Solution conformation of the antibody-bound tyrosine phosphorylation site of the nicotinic acetylcholine receptor beta-subunit in its phosphorylated and nonphosphorylated states

Phosphorylation of the acetylcholine receptor (AChR) seems to be responsible for triggering several effects including its desensitization and aggregation at the postsynaptic membrane and probably initiates a signal transduction pathway at the postsynaptic membrane. To study the structural and functional role of the tyrosine phosphorylation site of the AChR beta-subunit and contribute to the in-depth understanding of the structural basis of the ion channel function, we synthesized four peptides containing the phosphorylated and nonphosphorylated sequences (380-391) of the human and Torpedo AChR beta-subunits and studied their interaction with a monoclonal antibody (mAb 148) that is known to bind to this region and that is capable of blocking ion channel function. All four peptides were efficient inhibitors of mAb 148 binding to AChR, although the nonphosphorylated human peptide was considerably less effective than the three others. We then investigated the conformation acquired by all four peptides in their antibody-bound state, which possibly illustrates the local conformation of the corresponding sites on the intact AChR molecule. The phosphorylated human and Torpedo peptides adopted a distorted 3(10) helix conformation. The nonphosphorylated Torpedo peptide, which is also an efficient inhibitor, was also folded. In contrast, the nonphosphorylated human peptide (a less efficient inhibitor) presented an extended structure. It is concluded that the phosphorylation of the AChR at its beta-subunit Tyr site leads to a significant change in its conformation, which may affect several functions of the AChR.     

Comparative PCR-based fingerprinting of Vibrio cholerae isolated in Malaysia

Vibrio cholerae, the causative agent of cholera, is endemic in many parts of the world, especially in countries poor in resources. Molecular subtyping of V. cholerae is useful to trace the regional spread of a clone or multidrug-resistant strains during outbreaks of cholera. Current available PCR-based fingerprinting methods such as Random Amplified Polymorphic DNA (RAPD)-PCR, Enterobacterial Repetitive Intergenic Consensus Sequence (ERIC)-PCR, and Repetitive Extragenic Palindromic (REP)-PCR were used to subtype V. cholerae. However, there are problems for inter-laboratory comparison as these PCR methods have their own limitations especially when different PCR methods have been used for molecular typing. In this study, a Vibrio cholerae Repeats-PCR (VCR-PCR) approach which targets the genetic polymorphism of the integron island of Vibrios was used and compared with other PCR-based fingerprinting methods in subtyping. Forty-three V. cholerae of different serogroups from various sources were tested. The PCR-fingerprinting approaches were evaluated on typeability, reproducibility, stability and discriminatory power. Overall, Malaysian non-O1/non-O139 V. cholerae were more diverse than O1 strains. Four non-O1/non-O139 strains were closely related with O1 strains. The O139 strain in this study shared similarity with strains of both O1 and non-O1/non-O139 serogroups. ERIC-PCR was the most discriminative approach (D value = 0.996). VCR-PCR was useful in discriminating non-O1/non-O139 strains. RAPD-PCR and REP-PCR were less suitable for efficient subtyping purposes as they were not reproducible and lacked stability. The combination of the ERIC-PCR and VCR-PCR may overcome the inadequacy of any one approach and hence provide more informative data.     

Comparative Virulotyping of Salmonella typhi and Salmonella enteritidis

Members of Salmonella enterica are important foodborne pathogens of significant public health concern worldwide. This study aimed to determine a range of virulence genes among typhoidal (S. typhi) and non-typhoidal (S. enteritidis) strains isolated from different geographical regions and different years. A total of 87 S. typhi and 94 S. enteritidis strains were tested for presence of 22 virulence genes by employing multiplex PCR and the genetic relatedness of these strains was further characterized by REP-PCR. In S. typhi, invA, prgH, sifA, spiC, sopB, iroN, sitC, misL, pipD, cdtB, and orfL were present in all the strains, while sopE, agfC, agfA, sefC, mgtC, and sefD were present in 98.8, 97.7, 90.8, 87.4, 87.4 and 17.2 %, of the strains, respectively. No lpfA, lpfC, pefA, spvB, or spvC was detected. Meanwhile, in S. enteritidis, 15 genes, agfA, agfC, invA, lpfA, lpfC, sefD, prgH, spiC, sopB, sopE, iroN, sitC, misL, pipD, and orfL were found in all S. enteritidis strains 100 %, followed by sifA and spvC 98.9 %, pefA, spvB and mgtC 97.8 %, and sefC 90.4 %. cdtB was absent from all S. enteritidis strains tested. REP-PCR subtyped S. typhi strains into 18 REP-types and concurred with the virulotyping results in grouping the strains, while in S. enteritidis, REP-PCR subtyped the strains into eight profiles and they were poorly distinguishable between human and animal origins. The study showed that S. typhi and S. enteritidis contain a range of virulence factors associated with pathogenesis. Virulotyping is a rapid screening method to identify and profile virulence genes in Salmonella strains, and improve an understanding of potential risk for human and animal infections.     

