Accessibility and multivalency of immobilized Cibacron blue F3GA

The effect of immobilized dye concentration on protein complexation was observed using zonal chromatography. A monomeric protein, octopine dehydrogenase, was retained by a single interaction to a Sepharose CL-6B column containing 11.6 mM immobilized Cibacron blue F3GA. By contrast, a tetrameric protein, lactate dehydrogenase, was retained by the same column by multiple interactions. The degree of multiple interactions was found to systematically increase with increasing immobilized dye concentration. The concentration of immobilized dye accessible to protein was found to be inversely related to the concentration of ionic components in the solvent. Zonal chromatographic measurements of free dye and unconjugated matrix suggest that increasing the concentration of ionic components promotes the adsorption of immobilized dye to the adjacent matrix surface. Such adsorption markedly affects both the capacity of an immobilized dye column and the multiplicity of its interaction with oligomeric proteins.     

Liquid chromatography-mass spectrometric assay for quantitation of the short-chain fatty acid, 2,2-dimethylbutyrate (NSC 741804), in rat plasma

2,2-Dimethylbutyrate (DMB) is a potential treatment for thalassemia and hemoglobinopathies. To facilitate pharmacokinetic evaluation of DMB, we developed an LC-MS assay and quantitated DMB in plasma of rats after an oral dose of 500mg/kg. After acetonitrile protein precipitation, DMB and dimethylvaleric acid (DMV) internal standard were derivatized to benzylamides, chromatographed on a Hydro-RP column with acetonitrile, water, and 0.1% formic acid, and detected by electrospray positive-mode ionization mass spectrometry. The assay was accurate (97-107%) and precise (3.4-6.2%) between 100 and 10,000ng/mL. Recovery from plasma was >62%. Plasma freeze-thaw and room temperature stability were acceptable.     

穿山龙多糖的提取、纯化与抗氧化活性研究

采用热水煮提法和乙醇沉淀法从穿山龙中提取出水溶性粗多糖,再经DEAE-Sepharose CL-6B和Sepha-dex G-100柱层析分离纯化得到2个均多糖组分DMA和DMB。利用超氧阴离子实验,羟基阴离子实验和邻苯三酚自氧化实验研究DMA和DMB的抗氧化活性,结果表明DMB有较强的抗氧化活性,应该被作为潜在的抗氧化剂开发利用。     

Comparative study of electrokinetic potentials and binding affinity of lipopolysaccharides-chitosan complexes

Electrokinetic properties of complexes of chitosan (Ch) with lipopolysaccharides (LPSs) from Escherichia coli O55:B5, Yersinia pseudotuberculosis 1B 598, and Proteus vulgaris O25 (48/57) and their size distribution were investigated using zeta-potential distribution assay and quasi-elastic light scattering. The interaction of LPS from different microorganisms with chitosan at the same w/w ratio of components (1:1) resulted in the formation of complexes in which the negative charge of LPS was neutralized (LPS from E. coli) or overcompensated (Y. pseudotuberculosis and P. vulgaris). The changing in size of the endotoxin aggregates during binding with chitosan was observed. The binding constants of chitosan with LPSs were determined by a method with using the anionic dye Orange II. The LPS from E. coli possess higher affinity to chitosan in comparison with the two others samples of endotoxin.     

降钙素基因相关肽放射免疫测定中与内毒素交叉免疫的离体实验分析

本文用放射免疫法分析了内毒素对降钙素基因相关肽（CGRP）离体免疫测定的影响。高浓度的内毒素（终浓度大于5μg／ml）可通过与CGRP多克隆抗体竞争性结合,影响CGBP的测定,内毒素与CGRP的交叉兔疫比率为5．6×10=6。以C18柱纯化待测样本可以有效地清除混杂的内毒素。结果表明离体实验中高浓度内毒素可干扰CGRP的放射免疫测定,C18柱可清除样本中的内毒素从而避免其对CGRP放射免疫测定的影响。     

Tumor necrosis factor inhibits a polymorphonuclear leukocyte-dependent airway edema in guinea pigs

