Isolation and partial characterization of human erythrocyte membrane NADH: (Acceptor) oxidoreductase

Gengliosides generally provide a small portion of the complex carbohydrate content of cell surfaces. An exception is the central nervous system where they comprise up to 5–10% of the total lipid of some membranes. This tissue is unique in that the quantity of lipid-bound sialic acid exceeds that of the protein-bound fraction. Over 30 different molecular species have been characterized to date. These range in complexity from sialosylgalactosyl ceramide with 2 sugars to the pentasialoganglioside of fish brain with 9 carbohydrate units. Virtually all cellular and subcellular fractions of brain that have been carefully examined contain gangliosides to one degree or another, but the majority of brain ganglioside is located in the neurons. Their mode of distribution within the neuron has not been entirely clarified by subcellular studies. Calculations based on reported values for axon terminal density and synaptosomal ganglioside concentration in the rat reveal that nerve endings contribute less than 12% of total cerebral cortical ganglioside. It is concluded that the plasma membranes of neuronal processes contain most of the neuronal ganglioside. These and other considerations suggest the possibility that gangliosides may be distributed over the entire neuronal surface.     

Synthesis of a polymide containing a glucose unit in the main chain

A new type polyamide containing a glucose unit in the main chain has been synthesized by the polymerization of C₁, C₃, C₄ blocked C₆-carboxymethylglucosamine, prepared from chitin. The deblocking procedure gave the water-soluble polyamide, of MW 1.5 × 10⁴, which can be regarded as a model for the recognition site of lectin.     

壳聚糖固定化木瓜蛋白酶的研究

以壳聚糖为载体,成二醛为交联剂将木瓜蛋白酶固定化。5％戊二醛在4-6℃下处理载体5h,加酶液(3.5mg/mL蛋白,pH7.2)固定12h,活力回收达32％,作用于酪蛋白的半衰期为36天,其表观K_m(酪蛋白)值为0.075％(W/V),溶液酶的K_m值为0.086％;最适pH7.0～7.5,溶液酶为7.0～8.5。固定化酶在pH8.5以下,溶液酶在9.0以下活力稳定。固定化酶在45℃以下,溶液酶在75℃以下稳定。用6mol/L脲洗脱固定化酶4次(5.5h)活力仍有54.5％。用固定化酶处理啤酒浊度比对照下降了1.5-3.7倍,蛋白质含量下降了44％,冷藏(4℃)120天无冷混浊现象发生并保持了啤酒原有风味和理化性状。     

Dolichyl phosphate linked sugars as intermediates in the synthesis of yeast mannoproteins: an in vivo study

Freshly prepared protoplasts of Saccharomyces cerevisiae X 2180 incorporate [³H]mannose and [¹⁴C]glucose for about 30 min into glycolipids and mannoproteins. Among the radioactive glycolipids formed dolichyl phosphate mannose, dolichyl phosphate glucose and dolichyl pyrophosphate oligosaccharides have been identified. The oligosaccharides released by weak acid from the dolichyl pyrophosphate were treated with endo-N-acetylglucosaminidase H and separated by gel filtration on Bio-Gel P-4. The largest oligosaccharide obtained corresponded exactly in size to Glc₃Man₉GlcNAc₁ the compound formed also in animal tissues. Other oligosaccharides released from dolichyl pyrophosphate in addition to the glucose containing ones were mainly Man₉GlcNAc₁ and Man₈GlcNAc₁. No mannosyl oligosaccharide corresponding in size to the total inner core region found in native mannoproteins could be detected in a lipid-bound form.The radioactive dolichyl pyrophosphate oligosaccharides were formed transiently; after 40 min only about 40% of the maximal radioactivity was observed in this fraction. In the presence of cycloheximide this decrease did not take place.It is concluded that the dolichol pathway of N-glycosylation of glycoproteins in yeast cells is very similar, if not identical, to the reaction sequence worked out for animal cells.Dedicated to Professor Dr. Otto Kandler on his 60th birthday     

Method for Purification of Fluorescence-Labeled Oligosaccharides by Pyridylamination

We developed a convenient method for purification of PA-oligosaccharides to remove contaminants originating from natural fluorescent materials, and excess reagents as well as by-products of tagging reactions in glycan analysis. The method, using a C18-cartridge, is simple and powerful to remove them. Several examples of experiments that showed the usefulness of this purification method are described in this report.     

Stereochemistry of Cervicarcin

The relative stereochemistry of cervicarcin, an antitumor antibiotic, was determined as shown in 1, which represents the absolute stereochemistry also.     

Differences in the chitinolytic activity of mammalian chitinases on soluble and insoluble substrates

Chitin is an abundant polysaccharide used by many organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Acidic mammalian chitinase, a processive glycosyl hydrolase, is the primary enzyme involved in the degradation of environmental chitin in mammalian lungs. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion in mice increases morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a screening approach with commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with insoluble chitin, and identified a one‐pot fluorogenic assay that is sufficiently sensitive to quantify changes to activity due to the addition or removal of a carbohydrate‐binding domain. We show that the activity increases from our directed evolution screen were lost when insoluble substrates were used. In contrast, naturally occurring gain‐of‐function mutations gave similar results with oligomeric and insoluble substrates. We also show that activity differences between acidic mammalian chitinase and chitotriosidase are reduced with insoluble substrate, suggesting that previously reported activity differences with oligomeric substrates may have been driven by differential substrate specificity. These results highlight the need for assays against physiological substrates when engineering metabolic enzymes, and provide a new one‐pot assay that may prove to be broadly applicable to engineering glycosyl hydrolases.     

Chitin and chitosan biopolymer production from the Iranian medicinal fungus Ganoderma lucidum: Optimization and characterization

Abstract

Chitin and chitosan with unique properties and numerous applications can be produced from fungus. The production of chitin and chitosan from the mycelia of an Iranian Ganoderma lucidum was studied to improve cell growth and chitin productivity. Inoculum size and initial pH as two effective variables on the growth of G. lucidum and chitin production were optimized using response surface method (RSM) by central composite design (CCD). The results verified the significant effect of these two variables on the cell growth and chitin production. In optimum conditions, including pH?=?5.7 and inoculum size of 7.4%, the cell dry weight was 5.91?g/L and the amount of chitin production was 1.08?g/L with the productivity of 0.083?g/(L day). The produced chitin and chitosan were characterized using XRD and FTIR. Moreover, the antibacterial activity of the produced chitosan was investigated and compared with the commercial chitosan. The results showed that the produced chitin and chitosan had suitable quality and the Iranian G. lucidum would be a great source for safe and high-quality chitin and chitosan production.     

Role of LysM receptors in chitin-triggered plant innate immunity

Recent research findings clearly indicate that lysin motif (LysM)-containing cell surface receptors are involved in the recognition of specific oligosaccharide elicitors (chitin and peptidoglycan), which trigger an innate immunity response in plants. These receptors are either LysM-containing receptor-like kinases (LYKs) or LysM-containing receptor proteins (LYPs). In Arabidopsis, five LYKs (AtCERK1/AtLYK1 and AtLYK2–5) and three LYPs (AtLYP1–3) are likely expressed on the plasma membrane. In this review, we summarize recent research results on the role of these receptors in plant innate immunity, including the recent structural characterization of AtCERK1 and composition of the various receptor complexes in Arabidopsis.