Posthatching Development of Vascularization,Arteriovenous Anastomoses and Seromucous Glands in the Avian Nasal Mucosa

The development of blood vessels and growth of different tissue compartments in the avian nasal mucosa were studied in chickens from hatching to 5 months of age. The vascularity of the rostral and middle nasal conchae increased about 4-fold, while there was a progressive decrease in the relative size of the epithelium and connective tissue compartments, during the maturation period. Arteriovenous anastomoses (AVAs) were present already at hatching and their density increased 3-fold during the second week of posthatching development. The seromucous glands form by a process of invagination of the epithelium, which occurs predominantly during the first week after hatching. The glands continue to grow in size and an increasing fraction of the cells becomes mucous-secreting. The observations are discussed in relation to the role of the nasal mucosa in temperature regulation and conditioning of the inhaled air.     

Cloning of the mgtE Mg2+ transporter from Providencia stuartii and the distribution of mgtE in gram-negative and gram-positive bacteria.

The MM281 strain of Salmonella typhimurium possesses mutations in each of its three Mg2+ transport systems, requires 100 mM Mg2+ for growth, and was used to screen a genomic library from the gram-negative bacterium Providencia stuartii for clones that could restore the ability to grow without Mg2+ supplementation. The clones obtained also conferred sensitivity to Co2+, a phenotype similar to that seen with the S. typhimurium corA Mg2+ transport gene. The sequence of the cloned P. stuartii DNA revealed the presence of a single open reading frame, which was shown to express a protein with a gel molecular mass of 37 kDa in agreement with the deduced size of 34 kDa. Despite a phenotype similar to that of corA and the close phylogenetic relationship between P. stuartii and S. typhimurium, this new putative Mg2+ transporter lacks similarity to the CorA Mg2+ transporter and is instead homologous to MgtE, a newly discovered Mg2+ transport protein from the gram-positive bacterium Bacillus firmus OF4. The distribution of mgtE in bacteria was studied by Southern blot hybridization to PCR amplification products. In contrast to the ubiquity of the corA gene, which encodes the dominant constitutive Mg2+ influx system of bacteria, mgtE has a much more limited phylogenetic distribution.     

Characterization of a folding intermediate of human carbonic anhydrase II: probing local mobility by electron paramagnetic resonance.

The spin-labeling method was used to investigate human carbonic anhydrase, HCA II, undergoing unfolding induced by guanidine-HCI (Gu-HCI). The spin-probe, N-(2,2,5,5-tetramethyl-1-yloxypyrrolidinyl-3-yl)iodoacetamide, was attached covalently to the single cysteine (position 206) in the enzyme. The electron paramagnetic resonance spectrum of the folded structure showed the characteristic slow motional spectra. When the concentration of the denaturing agent, Gu-HCI, was gradually increased, new spectral components with narrower lines evolved to give complex electron paramagnetic resonance spectra, apparently containing superimposed contributions from several components of different mobility. By a differentiation technique, it was possible to follow the relative increase of the narrow components as a function of Gu-HCI concentration. The amplitude of difference spectra versus Gu-HCI concentration showed two distinct maxima, indicating the existence of a folding intermediate state/structure. The results were found to agree with optical absorption data, which showed similar transitions at the same Gu-HCI concentrations. From line-shape simulations assuming a Brownian diffusion model, the rotational diffusion constants for the spin-label in the folded, folding intermediate, and unfolded structures were determined. The relative abundances of the three conformations in the region 0-4 M Gu-HCI were obtained by least squares fitting of the simulated spectra to the experimental ones. The folding intermediate was found to have a maximum population of 39 +/- 4% at approximately 0.7 M Gu-HCI.     

Urinary excretion of mercury, copper and zinc in subjects exposed to mercury vapour

The excretion of mercury, copper and zinc in urine, and mercury in whole blood andplasma, was determined in 40 chloralkali workers exposed to mercury vapour and 40age-matched referents. The Hg concentrations in whole blood, plasma and urine werehigher in the exposed group (35 nmol l, 30 nmol l,and 11.5 nmol mmol creatinine, respectively) in comparison with thereference group (15 nmol l, 6.3 nmol l, and 1.8nmol mmol creatinine, respectively). The urinary copper excretionwas similar in the two groups, while U-Zn excretion was significantly higher (P = 0.04)in the exposed group, median 0.83 mmol mmol creatinine versus 0.76mnmol mmol creatinine in the reference group. In a subgroup of exposedworkers with current U-Hg above 11.5 nmol lmmolcreatinine (20 mg g creatinine) the medianU-Zn was 1.1 mmol mmol creatinine. In both groups smokers had highU-Zn levels than non smokers. When both U-Hg and smoking were taken into account in alinear regression model, there was a significant association between U-Hg and U-Zn inthe combined group of exposed and referents (P = 0.002). This study indicates thatmercury exposure in humans, as in animals, causes increased urinary excretion of zinc.The mechanisms may be induced synthesis of metallothionein in the kidneys, displacementof Zn from preexisting metallothionein by Hg, or a decreased reabsorption of zinc in thekidneys owing to a slight tubular dysfunction.     

Probing sequence-specific RNA recognition by the bacteriophage MS2 coat protein.

We present the results of in vitro binding studies aimed at defining the key recognition elements on the MS2 RNA translational operator (TR) essential for complex formation with coat protein. We have used chemically synthesized operators carrying modified functional groups at defined nucleotide positions, which are essential for recognition by the phage coat protein. These experiments have been complemented with modification-binding interference assays. The results confirm that the complexes which form between TR and RNA-free phage capsids, the X-ray structure of which has recently been reported at 3.0 A, are identical to those which form in solution between TR and a single coat protein dimer. There are also effects on operator affinity which cannot be explained simply by the alteration of direct RNA-protein contacts and may reflect changes in the conformational equilibrium of the unliganded operator. The results also provide support for the approach of using modified oligoribonucleotides to investigate the details of RNA-ligand interactions.     

