Microfluidic Interfaces for Chronic Bidirectional Access to the Brain.

Two-photon polymerization (TPP) is an additive manufacturing technique with micron-scale resolution that is rapidly gaining ground for a range of biomedical applications. TPP is particularly attractive for the creation of microscopic three-dimensional structures in biocompatible and noncytotoxic resins. Here, TPP is used to develop microfluidic interfaces which provide chronic fluidic access to the brain of preclinical research models.

Do not drop: mechanical shock in vials causes cavitation, protein aggregation, and particle formation.

Industry experience suggests that g-forces sustained when vials containing protein formulations are accidentally dropped can cause aggregation and particle formation. To study this phenomenon, a shock tower was used to apply controlled g-forces to glass vials containing formulations of two monoclonal antibodies and recombinant human growth hormone (rhGH). High-speed video analysis showed cavitation bubbles forming within 30 μs and subsequently collapsing in the formulations.

Navigating ambivalence.

This commentary explores the sometimes uncomfortable ambivalence that colors most people's experience of disability, either as an aspect of a person's own identity, or as an aspect of a person's interactions with a person with a disability. Disabilities are experienced by many as both a positive and a negative aspect of a person's identity. The commentary describes the work of two disability scholars who have recently explored how this ambivalence affects both legal schemes and parenting decisions, and argue for a 'destigmatizing' of this ambivalence.

Encapsulation into amphiphilic polyanhydride microparticles stabilizes Yersinia pestis antigens.

The design of biodegradable polymeric delivery systems based on polyanhydrides that would provide for improved structural integrity of Yersinia pestis antigens was the main goal of this study. Accordingly, the full-length Y. pestis fusion protein (F1-V) or a recombinant Y.

Insight into the induction mechanism of the GntR/HutC bacterial transcription regulator YvoA.

YvoA is a GntR/HutC transcription regulator from Bacillus subtilis implicated in the regulation of genes from the N-acetylglucosamine-degrading pathway. Its 2.4-A crystal structure reveals a homodimeric assembly with each monomer displaying a two-domain fold.

Twin-arginine-dependent translocation of SufI in the absence of cytosolic helper proteins.

The twin-arginine translocation (Tat) machinery present in bacterial and thylakoidal membranes is able to transport fully folded proteins. Folding of some Tat precursor proteins requires dedicated chaperones that also sequester the signal sequence during the maturation process. Whether or not signal sequence-binding chaperones are a general prerequisite for all Tat substrate proteins is not known.

Heterologous production, isolation, characterization and crystallization of a soluble fragment of the NADH:ubiquinone oxidoreductase (complex I) from Aquifex aeolicus.

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) is the first enzyme complex of the respiratory chains in many bacteria and most eukaryotes. It is the least understood of all, due to its enormous size and unique energy conversion mechanism. The bacterial complex is in general made up of 14 different subunits named NuoA-N.

A protein functional leap: how a single mutation reverses the function of the transcription regulator TetR.

Today's proteome is the result of innumerous gene duplication, mutagenesis, drift and selection processes. Whereas random mutagenesis introduces predominantly only gradual changes in protein function, a case can be made that an abrupt switch in function caused by single amino acid substitutions will not only considerably further evolution but might constitute a prerequisite for the appearance of novel functionalities for which no promiscuous protein intermediates can be envisaged. Recently, tetracycline repressor (TetR) variants were identified in which binding of tetracycline triggers the repressor to associate with and not to dissociate from the operator DNA as in wild-type TetR.

Expression and properties of arginyl-tRNA synthetase from jack bean (Canavalia ensiformis).

The coding region for arginyl-tRNA synthetase from jack bean (Canavalia ensiformis) has been sequenced and cloned into the bacterial expression vector pET32a. Transformation of BL21 cells and induction with IPTG results in the high level expression of the protein fused N-terminally with thioredoxin and bearing a His-tag. A substantial proportion of the enzyme is recovered in the soluble fraction of the cell lysate (10 mg per litre cell culture) and can be isolated with metal-affinity technology.

A cleavable N-terminal membrane anchor is involved in membrane binding of the Escherichia coli SRP receptor.

