatin.     

植物非结构性贮藏碳水化合物的生理生态学研究进展

非结构性碳水化合物是参与植物生命过程的重要物质。蔗糖不仅是植物体内碳水化合物运输的主要形式,而且可以在基因表达水平上对细胞内的代谢进行调节。果聚糖是植物营养组织碳水化合物的主要暂贮形式;淀粉是植物主要的长期贮存物质之一。植物体内非结构性碳水化合物的代谢在很大程度上影响着植株的生长发育和对环境因子的响应。综述了植物非结构性贮藏碳水化合物的生理生态学研究进展,着重介绍了蔗糖,果聚糖和淀粉代谢的生理过程及对环境因子（温度和水分）和人为因素的响应机制。     

Role of N glycosylation of hepatitis B virus envelope proteins in morphogenesis and infectivity of hepatitis delta virus

Hepatitis delta virus (HDV) particles are coated with the large (L), middle (M), and small (S) hepatitis B virus envelope proteins. In the present study, we constructed glycosylation-defective envelope protein mutants and evaluated their capacity to assist in the maturation of infectious HDV in vitro. We observed that the removal of N-linked carbohydrates on the S, M, and L proteins was tolerated for the assembly of subviral hepatitis B virus (HBV) particles but was partially inhibitory for the formation of HDV virions. However, when assayed on primary cultures of human hepatocytes, virions coated with S, M, and L proteins lacking N-linked glycans were infectious. Furthermore, in the absence of M, HDV particles coated with nonglycosylated S and L proteins retained infectivity. These results indicate that carbohydrates on the HBV envelope proteins are not essential for the in vitro infectivity of HDV.     

金针菇基因组测序与组装策略分析

采用二代和三代测序技术分别对金针菇单核体菌株“6-3”进行测序,应用4种组装策略进行基因组的de novo组装,对比组装效果。基因组组装的参数方面,仅使用二代测序组装的效果最差,长度大于10kb的Contig全长只有24.6Mb,Contig N50只有23kb,组装率只有59.27%。采用三代组装二代校正的组装策略效果最好,长度大于10kb的Contig全长为38.3Mb,Contig N50为2.8Mb,组装率高达92.16%。保守单拷贝基因拼接效果方面,4种组装策略获得基因组序列与BUSCO数据库里的担子菌的保守单拷贝基因比对,基因完整性均大于94%。在组装准确性方面,经过PCR扩增、Sanger测序验证,三代组装二代校正的基因组序列完整并且连续,同时序列上碱基的SNP、InDel数量最少。综上所述,三代组装二代校正得到的基因组序列具有Contig N50值大、组装率高、碱基准确性高的特点,是食用菌基因组测序较为理想的方案。     

Graphene Multilayer Supported Gold Nanoparticles for Efficient Electrocatalysts Toward Methanol Oxidation

This study reports a simple method of integrating electroactive gold nanoparticles (Au NPs) with graphene oxide (GO) nanosheet support by layer‐by‐layer (LbL) assembly for the creation of 3‐dimensional electrocatalytic thin films that are active toward methanol oxidation. This approach involves the alternating assembly of two oppositely charged suspensions of Au NPs with GO nanosheets based on electrostatic interactions. The GO nanosheets not only serve as structural components of the multilayer thin film, but also potentially improve the utilization and dispersion of Au NPs by taking advantages of the high catalytic surface area and the electronic conduction of graphene nanosheets. Furthermore, it is found that the electrocatalytic activity of the multilayer thin films of Au NPs with graphene nanosheet is highly tunable with respect to the number of bilayers and thermal treatment, benefiting from the advantageous features of LbL assembly. Because of the highly versatile and tunable properties of LbL assembled thin films coupled with electrocatalytic NPs, we anticipate that the general concept presented here will offer new types of electroactive catalysts for direct methanol fuel cells.     

Ultrathin hydrogel films for rapid optical biosensing

Novel biosensors have been designed by reporting an analyte-induced (de)swelling of a stimuli-responsive hydrogel (usually in a form of thin film) with a suitable optical transducer. These simple, inexpensive hydrogel biosensors are highly desirable, however, their practical applications have been hindered, largely because of their slow response. Here we show that quick response hydrogel sensors can be designed from ultrathin hydrogel films. By the adoption of layer-by-layer assembly, a simple but versatile approach, glucose-sensitive hydrogel films with thickness on submicrometer or micrometer scale, which is 2 orders of magnitude thinner than films used in ordinary hydrogel sensors, can be facilely fabricated. The hydrogel films can not only respond to the variation in glucose concentration, but also report the event via the shift of Fabry-Perot fringes using the thin film itself as Fabry-Perot cavity. The response is linear and reversible. More importantly, the response is quite fast, making it possible to be used for continuous glucose monitoring.