Recombination initiation: Easy as A, B, C, D… χ?

The octameric Chi (χ) sequence is a recombination hotspot in Escherichia coli. Recent studies suggest a singular mechanism by which χ regulates not only the nuclease activity of RecBCD enzyme, but also the ability of RecBCD to promote loading of the strand exchange protein, RecA, onto χ-containing DNA.     

TIMP-1, -2, -3, and -4 in idiopathic pulmonary fibrosis. A prevailing nondegradative lung microenvironment?

Fibroblast proliferation and extracellular matrix accumulation characterize idiopathic pulmonary fibrosis (IPF). We evaluated the presence of tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, and -4; collagenase-1, -2, and -3; gelatinases A and B; and membrane type 1 matrix metalloproteinase (MMP) in 12 IPF and 6 control lungs. TIMP-1 was found in interstitial macrophages and TIMP-2 in fibroblast foci. TIMP-3 revealed an intense staining mainly decorating the elastic lamina in vessels. TIMP-4 was expressed in IPF lungs by epithelial and plasma cells. TIMP-2 colocalized with Ki67 in fibroblasts, whereas TIMP-3 colocalized with p27 in inflammatory and epithelial cells. Collagenase-1 was localized in macrophages and alveolar epithelial cells, collagenase-2 was localized in a few neutrophils, and collagenase-3 was not detected. MMP-9 was found in neutrophils and subepithelial myofibroblasts. Myofibroblast expression of MMP-9 was corroborated in vitro by RT-PCR. MMP-2 was noticed in myofibroblasts, some of them close to areas of basement membrane disruption, and membrane type 1 MMP was noticed in interstitial macrophages. These findings suggest that in IPF there is higher expression of TIMPs compared with collagenases, supporting the hypothesis that a nondegrading fibrillar collagen microenvironment is prevailing.     

Review article. Starch: as simple as A, B, C?

Starch is the main carbohydrate reserve in plants and an important part ofour nutrition. Increasingly, it is being seen by industry as a useful rawmaterial to include in foodstuffs and with which to produce othercarbon-based polymers. Our understanding of starch biosynthesis andchemistry has advanced rapidly over the last few years, but our knowledgeof how this translates into structure and thence into physicochemicalproperties and function is still lacking. Here, we have reviewed thisinformation with an emphasis on genetics and physical properties,especially using data from the model crop, pea (Pisumsativum L.).     

Characterization of caveolins from human knee joint cartilage: expression of caveolin-1, -2, and -3 in chondrocytes and association with integrin β1

Interactions between the extracellular matrix (ECM) and chondrocytes are of great importance for structure and function of cartilage. The present study was undertaken to answer the question whether caveolins take part in integrin-mediated cell–ECM interactions in the human cartilage. In samples of human knee joint cartilage, we detected the caveolin subtypes -1, -2, and -3 by immunohistochemical methods. Double-label experiments revealed a colocalization of caveolin with β1-integrin. Results of immunoprecipitation and immunoblotting assays show that β1-integrins associate with all three caveolin subtypes in human chondrocytes and indicate that they are part of the same complexes. Furthermore, immunoelectron microscopy shows the localization of β1-integrin in caveolae-like structures of the cell membrane. The data stimulate further investigations on the role of the caveolin–integrin complex for integrin-mediated signaling pathways in chondrocytes. Accepted: 17 December 1999     

A biotin switch-based proteomics approach identifies 14-3-3ζ as a target of Sirt1 in the metabolic regulation of caspase-2

While lysine acetylation in the nucleus is well characterized, comparatively little is known about its significance in cytoplasmic signaling. Here we show that inhibition of the Sirt1 deacetylase, which is primarily cytoplasmic in cancer cell lines, sensitizes these cells to caspase-2-dependent death. To identify relevant Sirt1 substrates, we developed a proteomics strategy, enabling the identification of a range of putative substrates, including 14-3-3ζ, a known direct regulator of caspase-2. We show here that inhibition of Sirtuin activity accelerates caspase activation and overrides caspase-2 suppression by nutrient abundance. Furthermore, 14-3-3ζ is acetylated prior to caspase activation, and supplementation of Xenopus egg extract with glucose-6-phosphate, which promotes caspase-2/14-3-3ζ binding, enhances 14-3-3ζ-directed Sirtuin activity. Conversely, inhibiting Sirtuin activity promotes14-3-3ζ dissociation from caspase-2 in both egg extract and human cultured cells. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation.     

