Purification of the O-glycosylated protein p135 and identification as O-GlcNAc transferase.

We have previously shown that rat pancreatic islets contain a predominant 135 kDa O-glycosylated protein (p135) that is recognized by immunoprecipitation and Western blotting with anti-O-GlcNAc antibody. In this paper, we show that p135 is also detectable in other rat tissues including brain, heart, liver, spleen, and lung, but not kidney. To identify p135, the protein was purified from rat brain using a multistep procedure including selective absorption with anti-O-GlcNAc antibody. After electrophoresis, and Coomassie staining, the protein was excised from the gel for tryptic digestion. Next, O-methylisourea was used to convert lysine residues to homoarginine to increase the sequence coverage, and MALDI-TOF mass spectrometry detection was performed. MALDI-TOF identified p135 as rat O-GlcNAc transferase (OGT), an identity confirmed by LC/MS of individual peptides. The identification of p135 as OGT is consistent with previous reports of the tissue distribution of OGT, as well as reports that OGT is itself O-glycosylated.     

Probing the role of water in lamellar bone by dehydration in the environmental scanning electron microscope

Water, collagen and mineral are the three major components of bone. The structural organization of water and its functions within the bone were investigated using the environmental scanning electron microscope and by analyzing dimensional changes that occur when fresh equine osteonal bone is dehydrated and then rehydrated. These changes are attributed mainly to loss of bulk and weakly bound water. In longitudinal sections a contraction of 1.2% was observed perpendicular to the lamellae, whereas no contraction occurred parallel to the lamellae. In transverse sections a contraction of 1.4% was observed both parallel and perpendicular to the lamellae. SEM back scattered electron images showed that about half of an individual lamella is less mineralized, and thus has more water than the other half. We therefore propose that contractions perpendicular to lamellae are due to the presence of more water-filled rather than mineral-filled channels within the mineralized collagen fibril arrays. As these channels are also aligned with the crystal planes, the crystal arrays, oriented as depicted in the rotated plywood model for lamellar bone, facilitate or hinder contraction in different directions.     

Isolation and characterization of calcium-accumulating matrix vesicles from chondrocytes of chicken epiphyseal growth plate cartilage in primary culture

Matrix vesicles (MV) can be readily isolated from culture media of chicken growth plate hypertrophic chondrocytes grown in primary culture. The chondrocytes maintain normal morphology and synthesize type II collagen throughout the culture period. The culture-derived MV are morphologically indistinguishable from MV seen in situ and are rich in alkaline phosphatase. Formation of alkaline phosphatase-rich MV is strongly influenced by the stage of culture: large numbers are released shortly after cell seeding; marked decline is seen during cell spreading and rapid cell division; notable resurgence in alkaline phosphatase-rich MV production occurs as the cells attain confluency. Increasing the initial chondrocyte seeding density proportionately increases MV production. Cells derived from the hypertrophic region are much more capable of forming alkaline phosphatase-rich MV than those from the proliferating zone, indicating that MV formation is dependent on cellular differentiation. MV released by the cultured chondrocytes were compared in protein and phospholipid composition and in their ability to accumulate mineral ions, with plasma membrane fractions and collagenase-released MV obtained from the same tissue. Electrophoretic patterns of proteins, and the phospholipid profiles, suggest that significant modification of the plasma membrane occurs during MV formation. The vesicles are capable of accumulating large amounts of mineral ions from a metastable synthetic cartilage lymph when supplied with alkaline phosphatase substrates. This culture system thus appears to be a useful model for isolating native MV and characterizing factors required for vesicle formation and mineralization.     

Terrestrial venomous animals,the envenomings they cause,and treatment perspectives in the Middle East and North Africa

The Middle East and Northern Africa, collectively known as the MENA region, are inhabited by a plethora of venomous animals that cause up to 420,000 bites and stings each year. To understand the resultant health burden and the key variables affecting it, this review describes the epidemiology of snake, scorpion, and spider envenomings primarily based on heterogenous hospital data in the MENA region and the pathologies associated with their venoms. In addition, we discuss the venom composition and the key medically relevant toxins of these venomous animals, and, finally, the antivenoms that are currently in use to counteract them. Unlike Asia and sub-Saharan Africa, scorpion stings are significantly more common (approximately 350,000 cases/year) than snakebites (approximately 70,000 cases/year) and present the most significant contributor to the overall health burden of envenomings, with spider bites being negligible. However, this review also indicates that there is a substantial lack of high-quality envenoming data available for the MENA region, rendering many of these estimates speculative. Our understanding of the venoms and the toxins they contain is also incomplete, but already presents clear trends. For instance, the majority of snake venoms contain snake venom metalloproteinases, while sodium channel–binding toxins and potassium channel–binding toxins are the scorpion toxins that cause most health-related challenges. There also currently exist a plethora of antivenoms, yet only few are clinically validated, and their high cost and limited availability present a substantial health challenge. Yet, some of the insights presented in this review might help direct future research and policy efforts toward the appropriate prioritization of efforts and aid the development of future therapeutic solutions, such as next-generation antivenoms.     

