2-Phenylethylamine catabolism by Escherichia coli K12

Escherichia coli K12 grows on 2-phenylethylamine as sole carbon and energy source by converting it, via phenylacetaldehyde, to phenylacetic acid. Phenylacetaldehyde was formed by the action of an inducible amine oxidase and catalase activity was increased sixfold, presumably to ensure removal of the H2O2 that was expected to be a product of the amine oxidation. The phenylacetaldehyde was oxidized to phenylacetic acid by an inducible NAD+-dependent dehydrogenase. Mutants defective in phenylacetaldehyde dehydrogenase cannot grow on 2-phenylethylamine as carbon and energy source but can still use it as a nitrogen source.     

Conservation of δ-crystallin gene structure between ducks and chickens

A cloned chicken delta-crystallin cDNA was used to identify two putative delta-crystallin genes in the duck by Southern blot hybridization. A DNA fragment containing most of one of these genes was isolated from a library made in bacteriophage lambda Charon 28A containing genomic DNA from 14-day-old embryonic ducks. Electron microscopy, partial gene sequencing, primer extension analysis using duck mRNA, and comparison with the well-characterized chicken delta-crystallin genes suggest that our cloned duck delta-crystallin gene, like the chicken delta-crystallin genes, is 8-10 kb long and contains 17 exons. Hybridization and sequencing data show great similarity between the homologous 5' untranslated and coding exons of the duck and chicken delta-crystallin genes. Overall, the homologous introns also appear to have approximately 30% sequence similarity, and have been subject to deletion/insertion events. Our partial characterization of duck delta-crystallin gene sequences suggests that this avian and reptilian crystallin family has been conserved during evolution, as have the other crystallin gene families that are expressed in the eye lens.     

Animal model of acute pericarditis and its progression to pericardial fibrosis and adhesions: ultrastructural studies

To study the evolution of pericardial inflammation, we have developed a model of pericarditis in sheep by surgically injecting heat-killed staphylococci and Freund's adjuvant into the pericardial cavity under sterile conditions. The pericarditis evolved through the following phases: 1) inflammatory response, 2) mesothelial cell injury and desquamation, and 3) fibrotic phase. At 3-24 hr there was increased microvascular permeability, which resulted in the exudation of fluid, neutrophils, macrophages, and fibrin into the pericardial cavity and the pericardial interstitium. By 72 hr, large numbers of inflammatory cells were aggregated on the mesothelial surfaces and dispersed throughout the pericardial cavity, either as free-floating cells or located between strands of fibrin. At 6 days, fibrinolysis was apparent along the mesothelial surfaces; and newly formed collagen fibrils were deposited throughout the interstitial spaces and among the aggregated cells. These fibrils provided a matrix for the growth of new blood and lymphatic vessels into new connective tissue on both parietal and visceral pericardial surfaces. At 2 weeks, intrapericardial fibrosis had produced focal adhesions between the pericardial surfaces. At 1 month, extensive areas of the pericardial cavity were obliterated. By 9 months, there was a marked reduction in the numbers of cells and blood vessels and increased deposition of collagen and elastic fibers. The intrapericardial injection of heat-killed staphylococci and adjuvant provides a reproducible animal model to study the time course of pericardial inflammation.     

Pregnancy diagnosis in zoo animals by estrogen determination in feces

Estrogen concentration in feces was investigated in five different herbivorous species of zoo animals. Using a nonspecific estrogen radioimmunoassay, in four species (red buffalo, yak, Grevy's zebra, and Nubian ibex) pregnancy was revealed by measuring estrogen concentration in feces. In hippopotamus, the levels of fecal estrogens were not different between pregnant and nonpregnant animals.     

Clinical hypersensitivity to specific aerosol challenge in parenterally immunized calves.

This paper reports an experiment designed to demonstrate that the calf lung can be sensitized to a specific respirable challenge following parenteral immunization with a nonliving antigen (human serum albumin). The possibility that immune-mediated injury could subsequently interfere with nonspecific mucosal defenses was also investigated by infecting calves with Pasteurella haemolytica after the antigen challenge and assessing pulmonary clearance of the organism. The results indicated that specific aerosol challenge produces reversible signs of respiratory hypersensitivity and that persistence of incidental infection in the upper respiratory tract is potentiated. Since the calves were sensitized by an immunization regime which imitated conventional vaccination, this study highlights the potential dangers of inactivated parenteral respiratory vaccines.     

Generation of Tn5 insertions in streptococcal conjugative transposon Tn916.

A method has been developed for the introduction of Tn5 into Escherichia coli plasmid chimeras containing Streptococcus faecalis DNA. Tn5 could be introduced via a lambda::Tn5 delivery vehicle. The system proved to be particularly efficient and facilitated insertions at numerous sites on DNA containing the 16-kilobase conjugative transposon Tn916. It was possible to introduce some of the resulting Tn916::Tn5 derivatives back into S. faecalis by using a recently developed protoplast transformation procedure. A presumed zygotic induction resulted in insertion of the Tn916 derivatives at multiple sites in the S. faecalis chromosome.     