Intravenously administered endotoxin inhibits the polymorphonuclear leukocyte (PMN)-dependent airway edema produced in guinea pigs exposed to toluene diisocyanate (TDI). Tumor necrosis factor (TNF) is produced in vivo by peripheral blood monocytes and tissue macrophages stimulated with endotoxin and has been shown to activate PMN's and vascular endothelial cells. To determine whether the inhibition of airway edema is mediated by TNF, guinea pigs were treated with intravenous saline or 75,000 U/kg recombinant human TNF 1.5 h before exposure to air or 3 ppm TDI for 1 h. Animals were then injected intravenously with 50 mg/kg Evans blue dye as a marker of protein extravasation. Saline-treated animals exposed to TDI had a significant increase in tracheal Evans blue dye extravasation (85 +/- 6.5 micrograms dye/g trachea, mean +/- SE) compared with saline-treated animals exposed to air (31.3 +/- 2.5, P less than 0.001). The tracheal extravasation of Evans blue dye was significantly inhibited (P less than 0.05) in TDI-exposed animals treated with TNF (64.7 +/- 7.5). Neither heat-inactivated TNF (104.9 +/- 9.5) nor TNF neutralized with a monoclonal antibody against TNF (99.7 +/- 17.9) inhibited TDI-induced airway edema. In addition, treatment with 15,000 U/kg (99.9 +/- 21.3) or 150,000 U/kg (103.2 +/- 17.6) interleukin 1, a monokine also produced in response to endotoxin, did not prevent airway edema. These results suggest that TNF released in response to endotoxin mediates endotoxin's inhibition of a PMN-dependent airway edema.     

An Orally Active Allosteric GLP-1 Receptor Agonist Is Neuroprotective in Cellular and Rodent Models of Stroke

Diabetes is a major risk factor for the development of stroke. Glucagon-like peptide-1 receptor (GLP-1R) agonists have been in clinical use for the treatment of diabetes and also been reported to be neuroprotective in ischemic stroke. The quinoxaline 6,7-dichloro-2-methylsulfonyl-3-N-tert- butylaminoquinoxaline (DMB) is an agonist and allosteric modulator of the GLP-1R with the potential to increase the affinity of GLP-1 for its receptor. The aim of this study was to evaluate the neuroprotective effects of DMB on transient focal cerebral ischemia. In cultured cortical neurons, DMB activated the GLP-1R, leading to increased intracellular cAMP levels with an EC₅₀ value about 100 fold that of exendin-4. Pretreatment of neurons with DMB protected against necrotic and apoptotic cell death was induced by oxygen-glucose deprivation (OGD). The neuroprotective effects of DMB were blocked by GLP-1R knockdown with shRNA but not by GLP-1R antagonism. In C57BL/6 mice, DMB was orally administered 30 min prior to middle cerebral artery occlusion (MCAO) surgery. DMB markedly reduced the cerebral infarct size and neurological deficits caused by MCAO and reperfusion. The neuroprotective effects were mediated by activation of the GLP-1R through the cAMP-PKA-CREB signaling pathway. DMB exhibited anti-apoptotic effects by modulating Bcl-2 family members. These results provide evidence that DMB, a small molecular GLP-1R agonist, attenuates transient focal cerebral ischemia injury and inhibits neuronal apoptosis induced by MCAO. Taken together, these data suggest that DMB is a potential neuroprotective agent against cerebral ischemia.     

Naphthalene Black Staining of Granules of Eosinophilic Granulocytes: Proposed Mechanism of Action Using Chemical Blockade and Computer Graphics

Histochemical staining of the granules of eosinophilic granulocytes and subsequent blockade of the reaction by alkaline benzil was strongly suggestive that in its purified form, the diazo dye naphthalene black reacts with tissue sites containing high concentrations of arginine residues. Computer graphics modelling indicated that the sulfonate group of the dye reacts electrostatically with the guanidino functional group of arginine. This acid-base type reaction likely has a stoichiometric ratio of 2:1 in favor of the amino acid.     

Bacterial endotoxins: biological properties and mechanisms of action

Endotoxins (lipopolysaccharides, LPS) are agents of pathogenicity of Gram-negative bacteria, implicated in the development of Gram-negative shock. Endotoxin reacts with lipopolysaccharide-sensitive cells producing endogenous mediators such as tumour necrosis factor alpha (TNFalpha). Macrophages are cells mediating the toxic activities of LPS and TNFalpha is the primary mediator of the lethal action of endotoxin. This review article discusses the various mechanisms by which endotoxin hypersensitivity in bacteria-sensitized animals develops. The paper concludes with a discussion on the possible protective effect of carnitine congeners against the lethal action of LPS.     

Improved microtechnique for endotoxin assay by the Limulus amebocyte lysate test

A simple microtechnique for the endotoxin assay with Limulus amebocyte lysate is described. Mixtures of 1 μl of reagent with 1 μl of test sample are incubated in 5-μl capillaries which are subsequently dipped into a dye solution. In the absence of a firm gel the dye enters the capillaries whereas a firm gel, formed in the presence of endotoxin, prevents the stain from entering the capillaries. The technique offers a great saving of reagent; unequivocal distinction between positive and negative results, a good reproducibility, and a sensitivity equal to or higher than those in previously reported techniques could be demonstrated.     

