Poly (lactic acid)-chitosan-collagen composite nanofibers as substrates for blood outgrowth endothelial cells.

In this work, the attachment, viability and functionality of rat Blood Outgrowth Endothelial Cells (rBOEC) and genetically modified rBOEC (rBOEC/eNOS-GFP), which over express endothelial nitric oxide synthase (eNOS), were investigated on Poly(lactic acid) (PLA)-chitosan and PLA-chitosan-collagen nanofibrous scaffolds. Both the cell types displayed good attachment, remained viable and functional on both scaffolds. Moreover, incorporation of collagen in the scaffold helped in sustaining the rBOEC for upto one week, although collagen was not found necessary for rBOEC/eNOS-GFP. We conclude that PLA-chitosan based nanofibrous scaffolds can be a potential candidate for BOEC based wound healing applications.

Musculoskeletal disorders in industrial workers of Delhi.

To study the musculoskeletal disorders in industrial workers in Delhi, 631 workers from 60 factories representing small and medium-sized enterprises located in Delhi were interviewed. Many (59.4%) of the workers had musculoskeletal disorders. Tailors, those working near furnaces, cooks, workers in buffing, checking and assembly work, and those working with chemicals had the most joint complaints. Cervical pain was more frequent in tailoring and packing work, whereas lumbar pain was more common in buffing, operators working on presses, those using hand and power tools, and those lifting heavy manual loads. Contract workers had less musculoskeletal morbidity than regular and temporary workers. Skilled workers also had less morbidity. Workers experiencing more job satisfaction reported fewer musculoskeletal disorders. The high prevalence of musculoskeletal disorders in workers needs urgent attention from the health and labor sectors. An ergonomic approach to prevention should be considered. The current manual load handling limits prescribed in the Indian Factory Rules potentially expose workers to back stress. It is also inappropriate to have separate load-lifting limits for men and women. Research is urgently required to determine the safe load handling limits for the Indian working population based on ergonomic principles. Until internationally acceptable safe limits are established, back pain should be a notifiable disease in India.

Isolation and characterization of an N-linked oligosaccharide that is significantly increased in sera from patients with non-small cell lung cancer.

The structures of N-linked oligosaccharides present in human sera from 12 healthy volunteers and from 14 patients with non-small cell lung cancer (NSCLC) were analyzed by our recently developed partially automated systematic method. Thirty different structures of oligosaccharides were deduced, and these accounted for 84.1% of the total N-linked oligosaccharides present in human sera. All of the quantified oligosaccharide levels in healthy human sera were within twice the standard deviation. The amount of a triantennary trigalactosylated structure with one outer arm fucosylation (A3G3Fo) was found to be markedly increased in NSCLC patients in comparison to that in healthy volunteers (p < 0.01). No significant positive correlation with other clinical data was found. Serum A3G3Fo levels can thus be a novel marker for the diagnosis of NSCLC.

beta1-4Galactosyltransferase activity of mouse brain as revealed by analysis of brain-specific complex-type N-linked sugar chains.

We previously reported two brain-specific agalactobiantennary N-linked sugar chains with bisecting GlcNAc and alpha1-6Fuc residues, (GlcNAcbeta1-2)(0)(or)(1)Manalpha1-3(GlcNAcbeta1-2M analpha1-6)(GlcNA cbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)Glc NAc [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. 114, 334-338]. Here, the reason for the absence of Gal on the sugar chains was analyzed through the detection of other complex type sugar chains. Analysis of N-linked sugar chains revealed the absence of Sia-Gal and Gal on the GlcNAc residues of brain-specific agalactobiantennary N-linked sugar chains. We therefore investigated the substrate specificity of galactosyltransferase activities in brain using pyridylamino derivatives of agalactobiantennary sugar chains with structural variations in the bisecting GlcNAc and alpha1-6Fuc residues as acceptor substrates. While the beta1-4galactosyltransferases in liver and kidney could utilize all four oligosaccharides as substrates, the beta1-4galactosyltransferase(s) in brain could not utilize the agalactobiantennary sugar chain with both bisecting GlcNAc and Fuc residues, but could utilize the other three acceptors. Similar results were obtained using glycopeptides with agalactobiantennary sugar chains and bisecting GlcNAc and alpha1-6Fuc residues as substrates. The beta1-4galactosyltransferase activity of adult mouse brain thus appears to be responsible for producing the brain-specific sugar chains and to be different from beta1-4galactosyltransferase-I. The agalactobiantennary sugar chain with bisecting GlcNAc and alpha1-6Fuc residues acts as an inhibitor against "brain type" beta1-4galactosyltransferase with a K(i) value of 0.29 mM.