Superoxide removal and radiation protection in bacteria

Previous work with procaryotic cells has identified one kind of lethal damage from ionizing radiation which occurs only within a specific range of low O2 concentrations, about 10(-6) to 10(-4) M. Within this range, protection can occur in three ways: through the enzymatic decomposition of hydrogen peroxide (H2O2) by added catalase, through the enzymatic degradation of superoxide anion radicals (.O2-) by added superoxide dismutase (SOD), and through scavenging hydroxyl radicals (.OH) by various additives. These results indicate that three radiolytic products, H2O2, .OH, and .O2- (and/or the conjugate acid, the perhydroxyl radical, .HO2) are involved in this single kind of radiation-induced damage. Although the radiolytic productions of H2O2 and .O2- are strongly enhanced in higher O2 concentrations, neither enzyme protects when these air-equilibrated bacteria are irradiated. These experiments address this apparent contradiction and focus on the specific issue of why the addition of SOD protects at low but not at high O2 concentrations. We propose that, at a given O2 concentration, .O2- (and/or .HO2) may either react (with some cellular component?) to cause damage or react (with itself) to form hydrogen peroxide (H2O2). The specific O2 concentration during irradiation would determine the relative rates of these competing reactions and therefore the O2 concentration itself would establish whether or not we will observe damage from .O2-.     

Phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in isolated hepatocytes

Hepatocytes, isolated from rats fed a low-protein diet, were incubated with [32P]Pi and the phosphoproteins analysed. Immunoprecipitation using antibody against El of branched-chain 2-oxo acid dehydrogenase complex demonstrated phosphorylation of the alpha-subunit of El. Analysis of the tryptic phosphopeptides from the alpha-subunit indicated that two sites were phosphorylated. 4-methyl 2-oxopentanoate and DL-2-chloro 4-methylpentanoate decreased labelling of both sites. No major direct effects of several hormones on phosphorylation of branched-chain 2-oxo acid dehydrogenase was observed.     

Biochemical and biophysical comparison of two mucins from human submandibular-sublingual saliva

A high-molecular-weight mucin-glycoprotein (MG1) was isolated from human submandibular-sublingual saliva and was comprised of 14.9% protein, 29.0% N-acetylglucosamine, 9.4% N-acetylgalactosamine, 10.5% fucose, 24.2% galactose, 0.9% mannose, 4.0% N-acetylneuraminic acid, and 7.0% sulfate. Carbohydrate units were O-glycosidically linked and ranged in size from 4 to 16 residues. The biophysical properties of MG1 were compared to those of a smaller mucin (MG2) also isolated from submandibular-sublingual saliva. Fluorescence spectroscopy demonstrated that MG1 bound both 1-anilino-8-naphthalenesulfonate (ANS) and N-phenyl-1-naphthylamine (NPNA) in stable hydrophobic binding sites (melting temperature, 47 +/- 2 degrees C), whereas MG2 did not bind these hydrophobic probes. These hydrophobic domains occurred on nonglycosylated or naked portions of MG1 since Pronase treatment eliminated ANS binding. Reduction of disulfide bridges in MG1 increased the number of available hydrophobic binding sites. High ionic strength (0 to 2 M NaCl) had no effect on ligand binding, whereas lowering pH (9 to 2) increased ANS binding without affecting NPNA complexation. Circular dichroism (CD) data suggested that MG1's carbohydrate chains dominated its spectrum. In contrast, the peptide backbone dominated the CD spectrum of MG2. Collectively, the results of this study indicate that human submandibular-sublingual saliva contains two structurally distinct mucins.     

Association of viral and plasmid DNA with the nuclear matrix during productive infection

The association of simian virus 40 (SV40) DNA or plasmid DNA in subcellular fractions from either infected or transfected cells was examined. In lytically infected cells, approx. 25% of viral specific DNA during the infection cycle was retained in nuclei after washing with low ionic strength buffer and 1% Triton X-100. Viral replicating DNA found in the nuclear matrix was capable of performing limited DNA synthesis by the endogenous DNA polymerase in vitro. Viral DNA synthesized in vitro hybridized preferentially to SV40 Hind-III B and C fragments which are in proximity to the origin of replication. In plasmid-transfected COS-7 cells (SV40-transformed cells), the amount of plasmid DNA found in the nuclear matrix was related to its replication efficiency in cells. More than 80% of the plasmid DNA was tightly associated with subnuclear structures. Little or no plasmid DNA was found in the cytoplasmic fraction. The results suggest that, in extrachromosomal model systems, the association of DNA with nuclear matrix is important for the regulation of DNA replication.