Different from eukaryotes, the bacterial signal recognition particle (SRP) receptor lacks a membrane-tethering SRP receptor (SR) beta subunit and is composed of only the SR alpha homologue FtsY. FtsY is a modular protein composed of three domains. The N- and G-domains of FtsY are highly similar to the corresponding domains of Ffh/SRP54 and SR alpha and constitute the essential core of FtsY.

PRMT6-mediated methylation of R2 in histone H3 antagonizes H3 K4 trimethylation.

The arginine methyltransferase PRMT6 (protein arginine methyltransferase 6) has been shown recently to regulate DNA repair and gene expression. As arginine methylation of histones is an important mechanism in transcriptional regulation, we asked whether PRMT6 possesses activity toward histones. We show here that PRMT6 methylates histone H3 at R2 and histones H4/H2A at R3 in vitro.

The toposome, essential for sea urchin cell adhesion and development, is a modified iron-less calcium-binding transferrin.

We describe the structure and function of the toposome, a modified calcium-binding, iron-less transferrin, the first member of a new class of cell adhesion proteins. In addition to the amino acid sequence of the precursor, we determined by Edman degradation the N-terminal amino acid sequences of the mature hexameric glycoprotein present in the egg as well as that of its derived proteolytically modified fragments necessary for development beyond the blastula stage. The approximate C-termini of the fragments were determined by a combination of mass spectrometry and migration in reducing gels before and after deglycosylation.

A derivative of lipid A is involved in signal recognition particle/SecYEG-dependent and -independent membrane integrations.

A cell-free system was developed that allows the correct integration of single and multispanning membrane proteins of Escherichia coli into proteoliposomes. We found that physiological levels of diacylglycerol were required to prevent spontaneous integration into liposomes even of the polytopic mannitol permease. Using diacylglycerol-containing proteoliposomes, we identified a novel integration-stimulating factor.

Membrane binding of the bacterial signal recognition particle receptor involves two distinct binding sites.

Cotranslational protein targeting in bacteria is mediated by the signal recognition particle (SRP) and FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRalpha subunit of eukaryotes, which is tethered to the membrane via its interaction with the membrane-integral SRbeta subunit. Despite the lack of a membrane-anchoring subunit, 30% of FtsY in Escherichia coli are found stably associated with the cytoplasmic membrane.

A functional mobA gene for molybdopterin cytosine dinucleotide cofactor biosynthesis is required for activity and holoenzyme assembly of the heterotrimeric nicotine dehydrogenases of Arthrobacter nicotinovorans.

Two Arthrobacter nicotinovorans molybdenum enzymes hydroxylate the pyridine ring of nicotine. Molybdopterin cytosine dinucleotide (MCD) was determined to be a cofactor of these enzymes. A mobA gene responsible for the formation of MCD could be identified and its function shown to be required for assembly of the heterotrimeric molybdenum enzymes.

An alpha/beta-fold C--C bond hydrolase is involved in a central step of nicotine catabolism by Arthrobacter nicotinovorans.

The enzyme catalyzing the hydrolytic cleavage of 2,6-dihydroxypseudooxynicotine to 2,6-dihydroxypyridine and gamma-N-methylaminobutyrate was found to be encoded on pAO1 of Arthrobacter nicotinovorans. The new enzyme answers an old question about nicotine catabolism and may be the first C--C bond hydrolase that is involved in the biodegradation of a heterocyclic compound.

Is the nephritogenic antigen in post-streptococcal glomerulonephritis pyrogenic exotoxin B (SPE B) or GAPDH?

Background: Acute glomerulonephritis can follow infection by group A streptococci. An immune-complex pathogenesis is accepted, but the causative antigen(s) is still controversial. In recent years, 2 streptococcal antigens, the cationic cysteine proteinase exotoxin B (SPE B) and the plasmin receptor, a glyceraldehyde phosphate dehydrogenase (Plr, GAPDH) have attracted attention because: (1) they were localized in glomeruli in patients with acute post-streptococcal glomerulonephritis (APSGN); and (2) serum antibody to these antigens was associated with nephritogenic streptococcal infections.

