A practical approach to crosslinking

The various aspects of chemical crosslinking are addressed. Crosslinker reactivity, specificity, spacer arm length and solubility characteristics are detailed. Considerations for choosing one of these crosslinkers for a particular application are given as well as reaction conditions and practical tips for use of each category of crosslinkers.Abbreviations ABH azidobenzoyl hydrazide - ANB- NOS N-5-azido-2-nitrobenzoyloxysuccinimide - ASIB 1-(p-azidosalicylamido)-4-(iodoacetamido)butane - ASBA 4-(p-azidosalicylamido)butylamine - APDP N-[4-(p-azidosalicylamido) butyl]-3(2-pyridyldithio)propionamide - APG p-azidophenyl glyoxal monohydrate - BASED bis-[-(4-azidosalicylamido)ethyl] disulfide - BMH bismaleimidohexane - BS³ bis(sulfosuccinimidyl) suberate - BSOCOES bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone - DCC N,N-dicyclohexylcarbodiimide - DFDNB 1,5-difluoro-2,4-dinitrobenzene - DMA dimethyl adipimidate·2HCl - DMP dimethyl pimelimidate·2HCl - DMS dimethyl suberimidate·2HCl - DPDPB 1,4-di-(3,2-pyridyldithio)propionamido butane - DMF dimethylformamide - DMSO dimethylsulfoxide - DSG disuccinimidyl glutarate - DSP dithiobis(succinimidylpropionate) - DSS disuccinimidyl suberate - DST disuccinimidyl tartarate - DTSSP 3,3-dithiobis (sulfosuccinimidylpropionate) - DTBP dimethyl 3,3-dithiobispropionimidate·2HCl - EDC or EDAC 1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride - EDTA ethylenediaminetetraacetic acid disodium salt, dihydrate - EGS ethylene glycolbis(succinimidylsuccinate) - GMBS N--maleimidobutyryloxysuccinimide ester - HSAB N-hydroxysuccinimidyl-4-azidobenzoate - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester - MES 4-morpholineethanesulfonic acid - NHS N-hydroxysuccinimide - NHS-ASA N-hydroxysuccinimidyl-4-azidosalicylic acid - PMFS phenylmethylsulfonyl fluoride - PNP-DTP p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate - SAED sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide) ethyl-1,3-dithiopropionate - SADP N-succinimdyl (4-azidophenyl)1,3-dithiopropionate - SAND sulfosuccinimidyl 2-(m-azido-o-nitrobenzamido)-ethyl-1,3-dithiopropionate - SANPAH N-succinimidyl-6(4-azido-2-nitrophenyl-amino)hexanoate - SASD sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3-dithiopropionate - SATA N-succinimidyl-S-acetylthioacetate - SDBP N-hydroxysuccinimidyl-2,3-dibromopropionate - SIAB N-succinimidyl(4-iodoacetyl)aminobenzoate - SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate - SMPB succinimidyl 4-(p-maleimidophenyl) butyrate - SMPT 4-succinimidyloxycarbonyl--methyl--(2-pyridyldithio)-toluene - sulfo-BSOCOES bis[2-sulfosuccinimidooxycarbonyloxy) ethyl]sulfone - sulfo-DST disulfosuccinimidyl tartarate - sulfo-EGS ethylene glycolbis(sulfosuccinimidylsuccinate) - sulfo-GMBS N--maleimidobutyryloxysulfosuccinimide ester - sulfo-MBS m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester - sulfo-SADP sulfosuccinimidyl(4-azidophenyldithio)propionate - sulfo-SAMCA sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate - sulfo-SANPAH sulfosuccinimidyl 6-(4-azido-2-nitrophenylamino)hexanoate - sulfo-SIAB sulfosuccinimidyl(4-iodoacetyl)aminobenzoate - sulfo-SMPB sulfo-succinimidyl 4-(p-maleimidophenyl)butyrate - sulfo-SMCC sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate - SPDP N-succinimidyl 3-(2-pyridyldithio)propionate     