Systematic analysis of N-linked sugar chains from whole tissue employing partial automation.

A partially automated technique for the isolation and characterization of N-linked sugar chains from glycoproteins of crude tissue samples is established. The N-linked sugar chains from the acetone-extracted tissues are made free by a process of hydrazinolysis and subsequently N-acetylated by GlycoPrep 1000 (Oxford Glycosystems). These free sugar chains are further converted to pyridylamino derivatives by GlycoTag (Takara). Characterization of these sugar chains is achieved by a combination of HPLC columns using a highly sensitive fluorescence detector at femtomole levels. Tissue sample can be successfully pyridylaminated and analyzed to give highly reproducible results with consistent yield, requiring fewer purification steps, minimum skills, and less time. Moreover, fixed tissues can also be analyzed employing this technique, giving a similar sugar chain pattern compared to normal tissue samples. Using this method we show that the pattern of N-linked sugar chains present in human sera or in one small region of brain is strikingly similar among the different individuals. However, the absence of a highlighted peak in one of the samples suggests this method can be extrapolated to identify changes, if any, associated with disorders such as inflammation or cancer. Furthermore, this two-dimensional display of sugar chains would discover the function-specific molecules as we see in proteins.

Expression, purification, and encephalitogenicity of recombinant human myelin oligodendrocyte glycoprotein.

Myelin oligodendrocyte glycoprotein (MOG), a putative autoantigen in multiple sclerosis (MS), is a quantitatively minor component of the CNS. In view of the difficulties associated with the purification of MOG from brain tissues, the extracellular domain of human MOG corresponding to the N-terminal 121 amino acids was expressed in Escherichia coli as a glutathione sulfotransferase fusion protein. The expressed protein was localized to inclusion bodies, and varying the growth parameters resulted in the solubilization of small amounts of GST-MOG that could be affinity purified on glutathione agarose columns. The fusion protein found in the inclusion bodies could be solubilized with urea. The solubilized fusion protein was cleaved with thrombin, and the extracellular domain was purified by CM Sephadex 50 chromatography to homogeneity. Injection of recombinant human MOG into different strains of mice resulted in the induction of an MS-like disease, characterized by severe neurological impairment and extensive CNS demyelinated lesions. Recombinant MOG produced in E. coli should prove to be useful as a highly purified biological reagent for immunological, pathological, functional, and structural studies.

Demyelinating antibodies to myelin oligodendrocyte glycoprotein and galactocerebroside induce degradation of myelin basic protein in isolated human myelin.

Although the specificity of multiple sclerosis (MS) brain immunoglobulins (Igs) remains unknown, the incubation of these Igs with human myelin can lead to myelin basic protein (MBP) degradation mediated by neutral proteases. In this study, we demonstrate that monoclonal antibodies (mAbs) specific to myelin components such as the CNS-specific myelin oligodendrocyte glycoprotein (MOG) and galactocerebroside (GalC) are found to induce a significant loss of MBP mediated by neutral proteases in myelin. By contrast, antibodies to periaxonal and structural components of myelin, such as MBP and myelin-associated glycoprotein, are ineffective in inducing such MBP degradation. Among the 11 different anti-MOG mAbs directed to externally located epitopes of MOG, only two were found to induce a significant degradation of MBP, suggesting that antibody-induced MBP degradation is not only antigen specific but also epitope specific. Based on the inhibition of MBP degradation in the presence of EGTA and the analysis of the degradation products obtained following incubation of myelin with mAbs to GalC and MOG (8-18C5), the neutral protease involved in this antibody-induced degradation of MBP could be calcium-activated neutral protease. Taken together, these results suggest that antibodies to GalC and MOG can play a major role in destabilizing myelin through MBP breakdown mediated by neutral proteases and thus have an important role to play in the pathogenesis of MS.