Investigation of the spinnability of cellulose/alkaline ferric tartrate solutions

Dynamic rheology, UV/VIS spectrometry with temperature programming cuvette and reaction calorimetry were conducted on cellulose pulp/FeTNa (NaOH solution containing ferric tartaric acid complex) solutions to investigate their thermostability and spinnability. Color of cellulose/FeTNa solutions changed above 90 °C due to the decomposition of the complex. Thermal activity of cellulose/FeTNa solution started above 130 °C induced by water vapor evolution and complex decomposition. Regeneration of cellulose/FeTNa solutions in a non-solvent (acetic acid and Na-gluconate mixture) resulted in transition from cellulose I into cellulose II structure as revealed by WAXS measurements. Wet-spinning attempts of cellulose/FeTNa solutions yielded fiber-like shaped bodies with a brittle structure.     

Alterations in cerebrospinal fluid glycerophospholipids and phospholipase A2 activity in Alzheimer's disease

Our aim is to study selected cerebrospinal fluid (CSF) glycerophospholipids (GP) that are important in brain pathophysiology. We recruited cognitively healthy (CH), minimally cognitively impaired (MCI), and late onset Alzheimer''s disease (LOAD) study participants and collected their CSF. After fractionation into nanometer particles (NP) and supernatant fluids (SF), we studied the lipid composition of these compartments. LC-MS/MS studies reveal that both CSF fractions from CH subjects have N-acyl phosphatidylethanolamine, 1-radyl-2-acyl-sn-glycerophosphoethanolamine (PE), 1-radyl-2-acyl-sn-glycerophosphocholine (PC), 1,2-diacyl-sn-glycerophosphoserine (PS), platelet-activating factor-like lipids, and lysophosphatidylcholine (LPC). In the NP fraction, GPs are enriched with a mixture of saturated, monounsaturated, and polyunsaturated fatty acid species, while PE and PS in the SF fractions are enriched with PUFA-containing molecular species. PC, PE, and PS levels in CSF fractions decrease progressively in participants from CH to MCI, and then to LOAD. Whereas most PC species decrease equally in LOAD, plasmalogen species account for most of the decrease in PE. A significant increase in the LPC-to-PC ratio and PLA₂ activity accompanies the GP decrease in LOAD. These studies reveal that CSF supernatant fluid and nanometer particles have different GP composition, and that PLA₂ activity accounts for altered GPs in these fractions as neurodegeneration progresses.     

The protective effect of alpha lipoic acid against traumatic brain injury in rats

Traumatic brain injury (TBI) was induced by a weight-drop device using 300 g–1 m weight-height impact. The study groups were: control, alpha-lipoic acid (LA) (100 mg/kg, po), TBI, and TBI + LA (100 mg/kg, po). Forty-eight hours after the injury, neurological scores were measured and brain samples were taken for histological examination or determination of thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) levels, myeloperoxidase (MPO) and Na⁺-K⁺ ATPase activities, whereas cytokines (TNF-α, IL-1β) were determined in blood. Brain oedema was evaluated by wet–dry weight method and blood–brain barrier (BBB) permeability was evaluated by Evans Blue (EB) extravasation. As a result, neurological scores mildly increased in trauma groups. Moreover, TBI caused a significant decrease in brain GSH and Na⁺-K⁺ ATPase activity, which was accompanied with significant increases in TBARS level, MPO activity and plasma proinflammatory cytokines. LA treatment reversed all these biochemical indices as well as histopathological alterations. TBI also caused a significant increase in brain water content and EB extravasation which were partially reversed by LA treatment. These findings suggest that LA exerts neuroprotection by preserving BBB permeability and by reducing brain oedema probably by its anti-inflammatory and antioxidant properties in the TBI model.     

Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.