Isolation of an ammonium or methylammonium ion transport mutant of Escherichia coli and complementation by the cloned gene.

During nitrogen-limited growth, Escherichia coli expresses a specific ammonium or methylammonium ion transport system (Amt). Strains carrying defects in Amt have been isolated following Tn10 transposon mutagenesis. These mutants have less than 10% of the transport activity of the parental strain. Glutamate, glutamine, arginine, or high levels (20 mM) of ammonium will serve as the sole nitrogen source for growth of these strains, and glutamine synthetase is normally expressed and repressed by the nitrogen regulatory (Ntr) system. When transformed with plasmid pGln84, containing lacZ fused to an Ntr promoter (glnLp), the Amt mutants expressed a normal level of beta-galactosidase. Furthermore, P1 bacteriophage transduction of the amt mutation into an Ntr mutant, normally constitutive for Amt, gave Amt- transductants. Therefore, the mutations are unlikely to lie within genes affecting Ntr elements. Following transformation with plasmid libraries of E. coli genomic DNA constructed in pUC9, two plasmids conferring the Amt+ phenotype on the amt mutants were isolated. These plasmids were unable to complement the Amt- phenotype of Ntr- mutants. Restriction digestion of these plasmids revealed common fragments, and Southern blot analyses indicated that the Amt-complementing sequence and the site of Tn10 insertion in the genome occur in the same 3.4-kilobase HindIII-SalI fragment. Insertion of TnphoA into this fragment produced amt::phoA fusions which gave high levels of alkaline phosphatase under nitrogen-limiting conditions but low levels during ammonia excess. This suggests that the amt product contains domains which are exported to the periplasm.     

Fabrication of chitin-chitosan/nano TiO2-composite scaffolds for tissue engineering applications

In this study, we prepared chitin-chitosan/nano TiO(2) composite scaffolds using lyophilization technique for bone tissue engineering. The prepared composite scaffold was characterized using SEM, XRD, FTIR and TGA. In addition, swelling, degradation and biomineralization capability of the composite scaffolds were evaluated. The developed composite scaffold showed controlled swelling and degradation when compared to the control scaffold. Cytocompatibility of the scaffold was assessed by MTT assay and cell attachment studies using osteoblast-like cells (MG-63), fibroblast cells (L929) and human mesenchymal stem cells (hMSCs). Results indicated no sign of toxicity and cells were found attached to the pore walls within the scaffolds. These results suggested that the developed composite scaffold possess the prerequisites for tissue engineering scaffolds and it can be used for tissue engineering applications.     

Role of Matricellular Proteins in Disorders of the Central Nervous System

Matricellular proteins (MCPs) are actively expressed non-structural proteins present in the extracellular matrix, which rapidly turnover and possess regulatory roles, as well as mediate cell–cell interactions. MCPs characteristically contain binding sites for other extracellular proteins, cell surface receptors, growth factors, cytokines and proteases, that provide structural support for surrounding cells. MCPs are present in most organs, including brain, and play a major role in cell–cell interactions and tissue repair. Among the MCPs found in brain include thrombospondin-1/2, secreted protein acidic and rich in cysteine family (SPARC), including Hevin/SC1, Tenascin C and CYR61/Connective Tissue Growth Factor/Nov family of proteins, glypicans, galectins, plasminogen activator inhibitor (PAI-1), autotaxin, fibulin and perisostin. This review summarizes the potential role of MCPs in the pathogenesis of major neurological disorders, including Alzheimer’s disease, amyotrophic lateral sclerosis, ischemia, trauma, hepatic encephalopathy, Down’s syndrome, autism, multiple sclerosis, brain neoplasms, Parkinson’s disease and epilepsy. Potential therapeutic opportunities of MCP’s for these disorders are also considered in this review.     

