Turnover of Free Sialic Acid, CMP-Sialic Acid, and Bound Sialic Acid In Rat Brain

Abstract: Adult male rats were injected intraventricularly with N-[³H]acetylmannosamine. After different time intervals the rats were killed and free sialic acid, CMP-sialic acid, lipid- and protein-bound sialic acid were isolated from brain and the specific radioactivities determined. Maximal specific radioactivity was reached after approximately 4 h for CMP-sialic acid, after 10–12 h for free sialic acid and after approximately 42 h for lipid-and protein-bound sialic acid. After some days the specific radioactivities of all four pools were the same and decreased equally, with a calculated turnover rate of approximately 3.5 weeks. The conclusion was that this phenomenon was the result of reutilisation of sialic acid and/or precursors. Therefore, the calculated turnover is not the turnover of bound sialic acid, but merely the rate of leakage of sialic acid and/or precursors out of the brain, so that no real turnover can be measured by this method. The first few hours after injection the specific radioactivity of CMP-sialic acid rose above that of free sialic acid. It is supposed that a compartmentalization exists of free sialic acid. The newly synthesized sialic acid molecules are not secreted into the cytoplasmic pool but are preferentially used for the synthesis of CMP-sialic acid. The results and conclusions are discussed in view of the general problems concerning turnover measurements of glycoconjugates.     

Diversity of Microbial Sialic Acid Metabolism

Sialic acids are structurally unique nine-carbon keto sugars occupying the interface between the host and commensal or pathogenic microorganisms. An important function of host sialic acid is to regulate innate immunity, and microbes have evolved various strategies for subverting this process by decorating their surfaces with sialylated oligosaccharides that mimic those of the host. These subversive strategies include a de novo synthetic pathway and at least two truncated pathways that depend on scavenging host-derived intermediates. A fourth strategy involves modification of sialidases so that instead of transferring sialic acid to water (hydrolysis), a second active site is created for binding alternative acceptors. Sialic acids also are excellent sources of carbon, nitrogen, energy, and precursors of cell wall biosynthesis. The catabolic strategies for exploiting host sialic acids as nutritional sources are as diverse as the biosynthetic mechanisms, including examples of horizontal gene transfer and multiple transport systems. Finally, as compounds coating the surfaces of virtually every vertebrate cell, sialic acids provide information about the host environment that, at least in Escherichia coli, is interpreted by the global regulator encoded by nanR. In addition to regulating the catabolism of sialic acids through the nan operon, NanR controls at least two other operons of unknown function and appears to participate in the regulation of type 1 fimbrial phase variation. Sialic acid is, therefore, a host molecule to be copied (molecular mimicry), eaten (nutrition), and interpreted (cell signaling) by diverse metabolic machinery in all major groups of mammalian pathogens and commensals.     

型糖尿病患者红细胞膜中性糖、氨基糖及唾液酸含量的改变

 为阐明Ⅱ型糖尿病（ Ｎ Ｉ Ｄ Ｄ Ｍ ）患者红细胞膜的化学组成在非酶糖基化（ Ｎ Ｅ Ｇ）影响下发生的变化,采用毛细管气相色谱法和分光光度法测定了 ２０ 名正常人和 １９ 例Ⅱ型糖尿病患者红细胞膜的 ４ 种结合中性糖与 ２ 种氨基糖和唾液酸含量以及 ４ 种寡糖链上的末端中性糖和唾液酸含量．结果表明, Ｎ Ｉ Ｄ Ｄ Ｍ 患者红细胞膜几种结合单糖（ Ｇｌｃ, Ｆｕｃ, Ｇｌｃ Ａ, Ｇａｌ Ａ）与游离单糖（ Ｇｌｃ, Ｇａｌ, Ｍａｎ, Ｆｕｃ）含量以及唾液酸含量均较正常对照组明显降低（ Ｐ＜ ００１ 或 ００５）．据此推测,由于患者红细胞膜蛋白的重度糖基化导致某些膜结构蛋白的氧化损伤,细胞膜糖类含量的减少,可能是重度糖基化的继发后果．     