β- Alanine,a Possible Neurotransmitter in the Visual System?

Abstract— The chemically evoked efflux of endogenous amino acids from perfused rabbit superior colliculus (SC) slices was studied. Amino acids in the perfusates were determined fluorimetrically with a precolumn derivatisation method and subsequent separation on an HPLC column. Potassium-induced depolarisation caused a calcium-dependent release of β-alanine, GABA, glutamate, and aspartate. Veratridine-evoked efflux was essentially similar in selectivity and could be blocked by tetrodotoxin (TTX). The results are indicative of a neurotransmitter role of β-alanine, GABA, glutamate, and aspartate in the SC of the rabbit.     

Carnosine,homocarnosine and anserine: could they act as antioxidants in vivo?

Carnosine, homocarnosine and anserine have been proposed to act as antioxidants in vivo. Our studies show that all three compounds are good scavengers of the hydroxyl radical (.OH) but that none of them can react with superoxide radical, hydrogen peroxide or hypochlorous acid at biologically significant rates. None of them can bind iron ions in ways that interfere with 'site-specific' iron-dependent radical damage to the sugar deoxyribose, nor can they restrict the availability of Cu2+ to phenanthroline. Homocarnosine has no effect on iron ion-dependent lipid peroxidation; carnosine and anserine have weak inhibitory effects when used at high concentrations in some (but not all) assay systems. However, the ability of these compounds to interfere with a commonly used version of the thiobarbituric acid (TBA) test may have led to an overestimate of their ability to inhibit lipid peroxidation in some previous studies. By contrast, histidine stimulated iron ion-dependent lipid peroxidation. It is concluded that, because of the high concentrations present in vivo, carnosine and anserine could conceivably act as physiological antioxidants by scavenging .OH, but that they do not have a broad spectrum of antioxidant activity, and their ability to inhibit lipid peroxidation is not well established. It may be that they have a function other than antioxidant protection (e.g. buffering), but that they are safer to accumulate than histidine, which has a marked pro-oxidant action upon iron ion-dependent lipid peroxidation. The inability of homocarnosine to react with HOCl, interfere with the TBA test or affect lipid peroxidation systems in the same way as carnosine is surprising in view of the apparent structural similarity between these two molecules.     

Neurturin, RET, GFRα-1 and GFRα-2, but not GFRα-3, mRNA are expressed in mice gonads

The gonads are known to produce numerous hormones and also neurotrophins and their receptors. Here we demonstrate expression of glial-cell-line-derived neurotrophic factor (GDNF) family ligands and related receptors in adult mice gonads by in situ hybridization. GDNF mRNA was expressed in the ovary, but was not detectable in testis. Neurturin (NTN), another ligand in this family, gave rise to strong mRNA hybridization signals in a mosaic pattern in the seminiferous tubules of the testis at stages IX-XII and I-II of the cycle. NTN mRNA signals were also found in uterus and the oviduct. In testis, the transducing receptor RET as well as GDNF receptor alpha-1 (GFR)alpha-1 and GFRalpha-2 were distributed in complementary and overlapping patterns, the former at stages XI-XII-I and the latter at stages VII and VIII. GFRalpha-3 could not be detected. Expression of these trophic molecules suggests involvement of GDNF family ligands and related receptor components in reproduction.     

Ray Wu’s Golden Rules: How he achieved greatness in work, and in life

Overview When Ray Wu passed away on February 10, 2008, he was 79, working full-time in his lab at Cornell University, and was at or near the top of his long and noteworthy career. He had developed the first method for DNA sequencing and pioneered recombinant DNA technology. He had identified key genes related to stress tolerance in plants and was poised to apply these to boost crop yields of rice and other     

Proof of a 1-(3-chlorophenyl)piperazine (mCPP) intake: use as adulterant of cocaine resulting in drug-drug interactions?