Biochemical and functional characterization of membrane blebs purified from Neisseria meningitidis serogroup B

Studies with purified aggregates of endotoxin have revealed the importance of lipopolysaccharide-binding protein (LBP)-dependent extraction and transfer of individual endotoxin molecules to CD14 in Toll-like receptor 4 (TLR4)-dependent cell activation. Endotoxin is normally embedded in the outer membrane of intact Gram-negative bacteria and shed membrane vesicles ("blebs"). However, the ability of LBP and CD14 to efficiently promote TLR4-dependent cell activation by membrane-associated endotoxin has not been studied extensively. In this study, we used an acetate auxotroph of Neisseria meningitidis serogroup B to facilitate metabolic labeling of bacterial endotoxin and compared interactions of purified endotoxin aggregates and of membrane-associated endotoxin with LBP, CD14, and endotoxin-responsive cells. The endotoxin, phospholipid, and protein composition of the recovered blebs indicate that the blebs derive from the bacterial outer membrane. Proteomic analysis revealed an unusual enrichment in highly cationic (pI > 9) proteins. Both purified endotoxin aggregates and blebs activate monocytes and endothelial cells in a LBP-, CD14-, and TLR4/MD-2-dependent fashion, but the blebs were 3-10-fold less potent when normalized for the amount of endotoxin added. Differences in potency correlated with differences in efficiency of LBP-dependent delivery to and extraction of endotoxin by CD14. Both membrane phospholipids and endotoxin are extracted by LBP/soluble CD14 (sCD14) treatment, but only endotoxin.sCD14 reacts with MD-2 and activates cells. These findings indicate that the proinflammatory potency of endotoxin may be regulated not only by the intrinsic structural properties of endotoxin but also by its association with neighboring molecules in the outer membrane.     

Demethyleneberberine induces cell cycle arrest and cellular senescence of NSCLC cells via c-Myc/HIF-1α pathway

BackgroundDemethyleneberberine (DMB) is a natural active component of medicinal plant Cortex phellodendri chinensis with favorable bioactivity. However, the role of DMB in suppressing non-small cell lung cancer (NSCLC) remains unknown.PurposeIn this study, we aimed to examine the effect and underlying mechanism of DMB in suppressing NSCLC.MethodsCCK8 assay and colony formation assay were utilized to assess the efficiency of DMB on the viability and colony formation capacity of NSCLC cells. Flow cytometry and β-Galactosidase Staining Kit were utilized to determine the efficiency of DMB on the cell cycle and cellular senescence of NSCLC cells. RT-qPCR and Western blot were used to detect the effect of DMB on cell cycle and cellular senescence related gene and protein expression of NSCLC cells. In vivo tumor model was established to evaluate the anti NSCLC effect of DMB. In addition, RNA-seq analysis was performed to detect the differential gene expression after DMB treatments.ResultsIn this study, we revealed that DMB exhibits efficient inhibitory effect on NSCLC cell proliferation and tumor xenografts growth in vivo. We also demonstrated that DMB could inhibit cell migration by suppressing epithelial-mesenchymal transition (EMT) and trigger cell cycle arrest by down-regulating the expression of cell cycle related genes in NSCLC cells. In addition, DMB treatment efficiently induces cellular senescence of NSCLC cells. From the RNA-seq analysis, we found that DMB accelerates senescence through suppressing HIF-1α expression, which was further elucidated by overexpressing HIF-1α in NSCLC to reduce the inhibitory effect of DMB. Furthermore, we also revealed that DMB decreases the expression of c-Myc, an up-stream protein of HIF-1α.ConclusionsTaken together, we first report that DMB inhibits NSCLC progress through inducing cell cycle arrest and triggering cellular senescence by downregulating c-Myc/HIF-1α pathway.     

贵州汉族人群HLA-DM基因多态性分析

为了探讨贵州汉族人群HLA-DM基因多态性的分布情况, 采用PCR-RFLP法对125 例贵州汉族人进行HLA-DM基因分型。结果显示, 贵州汉族人群DMA*0101~0103等位基因频率依次是0.720、0.244、0.036, DMB*0101~0104等位基因频率分布依次是0.620、0.156、0.188和0.036; 贵州汉族人群中DMA的基因型以DMA*0101/0101和0101/0102为主, 而DMB的基因型以DMB*0101/0101、0101/0102和0101/0103为主。结果表明, HLA-DM基因多态性具有地区性、民族性的遗传特征。     

The end of the cob operon: evidence that the last gene (cobT) catalyzes synthesis of the lower ligand of vitamin B12, dimethylbenzimidazole.

