Synthesis, biological studies of linear and branched arabinofuranoside-containing glycolipids and their interaction with surfactant protein A

Oligoarabinofuranoside-containing glycolipids relevant to mycobacterial cell wall components were synthesized in order to understand the functional roles of such glycolipids. A series of linear tetra-, hexa-, octa- and a branched heptasaccharide oligoarabinofuranosides, with 1?→?2 and 1?→?5 α-linkages between the furanoside residues, were synthesized by chemical methods from readily available monomer building blocks. Upon the synthesis of glycolipids, constituted with a double alkyl chain-substituted sn-glycerol core and oligosaccharide fragments, biological studies were performed to identify the effect of synthetic glycolipids on the biofilm formation and sliding motilities of Mycobacterium smegmatis. Synthetic glycolipids and arabinofuranosides displayed an inhibitory effect on the growth profile, but mostly on the biofilm formation and maturation. Similarly, synthetic compounds also influenced the sliding motility of the bacteria. Further, biophysical studies were undertaken, so as to identify the interactions of the glycolipids with a pulmonary surfactant protein, namely surfactant protein A (SP-A), with the aid of the surface plasmon resonance technique. Specificities of each glycolipid interacting with SP-A were thus evaluated. From this study, glycolipids were found to exhibit higher apparent association constants than the corresponding oligosaccharide portion alone, without the double alkyl group-substituted glycerol core.     

A novel spectrofluorimetric method for the assay of pseudoephedrine hydrochloride in pharmaceutical formulations via derivatization with 4-chloro-7-nitrobenzofurazan

A new highly sensitive and specific spectrofluorimetric method has been developed to determine a sympathomimetic drug pseudoephedrine hydrochloride. The present method was based on derivatization with 4-chloro-7-nitrobenzofurazan in phosphate buffer at pH 7.8 to produce a highly fluorescent product which was measured at 532 nm (excitation at 475 nm). Under the optimized conditions a linear relationship and good correlation was found between the fluorescence intensity and pseudoephedrine hydrochloride concentration in the range of 0.5-5 μg mL(-1). The proposed method was successfully applied to the assay of pseudoephedrine hydrochloride in commercial pharmaceutical formulations with good accuracy and precision and without interferences from common additives. Statistical comparison of the results with a well-established method showed excellent agreement and proved that there was no significant difference in the accuracy and precision. The stoichiometry of the reaction was determined and the reaction pathway was postulated.     

Beta2-integrin, LFA-1, and TCR/CD3 synergistically induce tyrosine phosphorylation of focal adhesion kinase (pp125(FAK)) in PHA-activated T cells

Complete T cell activation requires not only a first signal via TCR/CD3 engagement but also a costimulatory signal through accessory receptors such as CD2, CD28, or integrins. Focal adhesion kinase, pp125(FAK) (FAK), was previously shown to be localized in focal adhesions in fibroblasts and to be involved in integrin-mediated cellular activation. Although signaling through beta1- or beta3-integrins induces tyrosine phosphorylation of FAK, there has been no evidence that activation of T cells through the beta2-integrin, LFA-1, involves FAK. We report here that crosslinking of LFA-1 induces tyrosine phosphorylation of FAK in PHA-activated T cells. Moreover, cocrosslinking with anti-LFA-1 mAb and suboptimal concentration of anti-CD3 mAb markedly increases tyrosine phosphorylation of FAK in an antibody-concentration-dependent and time-kinetics-dependent manner compared with stimulation through CD3 alone, which correlates well with enhanced proliferation of PHA-activated T cells. Furthermore, LFA-1beta costimulation with CD3 induces tyrosine phosphorylation of Syk associated with FAK. These results indicate, for the first time, that signals mediated by LFA-1 can regulate FAK, suggesting that LFA-1-mediated T cell costimulation may be involved in T cell activation at least partially through FAK.     

Molecular modeling of human acidic mammalian chitinase in complex with the natural-product cyclopentapeptide chitinase inhibitor argifin

Human acidic mammalian chitinase (hAMCase) is an attractive target for developing anti-asthma medications. We used a variety of computational methods to investigate the interaction between hAMCase and the natural-product cyclopentapeptide chitinase inhibitor argifin. The three-dimensional structure of hAMCase was first constructed using homology modeling. The interaction mode and binding free energy between argifin and hAMCase were then examined by the molecular-docking calculation and the molecular mechanics Poisson–Boltzmann surface area method combined with molecular dynamics simulation, respectively. The results suggested that argifin binds to hAMCase in a similar fashion to the interaction mode observed in the crystal structure of argifin-human chitotriosidase complex, and possesses inhibitory activity against hAMCase in the micromolar range. We further designed argifin derivatives expected to be selective for hAMCase.     

Association of the Common Catalase Gene Polymorphism rs1001179 With Glycated Hemoglobin and Plasma Lipids in Hyperlipidemic Patients

Catalase represents perhaps the most effective antioxidant defense in the body under conditions of increased oxidative stress, and rs1001179 (CAT-262C >T) is its most extensively studied gene polymorphism. Using an established PCR–RFLP method for genotyping, we examined the association of rs1001179 with glycated hemoglobin (HbA1c) and plasma lipids using univariate analyses with age, sex, body mass index (BMI), smoking, and alcohol abuse as covariates, in a group of dyslipidemic patients from northern Greece. Our results suggest that the TT genotype is a risk factor for increased HbA1c and plasma triglycerides, and that this association is modulated by the BMI and/or age of the patients.     

Loss of calcitonin binding in rat is not related to calcitonin receptor gene abnormality.