Since 2005, increasing numbers of seizures of the designer drug of abuse 1-(3-chlorophenyl)piperazine (mCPP) have been reported. This paper describes the unequivocal proof of a mCPP intake. Differentiation from the intake of its precursor drugs trazodone and nefazodone was performed by a systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation. The found mCPP/hydroxy-mCPP ratio indicated altered metabolism of this cytochrome (CYP) 2D6 catalyzed reaction compared to published studies using the same procedure. Although this might be ascribed to a poor metabolizer (PM) phenotype, genotyping revealed no PM genotype but indications for an intermediate metabolizer genotype. However, a PM phenotype could also be caused by drug-drug interactions with CYP2D6 inhibitors or substrates such as the co-consumed cocaine and diltiazem and/or diltiazem metabolites, respectively. In conclusion, the presented data indicate a possible relevance of CYP2D6 polymorphism and/or drug interactions to mCPP toxicokinetics, which is important for risk assessment of this new designer drug of abuse, in particular if it is used as adulterant of CYP2D6 substrates such as cocaine.     

MMP-2, -9, -14及其抑制剂TIMP-1, -2, -3在恒河猴周期黄体中的时空表达

用原位杂交和免疫组织化学方法研究了基质金属蛋白酶MMP-2, -9, -14及其组织抑制因子TIMP-1, -2, -3在恒河猴周期黄体发育不同阶段的协同表达. 结果显示: MMP-2 mRNA及其蛋白主要表达在早中期发育黄体的内皮细胞上, 在晚期黄体发生萎缩时则大量表达于黄体细胞; MMP-9, -14及其TIMP-1, -2, -3主要表达于黄体细胞; MMP-14 mRNA在早期和晚期黄体中高表达, MMP-9蛋白只在晚期黄体中高表达; TIMP-3蛋白在早、中、晚三期黄体中表达均较高, 但很明显晚期表达降低. 结果提示: MMP/TIMP系统参与灵长类黄体发育的调控, MMP-2, -14及其TIMP-1, -3可能参与黄体的形成和功能维持, 同时MMP-2, -9, -14及其TIMP-1, -2, -3在黄体萎缩期的协同表达, 提示它们可能在黄体发生萎缩时发挥作用.     

Amino acid composition of α1-, α2-, and α3-caseins

Amino acid analyses have been made of a purified α-casein, of its principal component, α₁-casein, and of two other purified components, α₂- and α₃-caseins. α₁-Casein resembles α-casein in content of most amino acids, but both differ from α₂- and α₃-caseins which have their own characteristic amino acid compositions.     

Haptoglobin-α1, -α2, vitamin D-binding protein and apolipoprotein C-III as predictors of etanercept drug response in rheumatoid arthritis

IntroductionThe introduction of tumor necrosis factor-alpha (TNF-α) antagonists has substantially improved patient’s clinical outcome in rheumatoid arthritis (RA). However, nearly 20% to 40% of RA patients do not respond to anti-TNF-α treatment strategies. To identify valid predictors of TNF-α antagonist response in RA, serum proteome profiles from responders (R) and non-responders (NR) to etanercept, a soluble recombinant TNF-α receptor/IgG Fc fusion protein receptor, were compared in a prospective cohort study.MethodsIn this clinical study 50 RA patients with inadequate response to conventional DMARDs were included and treated with etanercept. The primary efficacy endpoint was response according to the European League against Rheumatism (EULAR) improvement criteria. Serum samples collected prior to initiation and after six months of etanercept therapy were cleared of the most abundant major proteins by immunoaffinity chromatography. After separation by two-dimensional differential gel electrophoresis (2D-DIGE) and identification by mass spectrometry (MS) data were validated by Western blot analysis.ResultsAfter six months of etanercept treatment 62% (n = 31) of RA patients achieved response. Haptoglobin-α1 (Hp-α1) and -α2 (Hp-α2) and vitamin D-binding protein (VDBP) were found to be significantly upregulated in responder sera (P ≤0.02) at study entry. In contrast, apolipoprotein C-III (ApoC-III) showed significantly higher levels in non-responders (P = 0.0162). At study end ApoA-II, Hp-α1, Hp-α2 and VDBP were identified to be expressed at significantly higher levels (P <0.05) in responder sera.ConclusionsBy application of clinical proteomics in immunodepleted sera we could identify and validate for the first time Hp-α1, -α2, VDBP and ApoC-III as potential biomarkers for prediction of etanercept drug response in RA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0553-1) contains supplementary material, which is available to authorized users.     