Phenylalanine ammonia-lyase modified with polyethylene glycol: potential therapeutic agent for phenylketonuria.

Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by the defects in the phenylalanine hydroxylase (PAH) gene. Individuals homozygous for defective PAH alleles show elevated levels of systemic phenylalanine and should be under strict dietary control to reduce the risk of neuronal damage associated with high levels of plasma phenylalanine. Researchers predict that plant phenylalanine ammonia-lyase (PAL), which converts phenylalanine to nontoxic t-cinnamic acid, will be an effective therapeutic enzyme for the treatment of PKU.

A novel gamma-N-methylaminobutyrate demethylating oxidase involved in catabolism of the tobacco alkaloid nicotine by Arthrobacter nicotinovorans pAO1.

Nicotine catabolism, linked in Arthrobacter nicotinovorans to the presence of the megaplasmid pAO1, leads to the formation of gamma-N-methylaminobutyrate from the pyrrolidine ring of the alkaloid. Until now the metabolic fate of gamma-N-methylaminobutyrate has been unknown. pAO1 carries a cluster of ORFs with similarity to sarcosine and dimethylglycine dehydrogenases and oxidases, to the bifunctional enzyme methylenetetrahydrofolate dehydrogenase/cyclohydrolase and to formyltetrahydrofolate deformylase.

Identification and immunoreactivity of proteins released from Streptococcus agalactiae.

The aim of the present study was to identify released proteins of Streptococcus agalactiae and to investigate their immunoreactivity with human sera to determine whether such proteins might be viable as carrier proteins in conjugate vaccines. Infections with S. agalactiae are the leading cause of sepsis and meningitis in neonates.

Composition and activity of the Rhodobacter capsulatus degradosome vary under different oxygen concentrations.

The influence of changes in temperature or oxygen tension during growth of Rhodobacter capsulatus on the composition and activity of the degradosome, an RNA-processing protein complex, was investigated. Only minor differences in the amount of specific proteins of the complex were observed after a decrease or increase of the temperature, but dramatic variations were detectable during growth at different oxygen concentrations. In particular, the amount of the transcription factor Rho, which was previously shown to be associated with the R.

Identification of beta-subunit of bacterial RNA-polymerase--a non-species-specific bacterial protein--as target of antibodies in primary biliary cirrhosis.

Several observations suggest that bacteria induce autoimmunity in primary biliary cirrhosis (PBC). Since no PBC-specific bacterial species could be identified, it can be speculated that the triggers are non-species-specific bacterial proteins. This hypothesis would imply that several or even all bacterial species can trigger PBC.

Two W-containing formate dehydrogenases (CO2-reductases) involved in syntrophic propionate oxidation by Syntrophobacter fumaroxidans.

Two formate dehydrogenases (CO2-reductases) (FDH-1 and FDH-2) were isolated from the syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans. Both enzymes were produced in axenic fumarate-grown cells as well as in cells which were grown syntrophically on propionate with Methanospirillum hungatei as the H2 and formate scavenger. The purified enzymes exhibited extremely high formate-oxidation and CO2-reduction rates, and low Km values for formate.

Characterization of the chlorate reductase from Pseudomonas chloritidismutans.

A chlorate reductase has been purified from the chlorate-reducing strain Pseudomonas chloritidismutans. Comparison with the periplasmic (per)chlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of P. chloritidismutans reduced only chlorate and bromate.

Specific stimulation of peripheral blood mononuclear cells from patients with acute myocarditis by peptide-bound flavin adenine dinucleotide (FAD), a naturally occurring autologous hapten.

The tryptic FAD-peptide carrying the flavin in 8alpha-(N3)histidyl linkage as natural hapten was isolated by HPLC from the bacterial enzyme 6-hydroxy-d-nicotine oxidase. The same flavin protein linkage is found in the mitochondrial succinate dehydrogenase flavoprotein subunit, the predominant flavoprotein with covalently bound FAD in mitochondria of cardiomyocytes. Peripheral blood mononuclear cells (PBMC) were isolated from four patients with acute myocarditis, seven patients with dilated cardiomyopathy (DCM) and from four healthy control individuals.