Cobalamin producers and prokaryotic consumers in the Northwest Atlantic

Cobalamin availability can influence primary productivity and ecological interactions in marine microbial communities. The characterization of cobalamin sources and sinks is a first step in investigating cobalamin dynamics and its impact on productivity. Here, we identify potential cobalamin sources and sinks on the Scotian Shelf and Slope in the Northwest Atlantic Ocean. Functional and taxonomic annotation of bulk metagenomic reads, combined with analysis of genome bins, were used to identify potential cobalamin sources and sinks. Cobalamin synthesis potential was mainly attributed to Rhodobacteraceae, Thaumarchaeota, and cyanobacteria (Synechococcus and Prochlorococcus). Cobalamin remodelling potential was mainly attributed to Alteromonadales, Pseudomonadales, Rhizobiales, Oceanospirilalles, Rhodobacteraceae, and Verrucomicrobia, while potential cobalamin consumers include Flavobacteriaceae, Actinobacteria, Porticoccaceae, Methylophiliaceae, and Thermoplasmatota. These complementary approaches identified taxa with the potential to be involved in cobalamin cycling on the Scotian Shelf and revealed genomic information required for further characterization. The Cob operon of Rhodobacterales bacterium HTCC2255, a strain with known importance in cobalamin cycling, was similar to a major cobalamin producer bin, suggesting that a related strain may represent a critical cobalamin source in this region. These results enable future inquiries that will enhance our understanding of how cobalamin shapes microbial interdependencies and productivity in this region.     

Genetic influences in self-reported symptoms of obstructive sleep apnoea and restless legs: a twin study.

Sleep disorders, such as obstructive sleep apnoea (OSA) and restless legs syndrome (RLS), are very common. The relative importance of genetic and nongenetic (environmental) influences on the symptomatology of these conditions has not been well studied. This study uses the twin design to examine this by evaluating OSA and RLS symptoms in monozygotic (MZ) and dizygotic (DZ) twins. Six thousand six hundred unselected female twin pairs, identified from a national volunteer twin register, were asked to complete a medical questionnaire. This questionnaire included questions on OSA and RLS symptoms, as well as questions on subject demographics, past medical history, smoking history and menopausal status. Responses were obtained from 4503 individuals (68% response rate). A total of 1937 twin pairs were evaluable: 933 MZ pairs (mean [range] age 51 [20-76] years) and 1004 DZ pairs (age 51 [20-80] years). Concordance rates were higher for MZ than DZ twins for OSA and RLS symptoms. Multifactorial liability threshold modeling suggests that additive genetic effects combined with unique environmental factors provide the best model for OSA and RLS symptoms. Heritability was estimated to be 52% (95% confidence interval 36% to 68%) for disruptive snoring, 48% (37% to 58%) for daytime sleepiness, 54% (44% to 63%) for restless legs, and 60% (51% to 69%) for legs jerking. These estimates dropped only slightly after adjustment for potential confounding influences on the symptoms of snoring and daytime sleepiness. These results suggest a substantial genetic contribution to the symptomatology of OSA and RLS. More research is needed to identify the genes responsible, and may ultimately lead to new therapies.     

Genomic organization of GB viruses A and B: two new members of the Flaviviridae associated with GB agent hepatitis.

The genomes of two positive-strand RNA viruses have recently been cloned from the serum of a GB agent-infected tamarin by using representational difference analysis. The two agent, GB viruses A and B (GBV-A and GBV-B, respectively), have genomes of 9,493 and 9,143 nucleotides, respectively, and single large open reading frames that encode potential polyprotein precursors of 2,972 and 2,864 amino acids, respectively. The genomes of these agents are organized much like those of other pestiviruses and flaviviruses, with genes predicted to encode structural and nonstructural proteins located at the 5' and 3' ends, respectively. Amino acid sequence alignments and subsequent phylogenetic analysis of the RNA-dependent RNA polymerases (RdRps) of GBV-A and GBV-B show that they possess conserved sequence motifs associated with supergroup II RNA polymerases of positive-strand RNA viruses. On the basis of similar analyses, the GBV-A- and GBV-B-encoded helicases show significant identity with the supergroup II helicases of positive-strand RNA viruses. Within the supergroup II RNA polymerases and helicases, GBV-A and GBV-B are most closely related to the hepatitis C virus group. Across their entire open reading frames, the GB agents exhibit 27% amino sequence identity to each other, approximately 28% identity to hepatitis C virus type 1, and approximately 20% identity to either bovine viral diarrhea virus or yellow fever virus. The degree of sequence divergence between GBV-A and GBV-B and other Flaviviridae members demonstrates that the GB agents are representatives of two new genera within the Flaviviridae family.     

Effects of phosphorylation by CAK on cyclin binding by CDC2 and CDK2.