Serum Fluoride and Sialic Acid Levels in Osteosarcoma

Osteosarcoma is a rare malignant bone tumor most commonly occurring in children and young adults presenting with painful swelling. Various etiological factors for osteosarcoma are ionizing radiation, family history of bone disorders and cancer, chemicals (fluoride, beryllium, and vinyl chloride), and viruses. Status of fluoride levels in serum of osteosarcoma is still not clear. Recent reports have indicated that there is a link between fluoride exposure and osteosarcoma. Glycoproteins and glycosaminoglycans are an integral part of bone and prolonged exposure to fluoride for long duration has been shown to cause degradation of collagen and ground substance in bones. The present study was planned to analyze serum fluoride, sialic acid, calcium, phosphorus, and alkaline phosphatase levels in 25 patients of osteosarcoma and age- and sex-matched subjects with bone-forming tumours other than osteosarcoma and musculo-skeletal pain (controls, 25 each). Fluoride levels were analyzed by ISE and sialic acid was analyzed by Warren’s method. Mean serum fluoride concentration was found to be significantly higher in patients with osteosarcoma as compared to the other two groups. The mean value of flouride in patients with other bone-forming tumors was approximately 50% of the group of osteosarcoma; however, it was significantly higher when compared with patients of group I. Serum sialic acid concentration was found to be significantly raised in patients with osteosarcoma as well as in the group with other bone-forming tumors as compared to the group of controls. There was, however, no significant difference in the group of patients of osteosarcoma when compared with group of patients with other bone-forming tumors. These results showing higher level of fluoride with osteosarcoma compared to others suggesting a role of fluoride in the disease.     

A Sialic Acid Binding Site in a Human Picornavirus

The picornaviruses coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac) for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 Å resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC.     

PLT,MPV,PDW对单采血小板产品聚集的影响

目的：探讨血小板含量（PLT）,血小板平均分布宽度（PDW）,血小板平均体积（MPV）对单采血小板聚集的影响,方法：随机抽取68例血小板捐献者,均成功捐献单采血小板,分为聚集组和对照组。献血前抽取静脉血,采用麦道尼克CA620血细胞分析仪进行血细胞分析,检测PLT,MPV,PDW。结果：聚集组（%）PDW 17.5±1.8,高于对照组（%）PDW12.1±0.9（P〈0.05）。聚集组（fl）MPV 11.0±0.9高于对照组（fl）MPV 7.8±0.8（P〈0.05）,差异有统计学意义,聚集组PLT（187±13.3）×109,对照组PLT（195±11.0）×109,二者无明显统计学差异（P〉0.05）。MPV,PDW分别与PLT进行相关性分析,PLT与MPV无显著相关（r=0.132,P〉0.05）;PLT与PDW无显著相关（r=0.147,P〉0.05）。结论：单采血小板产品出现聚集,其捐献者PDW,MPV高于单采产品正常捐献者,与献血者PLT计数无明显关系。     

Changes in the Level of Sialic Acid in Plasma,Brain and Liver of Inherently Scorbutic Rats during Vitamin C and E Deficiencies

The plasma level of sialic acid (NeuAc) in inherently scorbutic [Osteogenic Disorder Shionogi (ODS)] rats was increased by 21 days of vitamin C deficiency and simultaneous vitamins C and E deficiency. The brain content of NeuAc was decreased by deficiencies of these vitamins. The NeuAc level in the liver was not affected significantly by these deficiencies.     

Alkali-Labile, Sodium Borohydride-Reducible Ganglioside Sialic Acid Residues in Brain

Abstract: We have shown that ganglioside internal esters, reduced with sodium borohydride and hydrolyzed with mild acid, form nonulosamine and glycosan, whereas ester-free gangliosides yield only sialic acid when similarly treated. In an effort to demonstrate the occurrence of ganglioside internal esters in brain tissue, brain homogenates and brain ganglioside fractions were treated with NaB³H₄. The gangliosides were then hydrolyzed with mild acid and unlabeled carrier nonulosamine and its glycosan were added. The nonulosamine was purified to constant specific radioactivity. Homogenates and ganglioside fractions, initially treated with alkali and then similarly reduced and analyzed, provided control values. Ganglioside fractions directly reduced consistently gave nonulosamine with higher specific radioactivities than controls. A larger quantity of tissue was processed to allow the isolation of chemically measurable amounts of nonulosamine. The amount of nonulosamine formed by reduction of the crude ganglioside fraction was estimated by isotope dilution analysis. The quantity of nonulosamine formed from reduced untreated ganglioside fractions was about sevenfold that formed from alkali-treated fractions. These data provide evidence for the existence in brain tissue of ganglioside sialic acid residues in which the carboxyl group is bound in a structure that is alkali-labile and reducible with sodium borohydride.     

Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form.     

Effect of Erucic Acid on Platelets in Patients with Adrenoleukodystrophy

In a clinical trial for the management of adrenoleukodystrophy, we analyzed the effect of erucic acid (a component of Lorenzo's oil) on platelet number, fatty acid composition, and function. Analysis of variance was performed to compare platelet counts before starting treatment with Lorenzo's oil and at 6 and 12 months. We measured platelet fatty acid composition in subjects and control patients and correlated these values with their platelet counts using discriminant analysis. After 6 months, the mean platelet count decreased from 247,000/mm³to 169,000/mm³(±1 standard deviation 58,000,n= 39),P< 0.0001 compared to 18 subjects on a control diet having a mean baseline platelet count of 259,000/mm³(±1 standard deviation 67,000,n= 19) and at 6 months 267,000/mm³(±1 standard deviation 71,000). We found atP< 0.05 that the platelet counts showed a strong inverse relationship with erucic acid levels and other omega 9 fatty acids that form from the administration of the erucic acid component of Lorenzo's oil. Morphologic and platelet sizing measurements suggest that the physical properties of platelets may also be affected by erucic acid. Our studies show that the ingestion of erucic acid affects platelet biology. This indicates that platelet counts and properties are influenced by monounsaturated fatty acids, in addition to the well-known effects of polyunsaturated fatty acids. In areas of the world where erucic acid is widely ingested, the biology of platelets in these populations may be affected.     

Aggregation of Deoxyribonucleic Acid and the Helping Effect in Transformation

Deoxyribonucleic acid aggregates in the presence of a component of Neopeptone at low ionic strength and thereby loses its ability to bind to competent bacteria. The reversible and concentration-dependent nature of this aggregation is the basis of the helping effect in transformation.     

Sirtuin Inhibition Induces Apoptosis-like Changes in Platelets and Thrombocytopenia

Sirtuins are evolutionarily conserved NAD⁺-dependent acetyl-lysine deacetylases that belong to class III type histone deacetylases. In humans, seven sirtuin isoforms (Sirt1 to Sirt7) have been identified. Sirtinol, a cell-permeable lactone ring derived from naphthol, is a dual Sirt1/Sirt2 inhibitor of low potency, whereas EX-527 is a potent and selective Sirt1 inhibitor. Here we demonstrate that Sirt1, Sirt2, and Sirt3 are expressed in enucleate platelets. Both sirtinol and EX-527 induced apoptosis-like changes in platelets, as revealed by enhanced annexin V binding, reactive oxygen species production, and drop in mitochondrial transmembrane potential. These changes were associated with increased phagocytic clearance of the platelets by macrophages. Expression of acetylated p53 and the conformationally active form of Bax were found to be significantly higher in both sirtinol- and EX-527-treated platelets, implicating the p53-Bax axis in apoptosis induced by sirtuin inhibitors. Administration of either sirtinol or EX-527 in mice led to a reduction in both platelet count and the number of reticulated platelets. Our results, for the first time, implicate sirtuins as a central player in the determination of platelet aging. Because sirtuin inhibitors are being evaluated for their antitumor activity, this study refocuses attention on the potential side effect of sirtuin inhibition in delimiting platelet life span and management of thrombosis.     

Substituted Sialic Acid Prosthetic Groups as Determinants of Viral Hemagglutination

Inhibitors of hemagglutination by type A2 influenza virus and a recently isolated strain of type B influenza virus were separated by sucrose density gradient centrifugation and agarose gel filtration from horse serum. Using selected reagents, it was demonstrated that the active substituent on the horse serum inhibitor of A2 influenza virus was 4-O-acetyl-N-acetylneuraminic acid; however, the active substituent on the inhibitor of the influenza B virus was shown to be N-acetylneuraminic acid (NANA). Sodium metaperiodate treatment of a component of horse serum resulted in a 10 to 15-fold enhancement of inhibitory activity against the type B virus, whereas the A2 inhibitor was completely destroyed. Since this enhancement did not occur with influenza B viruses isolated prior to 1965, it was considered that this sensitivity to an oxidized NANA glycoside may have been a reflection of an antigenic change which occurred at that time. The use of different virus strains and selected chemical reagents to define the important sialic acid prosthetic groups active in inhibition was described.