The cob operon of Salmonella typhimurium includes 20 genes devoted to the synthesis of adenosyl-cobalamin (coenzyme B12). Mutants with lesions in the promoter-distal end of the operon synthesize vitamin B12 only if provided with 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B12. In the hope of identifying a gene(s) involved in synthesis of DMB, the DNA base sequence of the end of the operon has been determined; this completes the sequence of the cob operon. The cobT gene is the last gene in the operon. Four CobII (DMB-) mutations mapping to different deletion intervals of the CobII region were sequenced; all affect the cobT open reading frame. Both the CobT protein of S. typhimurium and its Pseudomonas homolog have been shown in vitro to catalyze the transfer of ribose phosphate from nicotinate mononucleotide to DMB. This reaction does not contribute to DMB synthesis but rather is the first step in joining DMB to the corrin ring compound cobinamide. Thus, the phenotype of Salmonella cobT mutants conflicts with the reported activity of the affected enzyme, while Pseudomonas mutants have the expected phenotype. J. R. Trzebiatowski, G. A. O'Toole, and J. C. Escalante Semerena have suggested (J. Bacteriol. 176:3568-3575, 1994) that S. typhimurium possesses a second phosphoribosyltransferase activity (CobB) that requires a high concentration of DMB for its activity. We support that suggestion and, in addition, provide evidence that the CobT protein catalyzes both the synthesis of DMB and transfer of ribose phosphate. Some cobT mutants appear defective only in DMB synthesis, since they grow on low levels of DMB and retain their CobII phenotype in the presence of a cobB mutation. Other mutants including those with deletions, appear defective in transferase, since they require a high level of DMB (to activate CobB) and, in combination with a cobB mutation, they eliminate the ability to join DMB and cobinamide. Immediately downstream of the cob operon is a gene (called ORF in this study) of unknown function whose mutants have no detected phenotype. Just counterclockwise of ORF is an asparagine tRNA gene (probably asnU). Farther counterclockwise, a serine tRNA gene (serU or supD) is weakly cotransducible with the cobT gene.     

Quantitative determination of sulfhydryl groups with "mercurochrome" (author's transl)

Dibrommercuryfluoresceine (DBMF) reacts stoichiometrically and quantitatively with the thiol group of cysteine, glutathione and thioglycolic acid respectively, at pH 7.0. Polarographical and spectrometrical titrations clearly show that in the spectra of the investigated mercaptides the wave length of the first absorption maximum of DMBF (507 nm) remains unchanged but the molar extinction coefficient increases by approximately 20%. Serum albumin, ovalbumin, beta-lactoglobulin and glyceraldehydephosphatedihydrogenase, after incubation with DBMF, form adducts with the dye from which the pure mercaptide complexes were separated by means of column chromatogrphy. These complexes were separated by means of column chromatography. These complexes show a bathochromic shift (520 nm) of the dye band which is decreased now by 50%. The molar extinction coefficient epsilon 520 has been determined from 32,000 to 33,850. On the basis of these values SH-contents of the four proteins were obtained which are in good accordance with data previously published in the literature. No selective reaction, f.i. with more accessible or/and reactive SH-groups was observed. After 30 min incubation with DBMF and washing with isotonic phosphate buffer, native animal tumor cells show in the main absorption band the bathochromically shifted dye maximum. A first temptative estimation of the protein SH-groups yielded 1.7-2.1 X 10(-14) mole SH/single cell. This result lies between the SH-content determined microspectrometrically on cells stained with DDD-Fast Blue B (1.1-1.55 X 10(-14)) and macroscopically on cell homogenates with DTNB (3.1 X 10(-14)). Up to now, no certain information can be given whether or to what extent unspecific absorption effects possibly might be involved in the data obtained with DBMF treated cells, but interaction with nucleic acids can be excluded with certainty on the basis of relevant model experiments.     

Endotoxin as modifier of particulate matter toxicity: a review of the literature

It is well known that particulate matter (PM) and endotoxin are able to trigger inflammatory responses in the lung. Most studies have focused on the components separately and on the identification of chemical components associated with PM. However, since biological components may represent around 20% of airborne PM, and endotoxin may reach concentrations as high as 30 EU/mg, recent studies have focused attention on the characterization of endotoxin present in PM and health effects. Most of the literature has suggested that endotoxin adsorbed in PM is able to elicit immunological responses associated with increase in pro-inflammatory cytokine expression. The aim of this paper is to provide an up to date review of the findings involving toxicity effects of endotoxin associated with PM.     

