Antimicrobial resistance and resistance plasmids in Salmonella from Ontario, Canada.

Collections of 589 human and 204 animal strains of Salmonella isolated in Ontario during the summer of1974 were examined for susceptibility to 12 antimicrobial agents. Many isolates were found to be resistant to both chloramphenicol (12.4% of the human and 38.2% of the animal sample) and ampicillin. The chloramphenicol resistance almost always occurred in strains which were also resistant to ampicillin and was usually due to a self-transmissible plasmid with a resistance pattern of CmKmSmTc (chloramphenicol, kanamycin, streptomycin, and tetracycline) or CmTc. Ampicillin resistance in these strains was mediated by a variety of plasmids with patterns ApSu (ampicillin and sulfa drugs) and ApSmSu, many of which were nonself-transmissible. Ampicillin resistance in chloramphenicol-sensitive strains was transferable from 21% of the strains, and it was associated with resistance patterns which were different from the self-transferable ampicillin patterns from the chloramphenicol-resistance strains.     

Techniques for Preparing Seeds with Water-Impermeable Coats for Light and Electron Microscopy

An improved method for fixing and embedding seeds with impermeable coats for microscopic study has been developed. Entry portals are cut into seed coats to permit better penetration of fixative. This makes it possible to obtain semithin sections of whole seeds for light microscopy and thin sections of selected areas for electron microscopy. Seed tissues may thereby be studied relative to their position in the seed and to surrounding tissues. This permits studies of imbibition and developmental morphology of seeds in histological and cytological detail previously possible only with soft or dissected seeds.     

Radioiodinated iodobenzoyl conjugates of a monoclonal antibody Fab fragment. In vivo comparisons with chloramine-T-labeled Fab

A comparative investigation of the biodistributions of radioiodinated p- and m-iodobenzoyl conjugates of a monoclonal antibody Fab fragment, NR-LU-10 Fab, and the same antibody Fab fragment radioiodinated by the chloramine-T (ChT) method has been carried out in mice. Coinjected, dual-isotope studies in athymic mice with tumor xenografts have demonstrated that there are only minor differences in the in vivo distributions of the iodobenzoyl-labeled Fabs, except in the excretory organs, kidneys, and intestines, where major differences were observed. Similarly, coinjection of either the p-iodobenzoyl or m-iodobenzoyl conjugate of NR-LU-10 Fab with the Fab radioiodinated with ChT/radioiodide into BALB/c mice provided additional data that indicated that the two iodobenzoyl conjugates distributed similar in a number of selected tissues. The tissue-distribution differences of the regioisomeric iodobenzoyl conjugates in relation to the ChT-radioiodinated Fab were large for the stomach and neck, consistent with previous studies. The most notable difference between the two iodobenzoyl conjugates was the kidney activity, where the m-iodobenzoyl conjugate was similar to the directly labeled Fab, but the p-iodobenzoyl-conjugated Fab was higher by nearly a factor of 2.     

Type VI collagen microfibrils: evidence for a structural association with hyaluronan

Type VI collagen, a widespread structural component of connective tissues, has been isolated in abundance from fetal bovine skin by a procedure involving bacterial collagenase digestion under nonreducing, nondenaturing conditions and gel filtration chromatography. Rotary shadowing electron microscopic analysis revealed that the collagen VI was predominantly in the form of extensive intact microfibrillar arrays. These microfibrils were seen in association with hyaluronan, which was identified by its ability to bind the G1 fragment of cartilage proteoglycan. Treatment with highly purified hyaluronidase largely disrupted the collagen VI microfibrils into component tetramers, double tetramers, and short microfibrillar sections. Subsequent incubation of disrupted collagen VI in the presence of hyaluronan facilitated a partial repolymerization of the microfibrils. In vitro binding studies have also demonstrated that type VI collagen binds hyaluronan with a relatively high affinity. These studies demonstrate that a specific structural relationship exists between type VI collagen and hyaluronan. This association is likely to be of primary importance in the growth and remodeling processes of connective tissues.     

Interaction of endothelial cells with a laminin A chain peptide (SIKVAV) in vitro and induction of angiogenic behavior in vivo.