Building proteins from C alpha coordinates using the dihedral probability grid Monte Carlo method.

Dihedral probability grid Monte Carlo (DPG-MC) is a general-purpose method of conformational sampling that can be applied to many problems in peptide and protein modeling. Here we present the DPG-MC method and apply it to predicting complete protein structures from C alpha coordinates. This is useful in such endeavors as homology modeling, protein structure prediction from lattice simulations, or fitting protein structures to X-ray crystallographic data. It also serves as an example of how DPG-MC can be applied to systems with geometric constraints. The conformational propensities for individual residues are used to guide conformational searches as the protein is built from the amino-terminus to the carboxyl-terminus. Results for a number of proteins show that both the backbone and side chain can be accurately modeled using DPG-MC. Backbone atoms are generally predicted with RMS errors of about 0.5 A (compared to X-ray crystal structure coordinates) and all atoms are predicted to an RMS error of 1.7 A or better.     

Anion binding to resting and half-reduced Pseudomonas cytochrome c peroxidase

The anion-binding characteristics of resting and half-reduced Pseudomonas cytochrome c peroxidase (ferrocytochrome c-551: hydrogen peroxide oxidoreductase, EC 1.11.1.5) have been examined by EPR and optical spectroscopy with cyanide, azide and fluoride as ligands. The resting enzyme was found to be essentially inaccessible for ligation, which indicates that it has a closed conformation. In contrast, the half-reduced enzyme has a conformation in which the low-potential heme is easily accessible for ligands, a behavior parallel to that towards the substrate hydrogen peroxide (R?nnberg, M., Araiso, T., Ellfolk, N. and Dunford, H.B. (1981) Arch. Biochem. Biophys. 207, 197-204). Cyanide and azide caused distinct changes in the low-potential heme c moiety, and the gz values of the two low-spin derivatives were 3.14 and 3.22, respectively. Fluoride binds to the same heme, giving rise to a high-spin signal at g = 6. The dissociation constants of the anions differ widely from each other, the values for the cyanide, azide and fluoride being 23 microM, 2.5 mM and 0.13 M, respectively. In addition, a partial shift of the low-spin peak at g = 2.84 of the half-reduced species to 3.24 was observed even at low concentrations of fluoride.     

Photosystem I photochemistry at low temperature. Heterogeneity in pathways for electron transfer to the secondary acceptors and for recombination processes

Electron transport has been studied by flash absorption and EPR spectroscopies at 10–30 K in Photosystem I particles prepared with digitonin under different redox conditions. In the presence of ascorbate, an irreversible charge separation is progressively induced at 10 K between P-700 and iron-sulfur center A by successive laser flashes, up to a maximum which corresponds to about two-thirds of the reaction centers. In these centers, heterogeneity of the rate for center A reduction is also shown. In the other third of reaction centers, the charge separation is reversible and relaxes with a t_1/2 ≈ 120 μs. When the iron-sulfur centers A and B are prereduced, the 120 μs relaxation becomes the dominant process (70–80% of the reaction centers), while a slow component (t_1/2 = 50–400 ms) reflecting the recombination between P-700⁺ and center X⁻ occurs in a minority of reaction centers (10–15%). Flash absorption and EPR experiments show that the partner of P-700⁺ in the 120 μs recombination is neither X nor a chlorophyll but more probably the acceptor A⁻₁ as defined by Bonnerjea and Evans (Bonnerjea, J. and Evans, M.C.W. (1982) FEBS Lett. 148, 313–316). The role of center X in low-temperature electron flow is also discussed.     

Self-transmissible plasmid in Zymomonas mobilis carrying antibiotic resistance.

The cryptic plasmid pRUT41 from Zymomonas mobilis was examined for its biological properties. This plasmid was found to be conjugally transferred from Z. mobilis CP4 to Escherichia coli BM21 and to carry genes for antibiotic resistance (gentamicin, kanamycin, and streptomycin). Covalently closed circular plasmid DNA was isolated from eight transconjugants of E. coli BM21. These plasmids were identical in mobility on agarose gels and exhibited the same restriction patterns as the native pRUT41 plasmid isolated from Z. mobilis. The plasmid location of the antibiotic resistance genes was further confirmed by transforming E. coli BM21 with isolated pRUT41 plasmid from strain CP4 and with plasmids from the transconjugants of BM21. Resistance to streptomycin, kanamycin, and gentamicin was tightly linked and transferred together in all cases.     

Preparation of isolated liver endothelial cells and Kupffer cells in high yield by means of an enterotoxin

A new method for preparing non-parenchymal rat liver cells (NPC) is described. The liver cell suspension, prepared by perfusing the liver with collagenase, was treated with enterotoxin from Clostridium perfringens for 15 min. The enterotoxin made the parenchymal cells leaky, and these cells could be separated from the NPC by centrifugation in a solution containing Nycodenz (20%, w/v). During the centrifugation, the NPC floated, while the parenchymal cells sedimented. The yield of NPC per liver (200 g rat) was about 250 X 10(6) cells. The NPC were further separated into endothelial cells, Kupffer cells and stellate cells by centrifugal elutriation. This method was particularly useful for preparing endothelial cells in high yield (100 X 10(6) cells per liver). Intravenously injected formaldehyde-treated albumin was selectively taken up by the endothelial cells. Isolated endothelial cells in suspension as well as in surface culture maintained their ability to endocytose this ligand.