Myelin oligodendrocyte glycoprotein induces a demyelinating encephalomyelitis resembling multiple sclerosis.

Chronic relapsing experimental autoimmune encephalomyelitis, a demyelinating disease induced by injection of central nervous system (CNS) tissue, is widely used as a model for multiple sclerosis. However, it is unclear which Ag or combination of Ags in the CNS induce the demyelinating immune response. We now show in Lewis rats that a single injection of myelin oligodendrocyte glycoprotein, a specific CNS myelin component, or an appropriately derived myelin oligodendrocyte glycoprotein peptide produces a relapsing remitting neurologic disease with extensive plaque-like demyelination. Igs from affected animals reacted specifically with myelin oligodendrocyte glycoprotein and stimulated a myelin protease activity, leading to myelin basic protein degradation. The demonstrated involvement of myelin oligodendrocyte glycoprotein as a new demyelinating neural Ag may provide a deeper insight into the pathogenesis of multiple sclerosis and its treatment.

Antibody coated bacteria in urine of patients with recent spinal injury.

Twenty patients with an acute spinal injury were prospectively studied to assess the clinical importance of antibody coated bacteria (ACB) in the urine and the association among the different bacterial species with a positive antibody coated bacteria test. Clinical urinary tract infection was associated with a positive ACB test on 45% of occasions. Three hundred and ninety nine urine samples containing 541 bacterial isolates were assessed for the presence of ACB; 13% were found to be positive and 87% negative for ACB; 67% of urines contained a single bacterial isolate. Pseudomonas aeruginosa was most commonly associated with clinical urinary tract infection, found in 25% of episodes, followed by Proteus mirabilis (17.5%), Klebsiella sp (12.5%), and Proteus morganii (10%). Providencia stuartii, however, was most commonly associated with a positive ACB test (found in 17%). Other bacteria associated with a positive ACB test included Klebsiella sp (14%), Acinetobacter sp (12.5%), Pseudomonas aeruginosa (12%), Citrobacter sp (11.5%). A positive ACB test is not to be expected from a patient with spinal injury who has a catheter in place, and the test may provide a useful guide to identify those patients with an invasive infection. It is doubtful that a decision to treat or not treat bacteriuria could rest on the identification of the bacterial species alone.

Evidence for the incorporation of [32P]orthophosphate into leaf inositol phospholipids.

Leaf discs of brinjal, tomato, sugar cane and maize rapidly incorporated [32P]orthophosphate into total phospholipids. Analyses of the labelled lipid extracts by thin-layer chromatography, autoradiography and comparison with inositol phospholipid standards demonstrated the labelling of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate in addition to other phospholipids. The presence of polyphosphoinositides was further confirmed by deacylation of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate and separation of the water-soluble products, glycerophosphoinositol phosphate and glycerophosphoinositol bisphosphate by formate exchange chromatography. Incorporation of [32P]orthophosphate into inositol phospholipids was time-dependent, with monoester phosphate groups attaining isotopic equilibrium within 90 min of incubation. After 2 h, incorporation of label into phosphatidylinositol, phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate was about 15, 10 and 3%, respectively, of the total phospholipids. The ratio of radioactivity in phosphatidylinositol/phosphatidylinositol monophosphate/phosphatidylinositol bisphosphate was about 5:5:1 in brinjal leaves. However, this ratio may be an overestimate of the amounts of inositol phospholipids present, as other lysophospholipids may comigrate with standards.

Serial concentrations of C-reactive protein as an indicator of urinary tract infection in patients with spinal injury.

C-reactive protein (CRP) was measured serially in 16 patients with an acute spinal injury. Twelve episodes of acute urinary tract infection (UTI) occurred during the study period. These were all associated with an increased concentration of CRP greater than 50 mg/l, which returned to normal after successful treatment. Thirteen episodes of asymptomatic bacteriuria associated with increased concentrations of CRP greater than 20 mg/l occurred, indicating tissue damage. More commonly, significant bacteriuria was associated with normal concentrations of CRP, and presumably, simple colonisation of the urinary tract, which, we suggest, does not require treatment with antibiotics. Serial measurement of CRP in patients with spinal injury may help distinguish between urinary tract colonisation and infection and be useful in monitoring the response to the treatment of clinical UTI.