Structural transitions involved in a novel amyloid-like beta-sheet assemblage of tripeptide derivatives

Self-assembly of two tripeptide derivatives containing three nonpolar isoleucine moieties and polar oxyethylene groups are studied in methanol. Peptide A [CH3(OCH2CH2)3OCH2CO(Ile)3OCH3] and peptide B [CH3(OCH2CH2)3OCH2CO(Ile)3NH (CH2CH2O)3CH3] take a mixture of unordered and helical conformation at low concentration (8.5 x 10(-4) M). However, at high concentration (2 x 10(-3) M), both the peptide showed significant increase in the helical conformation. An interesting conformational transition of peptides A and B at various methanol contents was observed in the solvated films of these compounds by spectroscopic methods like the far-uv circular dichroism and Fourier transform infrared (FT-IR) techniques. Peptide B, which contains more polar oxyethylene groups than A, showed a highly cooperative conformational transition when the methanol content was decreased. This transition was characterized by a large increase of beta-sheet, retaining a alpha-helical contribution. Peptide A showed a conformational transition resulting in a beta-sheet in the aggregated state. From the CD spectra, the ratio in the ellipticity indicates that peptide B forms twisted antiparallel beta-sheet conformation, whereas peptide A takes a parallel beta-sheet conformation. The results obtained in this work indicates the role of polar derivatization on the conformational preference of peptides having similar sequence.     

Preparative methods of phosphorylated chitin and chitosan--an overview

Biomaterials such as chitin, chitosan and their derivatives have a significant and rapid development in recent years. Chitin and chitosan have become cynosure of all party because of an unusual combination of biological activities plus mechanical and physical properties. However, the applications of chitin and chitosan are limited due to its insolubility in most of the solvents. The chemical modification of chitin and chitosan are keen interest because of these modifications would not change the fundamental skeleton of chitin and chitosan but would keep the original physicochemical and biochemical properties. They would also bring new or improved properties. The chemical modification of chitin and chitosan by phosphorylation is expected to be biocompatible and is able to promote tissue regeneration. In view of rapidly growing interest in chitin and chitosan and their chemical modified derivatives, we are here focusing the recent developments on preparation of phosphorylated chitin and chitosan in different methods.     

Glutamine in the pathogenesis of acute hepatic encephalopathy

Hepatic encephalopathy (HE) is the major neurological disorder associated with liver disease. It presents in chronic and acute forms, and astrocytes are the major neural cells involved. While the principal etiological factor in the pathogenesis of HE is increased levels of blood and brain ammonia, glutamine, a byproduct of ammonia metabolism, has also been implicated in its pathogenesis. This article reviews the current status of glutamine in the pathogenesis of HE, particularly its involvement in some of the events triggered by ammonia, including mitochondrial dysfunction, generation of oxidative stress, and alterations in signaling mechanisms, including activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-κB). Mechanisms by which glutamine contributes to astrocyte swelling/brain edema associated with acute liver failure (ALF) will also be described.     

Lymphocyte toxicity of prion fragments

Prion protein fragments that are extracted from the brains of patients with Gerstmann-Straussler-Scheinker disease are known to have stimulating action on circulating leukocytes. In particular, the amyloidogenic hydrophobic prion peptide HuPrP (113-127) AGAAAAGAVVGGLGG has been reported to be associated with significant cellular toxicity. In this paper we show that the self assembled form of HuPrP (113-127) and its valine rich domains viz. GAVVGGLG [HuPrP (119-126)] and VVGGLGG [HuPrP (121-127)] are toxic to peripheral lymphocytes. To explore the cytotoxic mechanism of these fragments, we studied 3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazolium bromide (MTT) reduction, reactive oxygen species (ROS) generation, calcium influx and raft sequestration of' peptide treated lymphocytes. Langmuir monolayer studies on these peptides showed a maximum lipid perturbing property of HuPrP (121-127) as compared to the other two fragments. MTT reduction assays on lymphocytes treated with peptides indicated that the prion peptide fibrils are relatively more toxic than freshly solubilized peptide preparations. Lymphocytes treated with HuPrP (121-127), HuPrP (113-127) and HuPrP (119-126) fibrils underwent 60%, 30% and 40% cell death, respectively. Abeta(1-42), HuPrP (119-126) and HuPrP (121-127) fibrils caused 4 fold increases in intracellular ROS as compared with control cells. However, HuPrP (113-127) fibrils lacked such a significant ROS generating activity, indicating that a subtle difference in sequence leads to a difference in the toxic mechanism in the cell. HuPrP (119-126) and HuPrP (121-127) fibrils also produced maximum raft sequestration and calcium influx. Taken together, these data suggest that the assemblage of prion fragments has significant toxic activity on peripheral lymphocytes, a finding with implications for controlling reactive lymphocytes in prion infected subjects.