Molecular differentiation of two morphological variants of Gracilaria salicornia

Xia in 1986 combined Gracilaria salicornia, G. canaliculata (G. crassa), G. cacalia and G. minor into one species: G.salicornia. Two morphological variants of G. salicornia were collectedfrom different localities in Malaysia. Variant A collected from Morib,Selangor grew on the roots of Avicennia. The samples showed absenceof main axis; segmented constrictions throughout; cylindrical or slightlycompressed thalli. Variant B was collected from the mudflats of TanjungTuan, growing on rocks, coral or forming mats on the mud. Plants showedabsence of main axis; segments were not constricted throughout the plant(if present only slightly articulated at the upper part), branching wasdichotomous or irregular; cylindrical or slightly compressed thalli. Thetechnique of Random Amplified Polymorphic DNA analysis (RAPD) wasused to investigate molecular characteristics of the two variants. Out ofsixty Operon primers that were screened, four primers, OPA 1, OPA 10,OPA 11 and OPK 7 were able to give polymorphism. The fingerprintsgenerated were stable and reproducible on repeated analysis. The DNAfingerprints generated were visually analysed and clustering analysis wascarried out using GelCompar 4.0. The matrix of similarities was based onthe Dice coefficients (S_D) and the cluster analysis was carried outusing the unweighted pair group method using arithmetic averages(UPGMA). DNA analysis showed that two primers (OPA 01, CAGGCCCTTC and OPK 07, AGCGAGCAAG) were able to differentiate the two variants.     

Suppression of monocyte and neutrophil function by recombinant IL-2

Little IS known about the influence of IL-2 on phagocytes. We now describe the effects of human recombinant IL-2 on human neutrophil and monocyte functions related to mobility, phagocytosis, glucose uptake, respiration and degranulation. Neutrophil adherence and hexose monophosphate shunt activities were both suppressed after incubation with IL-2. IL-2 had no effect on neutrophil migration, phagocytosis, deoxyglucose uptake or degranulation, ionocytes demonstrated a greater sensitivity to IL-2 with suppression of monocyte adherence, random and stimulated migration, glucose uptake and hexose monophosphate shunt activity, even after addition of phorbol myristate acetate. Monocyte phagocytosis and degranulation were not affected. All of the effects observed were dose-dependent within a biologically active range for IL-2. These studies suggest that IL-2 may have an important down-regulatory role across a broad range of monocyte functions including movement, deoxyglucose uptake and respiration. However, its role in regulation of neutrophil function is limited to adherence and respiration. IL-2 may be a more versatile cytokine than has previously been appreciated.     

New Dibenzocyclooctadiene Lignans and Phenolics from Kadsura heteroclite with Anti-Inflammatory Activity

A chemical investigation of K. heteroclite led to isolation of two new dibenzocyclooctadienes ( 1 and 2 ) together with 14 known compounds ( 3 – 16 ) by using multiple chromatographic techniques. New compounds ( 1 and 2 ) were obtained and identified by spectroscopic methods (HR-ESI-MS, 1D and 2D NMR, and ECD) as well as by comparison of their experimental data with those reported in the literatures. All the isolates were evaluated for their ability to modulate TNF-α production in lipopolysaccharide (LPS) stimulated RAW264.7 cells. Among them, compound 5 displayed the most inhibition against tumor necrosis factor (TNF)-α production with IC₅₀ value of 6.16±0.14 μM. Whereas, compounds ( 1 , 3 , and 6 ) showed the significant inhibition (IC₅₀ values ranging from 9.41 to 14.54 μM), and compounds ( 2 , 4 , 9 , 10 , 13 , 15 , and 16 ) exhibited moderate inhibition (IC₅₀ values ranging from 19.27 to 40.64 μM) toward TNF-α production, respectively.     

Inhibitory effects of bisbenzylisoquinolines on synthesis of the inflammatory cytokines interleukin-1 and tumour necrosis factor-alpha

Synthesis of IL-1beta and TNFalpha by human monocytesmacrophages was significantly inhibited by eleven bisbenzylisoquinolines and one half-molecule (benzylisoquinoline), with IC(50) values in the muM range. The results indicate that these compounds may have value in the therapy of human diseases where these inflammatory cytokines have a central role in pathogenesis.     

Nitrogen concentrations in a mangrove creek with a large tidal range,Peninsular Malaysia

We report on various species of nitrogen in subsurface water samples taken at the mouth of Sungai Sementa Besar, a creek about 4.5 km long off the Straits of Malacca and draining a mangrove forest on the west coast of Peninsular Malaysia. This tide-dominated watercourse shows a range greater than 4.5 metres from low to high water during springs, only 1.0 m during neaps, and salinities at the mouth of 30 or greater. After heavy rains or when the tides inundate the forest floor, inorganic nitrogen values increase as much as tenfold at the mouth. Particulate and total organic nitrogen also increase at the mouth under these conditions. There were also comparative highs of organic inputs at other times.     

Clinical significance of a high mean corpuscular volume in nonanemic patients.

A prospective study of the clinical significance of macrocytosis (mean corpuscular volume 100 fL or more) was carried out for 9 months in a teaching hospital in 1975. Of the 140 patients with macrocytosis at the time of admission (0l7% of all hospital admissions) 46 (33%) had low activity of serum or erythrocyte folate, or both, and 16 (11%) had low serum vitamin B12 concentrations. Among the 78 patients with normal B12 and folate values the most commonly associated significant clinical conditions were alcoholism or hepatic disease (36 patients), malignant disease or the effects of chemotherapy (25 patients) and chronic obstructive lung disease (10 patients).