C-cell tumors occur frequently (50%) in old WAG/Rij rats. Interestingly, genetically transmitted loss of CT binding sites in the kidney has also been demonstrated in WAG/Rij rats. To determine if these issues are resulted from mutation of calcitonin receptor (CTR), we analyzed the CTR genomic abnormality in WAG/Rij rat. We demonstrated that both Wistar and WAG/Rij rats expressed type-C1a CTR by RT-PCR analysis and their mRNA expressions were approximately equal by Northern blotting analysis. Direct sequence of RT-PCR products for CTR showed no different nucleotide sequences between the two strains. There were three polymorphisms at the first transmembrane domain and the fourth intracellular membranes, which are different from Sprague-Dawley rat. We concluded that the loss of CT binding in WAG/Rij rat is not related to CTR gene abnormality. Abnormal system of CTR amino acid modification may be occurred in WAG/Rij rat.     

Thermal inactivation of glucose oxidase. Mechanism and stabilization using additives

Thermal inactivation of glucose oxidase (GOD; beta-d-glucose: oxygen oxidoreductase), from Aspergillus niger, followed first order kinetics both in the absence and presence of additives. Additives such as lysozyme, NaCl, and K2SO4 increased the half-life of the enzyme by 3.5-, 33.4-, and 23.7-fold respectively, from its initial value at 60 degrees C. The activation energy increased from 60.3 kcal mol-1 to 72.9, 76.1, and 88.3 kcal mol-1, whereas the entropy of activation increased from 104 to 141, 147, and 184 cal x mol-1 x deg-1 in the presence of 7.1 x 10-5 m lysozyme, 1 m NaCl, and 0.2 m K2SO4, respectively. The thermal unfolding of GOD in the temperature range of 25-90 degrees C was studied using circular dichroism measurements at 222, 274, and 375 nm. Size exclusion chromatography was employed to follow the state of association of enzyme and dissociation of FAD from GOD. The midpoint for thermal inactivation of residual activity and the dissociation of FAD was 59 degrees C, whereas the corresponding midpoint for loss of secondary and tertiary structure was 62 degrees C. Dissociation of FAD from the holoenzyme was responsible for the thermal inactivation of GOD. The irreversible nature of inactivation was caused by a change in the state of association of apoenzyme. The dissociation of FAD resulted in the loss of secondary and tertiary structure, leading to the unfolding and nonspecific aggregation of the enzyme molecule because of hydrophobic interactions of side chains. This confirmed the critical role of FAD in structure and activity. Cysteine oxidation did not contribute to the nonspecific aggregation. The stabilization of enzyme by NaCl and lysozyme was primarily the result of charge neutralization. K2SO4 enhanced the thermal stability by primarily strengthening the hydrophobic interactions and made the holoenzyme a more compact dimeric structure.     

Investigation of Beckett–Casy model 3: Synthesis of novel naltrexone derivatives with contracted and expanded D-rings and their pharmacology

Novel naltrexone derivatives 7 and 8 with contracted and expanded D-rings were synthesized to investigate the importance of orientation of lone electron pair on the nitrogen for binding abilities to the opioid receptor. Compound 7 showed almost no binding affinity, whereas compound 8 was comparable to naltrexone (6) in binding affinity. Conformational analyses and NOE experiments in D₂O of compounds 6–8 suggested that the lone electron pairs of compounds 6 and 8 with respective six- and seven-membered D-rings would project in the pseudo-axial orientation, whereas compound 7 with five-membered D-ring would have the lone electron pair directing in pseudo-equatorial position. These results strongly supported the proposal that the axial orientation of the lone electron pair on nitrogen would provide sufficient binding abilities to the opioid receptor and that the 15–16 ethylene moiety in the morphine structure would play a role in fixation of the lone electron pair in the axial direction rather than interaction with the putative cavity in the Beckett–Casy model.     

Patient mutations in human ATP:cob(I)alamin adenosyltransferase differentially affect its catalytic versus chaperone functions

Human ATP:cob(I)alamin adenosyltransferase (ATR) is a mitochondrial enzyme that catalyzes an adenosyl transfer to cob(I)alamin, synthesizing 5′-deoxyadenosylcobalamin (AdoCbl) or coenzyme B₁₂. ATR is also a chaperone that escorts AdoCbl, transferring it to methylmalonyl-CoA mutase, which is important in propionate metabolism. Mutations in ATR lead to methylmalonic aciduria type B, an inborn error of B₁₂ metabolism. Our previous studies have furnished insights into how ATR protein dynamics influence redox-linked cobalt coordination chemistry, controlling its catalytic versus chaperone functions. In this study, we have characterized three patient mutations at two conserved active site residues in human ATR, R190C/H, and E193K and obtained crystal structures of R190C and E193K variants, which display only subtle structural changes. All three mutations were found to weaken affinities for the cob(II)alamin substrate and the AdoCbl product and increase K_M(ATP). ³¹P NMR studies show that binding of the triphosphate product, formed during the adenosylation reaction, is also weakened. However, although the k_cat of this reaction is significantly diminished for the R190C/H mutants, it is comparable with the WT enzyme for the E193K variant, revealing the catalytic importance of Arg-190. Furthermore, although the E193K mutation selectively impairs the chaperone function by promoting product release into solution, its catalytic function might be unaffected at physiological ATP concentrations. In contrast, the R190C/H mutations affect both the catalytic and chaperoning activities of ATR. Because the E193K mutation spares the catalytic activity of ATR, our data suggest that the patients carrying this mutation are more likely to be responsive to cobalamin therapy.