The cyclin-dependent protein kinases (CDKs) are activated by association with cyclins and by phosphorylation at a conserved threonine residue by the CDK-activating kinase (CAK). We have studied the binding of various human CDK and cyclin subunits in vitro, using purified proteins derived from baculovirus-infected insect cells. We find that most CDK-cyclin complexes known to exist in human cells (CDC2-cyclin B, CDK2-cyclin A, and CDK2-cyclin E) form with high affinity in the absence of phosphorylation or other cellular components. One complex (CDC2-cyclin A) forms with high affinity only after CAK-mediated phosphorylation of CDC2 at the activating threonine residue. CDC2 does not bind with high affinity to cyclin E in vitro, even after phosphorylation of the CDC2 subunit. Thus, phosphorylation is of varying importance in the formation of high-affinity CDK-cyclin complexes.     

Protein 1a: A major wheat germ agglutinin binding protein on the surface of human granulocytes associated with the cytoskeleton

Lectin-receptors on leukocyte and endothelial surfaces are becoming more important in the light of increasing evidence which implicates lectin-carbohydrate interactions in diverse physiological phenomena. This study reports the identification of a major 118 kDa granulocyte surface protein, (Protein 1a) which binds the lectin wheat germ agglutinin (WGA), and is distinctly different from reported WGA binding granulocyte membrane proteins. Protein 1a has been isolated from the Triton-soluble and Triton-insoluble lysates of normal individuals and patients with Chronic Myeloid Leukemia (CML) using a combination of differential solubilization, lectin affinity, ion exchange chromatography and HPLC. The protein from the detergent lysates of both normal and CML granulocytes has similar pI values, lectin affinities, and hydrophobicity. However, its solubility in Triton is different in the two cell types. In 71% of CML cases examined, Protein 1a exhibits decreased Triton solubility suggesting its increased association with the cytoskeleton (CSK). Stimulation of normal granulocytes with WGA leads to the translocation of the soluble form of Protein 1a to the Triton-insoluble fraction. This cytoskeletal recruitment of Protein 1a is sustained only under conditions of excess WGA and occupied receptor. The CSK disruptive agent dihydrocytochalasin B (H₂CB) releases the insoluble form of the receptor into the Triton-soluble fraction. Investigation of a CSK-involving process such as ligand internalization revealed that CML granulocytes exhibit slower kinetics of internalization of fluorescent WGA molecules. Since Protein 1a is a major WGA receptor on the granulocyte surface, its decreased Triton solubility in CML granulocytes suggests that this may be one of the factors contributing to the defective receptor-mediated endocytosis of WGA by CML cells, arising as a consequence of altered membrane-CSK interaction — a nodal point in the signal transduction cascade.     

High rate anaerobic digestion of a petrochemical wastewater using biomass support particles

Anaerobic digestion of wastewater from a dimethyl terephthalate plant was studied in continuously stirred tank reactors with plastic net biomass support particles (BSP) at a level of 20% (v/v). The experimental results showed that the BSP system could treat the wastewater at a hydraulic retention time as low as 1.5 d, organic loading as high as 20 kg COD/m3/d and at acidic feed pH as low as 4.5 with 95% COD reduction and biogas production of about 8l/l/d, while the control system without support particles could not treat the wastewater above a 5-d hydraulic retention time, 5 kg COD/m3/d organic loading and a feed pH of 6.0. Thus, augmentation of BSP upgraded the performance of the conventional suspended growth system to an equivalent level to advanced reactors.     

Construction of a cDNA clone for a nuclear-coded subunit of cytochrome c oxidase from rat liver

A cDNA library for 6S-9S poly(A)-containing RNA from rat liver was constructed in E. coli. Initial screening of the clones was carried out using single stranded 32P-labeled cDNA prepared against poly(A)-containing RNA isolated from immunoadsorbed polyribosomes enriched for the nuclear-coded subunit messenger RNAs of cytochrome c oxidase. One of the clones, pCO89, was found to hybridize with the messenger RNA for subunit VIC. The DNA sequence of the insert in pCO89 was carried out and it has got extensive homology with the C-terminal 33 amino acids of subunit VIC from beef heart cytochrome c oxidase. In addition, the insert contained 146 bp, corresponding to a portion of the 3'-non-coding region. Northern blot analysis of rat liver RNA with the nick-translated insert of pCO89 revealed that the messenger RNA for subunit VI would contain around 510 bases.