Mutators in SACCHAROMYCES CEREVISIAE: MUT1–1, MUT1–2 and MUT2–1

The purpose of this study was to characterize two mutator stocks of yeast which were induced and selected on the basis of high spontaneous reversion rates of the suppressible "ochre" nonsense allele lys1-1. In the mutator stock VA-3, a single mutation, designated mut1-1, is responsible for the increase in the reversion rate of the ochre alleles lys1-1 and arg4-17. In stock VA-105, there are two separate mutator mutations. Tetrad analysis data showed these two loci are loosely linked. Based on complementation data, one of these mutations is at the same locus as mut1-1 and designated mut1-2. The second mutator of stock VA-105 was designated mut2-1. All three mutators are recessive. Both mut1-1 and mut1-2 give a high mutation rate for ochre nonsense suppressor (SUP) loci, but not for the ochre nonsense alleles. On the contrary, the mutation rates of the ochre alleles are greatly reduced. With the mutant mut2-1 there were mutations at both the lys1-1 site and its suppressors; mut2-1 is as effective as mut1-2 but not as effective as mut1-1 in inducing reversions of a missense mutant, his1-7. Neither mut1-1, mut1-2 nor mut2-1 were effective in inducing reversions of a putative frameshift mutation, hom3-10, or in inducing forward mutations to canavanine resistance.     

ProcessingO-glycan core 1, Galβ1-3GalNAcα-R. Specificities of core 2, UDP-GlcNAc: Galβ1-3GalNAc-R(GlcNAc to GalNAc) β6-N-acetylglucosaminyltransferase and CMP-sialic acid:Galβ1-3GalNAc-R α3-sialyltransferase

To elucidate control mechanisms ofO-glycan biosynthesis in leukemia and to develop biosynthetic inhibitors we have characterized core 2 UDP-GlcNAc:Gal1-3GalNAc-R(GlcNAc to GalNAc) 6-N-acetylglucosaminyl-transferase (EC 2.4.1.102; core 2 6-GlcNAc-T) and CMP-sialic acid: Gal1-3GalNAc-R 3-sialyltransferase (EC 2.4.99.4; 3-SA-T), two enzymes that are significantly increased in patients with chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML). We observed distinct tissue-specific kinetic differences for the core 2 6-GlcNAc-T activity; core 2 6-GlcNAc-T from mucin secreting tissue (named core 2 6-GlcNAc-T M) is accompanied by activities that synthesize core 4 [GlcNAc1-6(GlcNAc1-3)GalNAc-R] and blood group I [GlcNAc1-6(GlcNAc1-3)Gal-R] branches; core 2 6-GlcNAc-T in leukemic cells (named core 2 -GlcNAc-T L) is not accompanied by these two activities and has a more restricted specificity. Core 2 6-GlcNAc-T M and L both have an absolute requirement for the 4- and 6-hydroxyls ofN-acetylgalactosamine and the 6-hydroxyl of galactose of the Gal1-3GalNAc-benzyl substrate but the recognition of other substituents of the sugar rings varies, depending on the tissue. 3-sialytransferase from human placenta and from AML cells also showed distinct specificity differences, although the enzymes from both tissues have an absolute requirement for the 3-hydroxyl of the galactose residue of Gal1-3GalNAc-Bn. Gal1-3(6-deoxy)GalNAc-Bn and 3-deoxy-Gal1-3GalNAc-Bn competitively inhibited core 2 6-GlcNAc-T and 3-sialyltransferase activities, respectively.Abbreviations AFGP antifreeze glycoprotein - AML acute myeloid leukemia - Bn benzyl - CML chronic myelogenous leukemia - Fuc l-fucose - Gal, G d-galactose - GalNAc, GA N-acetyl-d-galactosamine - GlcNAc, Gn N-acetyl-d-glucosamine - HC human colonic homogenate - HO hen oviduct microsomes - HPLC high performance liquid chromatography - mco 8-methoxycarbonyl-octy - Me methyl - MES 2-(N-morpholino)ethanesulfonate - MK mouse kidney homogenate - onp o-nitrophenyl - PG pig gastric mucosal microsomes - pnp p-nitrophenyl - RC rat colonic mucosal microsomes - SA sialic acid - T transferase Enzymes: UDP-GlcNAc:Gal1-3GalNAc-R (GlcNAc to GalNAc) 6-N-acetylglucosaminyltransferase,O-glycan core 2 6-GlcNAc-transferase, EC 2.4.1.102; CMP-sialic acid: Gal1-3GalNAc-R 3-sialyltransferase,O-glycan 3-sialic acid-transferase, EC 2.4.99.4.