Specific inhibition of mitochondrial Ca++ transport by ruthenium red

The ability of rat liver mitochondria to transport calcium ions has been found to be inhibited specifically by the dye ruthenium red. Since this dye reacts specifically with mucopolysaccharides, and since energy conservation is not inhibited by this dye, it is concluded that mucopolysaccharides (in the form of mucoproteins or muco or glycolipids) are at the active center of the sites of mediation of mitochondrial Ca⁺⁺ transport.     

Identification of 5,6-dimethylbenzimidazole as the Co alpha ligand of the cobamide synthesized by Salmonella typhimurium. Nutritional characterization of mutants defective in biosynthesis of the imidazole ring.

The Co beta-cyano derivative of the cobamide isolated from Salmonella typhimurium was identified as Co alpha-(alpha-5,6-dimethylbenzimidazolyl)-Co beta-cyanocobamide, indicating that this bacterium synthesizes 5,6-dimethylbenzimidazole (DMB) de novo. We found that mutants deficient in the synthesis of DMB can incorporate benzimidazole without modification to form Co alpha-(alpha-benzimidazolyl)cobamide, a cobamide that is physiologically active. The analysis of the nutritional requirements of mutants deficient in DMB synthesis identified 4,5-dimethylphenylenediamine as a putative intermediate in the synthesis of the imidazole ring of DMB. Our results suggest that the CobII region of the cob operon of S. typhimurium only encodes functions involved in the synthesis of the imidazole ring of DMB.     

3,3-Dimethyl-1-butanol attenuates cardiac remodeling in pressure-overload-induced heart failure mice

Trimethylamine N-oxide (TMAO) is closely related to cardiovascular diseases, particularly heart failure (HF). Recent studies shows that 3,3-dimethyl-1-butanol (DMB) can reduce plasma TMAO levels. However, the role of DMB in overload-induced HF is not well understood. In this research study, we explored the effects and the underlying mechanisms of DMB in overload-induced HF. Aortic banding (AB) surgery was performed in C57BL6/J mice to induce HF, and a subset group of mice underwent a sham operation. After surgery, the mice were fed with a normal diet and given water supplemented with or without 1% DMB for 6 weeks. Cardiac function, plasma TMAO level, cardiac hypertrophy and fibrosis, expression of inflammatory, electrophysiological studies and signaling pathway were analyzed at the sixth week after AB surgery. DMB reduced TMAO levels in overload-induced HF mice. Adverse cardiac structural remodeling, such as cardiac hypertrophy, fibrosis and inflammation, was elevated in overload-induced HF mice. Susceptibility to ventricular arrhythmia also significantly increased in overload-induced HF mice. However, these changes were prevented by DMB treatment. DMB attenuated all of these changes by reducing plasma TMAO levels, hence negatively inhibiting the p65 NF-κB signaling pathway and TGF-β1/Smad3 signaling pathway. DMB plays an important role in attenuating the development of cardiac structural remodeling and electrical remodeling in overload-induced HF mice. This may be attributed to the p65 NF-κB signaling pathway and TGF-β1/Smad3 signaling pathway inhibition.     

A quinoxaline-based compound ameliorates bone loss in ovariectomized mice

DMB (6,7-dichloro-2-methylsulfonyl-3-Ntert-butylaminoquinoxaline) is a quinoxaline-based compound that has been investigated as a glucagon-like peptide-1 receptor (GLP-1R) agonist. To clarify anti-osteoporosis effect of DMB, an osteoporotic mice model was established by ovariectomy (OVX) operation. The OVX mice were given intraperitoneally DMB, exendin-4 (EX-4), or 17β-estradiol (E₂) for two months. Then bone mass and structure, and bone morphometric parameters were examined by micro-CT. Weight gain and food consumption, bone turnover markers, and biomechanical strength of the femur were tested, and bone histomorphometry was analyzed. The food intake and weight gain was obviously reduced by E₂ or EX-4, but not DMB. However, DMB or EX-4 treatment obviously inhibited skeletal deterioration and enhanced bone strength. The improvement involved in the increased osteoblast number and level of bone formation markers, and reduced osteoclasts number and level of bone resorption markers. In addition, DMB was found to stimulate osteoblastogenesis-related marker gene expression. These results demonstrated that DMB ameliorated bone loss mainly via induction of bone formation, which suggests that the small molecule compound might be applied to the management of postmenopausal osteoporosis.