Endothelial cells are known to bind to laminin, and two peptides derived from the laminin A (CTFALRGDNP) and B1 (CDPGYIGSR) chains block the capillary-like tube formation on a laminin-rich basement membrane matrix, Matrigel. In the present study, we have used various in vitro and in vivo assays to investigate the angiogenic-biologic effects of a third active site in the laminin A chain, CSRARKQAASIKVAVSADR (designated PA22-2) on endothelial cells. The SIKVAV-containing peptide was as active as the YIGSR-containing peptide for endothelial cell attachment but was less active than either the RGD-containing peptide or intact laminin. Endothelial cells seeded on this peptide appeared fibroblastic with many extended processes, unlike the normal cobblestone morphology observed on tissue culture plastic. In addition, in contrast to normal tube formation on Matrigel, short irregular structures formed, some of which penetrated the matrix and sprouting was more apparent. Analysis of endothelial cell conditioned media of cells cultured in the presence of this peptide indicated degradation of the Matrigel and zymograms demonstrated active collagenase IV (gelatinase) at 68 and 62 Kd. A murine in vivo angiogenesis assay and the chick yolk sac/chorioallantoic membrane assays with the peptide demonstrated increased endothelial cell mobilization, capillary branching, and vessel formation. These data suggest that the -SIKVAV-site may play an important role in initiating branching and formation of new capillaries from the parent vessels, a behavior that is observed in vivo in response to tumor growth or in the normal vascular response to injury.     

Biochemical Correlates of Epilepsy in the El Mouse: Analysis of Glial Fibrillary Acidic Protein and Gangliosides

The E1 (epileptic) mouse is considered a model for complex partial seizures in humans. Seizures in E1 mice begin around 7-8 weeks of age and persist throughout life. To determine if astrocytic gliosis was present in adult seizing E1 mice, the distribution of glial fibrillary acidic protein (GFAP) was studied in the hippocampus using an antibody to GFAP. The mean number of GFAP-positive cells per square millimeter of hippocampus was approximately 15- to 40-fold higher in adult E1 mice than in nonseizing control C57BL/6J (B6) mice or in young nonseizing E1 mice. Relative GFAP concentration (expressed per milligram of total tissue protein) in hippocampus and cerebellum was estimated by densitometric scanning of peroxidase-stained western blots. GFAP concentration was 2.7-fold greater in hippocampus of adult seizing E1 mice than in the control B6 mice. No differences in GFAP content were detected between the strains in the cerebellum. Because gangliosides can serve as cell surface markers for changes in neuronal cytoarchitecture, they were analyzed to determine if the gliotic response in E1 mice was associated with changes in neural composition. Although the total ganglioside concentration of hippocampus, cerebral cortex, and cerebellum was similar in adult E1 and control B6 mice, a synaptic membrane enriched ganglioside, GD1a, was elevated in the adult E1 cerebral cortex and hippocampus. The findings indicate that E1 mice express a type of gliosis that is not accompanied by obvious neuronal loss.     

Site-directed mutagenesis of recombinant human arylamine N-acetyltransferase expressed in Escherichia coli. Evidence for direct involvement of Cys68 in the catalytic mechanism of polymorphic human NAT2.

The single coding exons of the cloned genes encoding two human arylamine N-acetyltransferases (NAT1 and NAT2) were amplified by expression-cassette polymerase chain reaction and subcloned into the tac promoter-based phagemid vector pKEN2 for production of the recombinant proteins in Escherichia coli strain XA90. Induction of cultures grown from selected bacterial transformants resulted in the production of substantial quantities of soluble recombinant human NAT1 and NAT2 with identical electrophoretic, immunologic and catalytic properties to those expressed in mammalian cell culture or in human liver. Oligonucleotide-directed mutagenesis of recombinant human NAT2 was then employed to determine the relative importance of 3 highly conserved cysteine residues in the enzyme's catalytic mechanism. Substitution of cysteine with glycine at position 68 of the 290 amino acid protein molecule (Cys68----Gly) resulted in the production of normal quantities of immunoreactive NAT2 which was completely devoid of enzyme activity, suggesting that the sulfhydryl group of Cys68 is directly involved in the transfer of acetate from the essential cofactor CoASAc to acceptor amine substrates. On the other hand, the mutations producing Cys44----Gly and Cys223----Gly led to the production of enzymatically active NAT2 proteins with markedly reduced in vitro stability, suggesting that substitution of either of these amino acids may cause alterations in the tertiary structure of the native enzyme.     

The effect of palate repair on otitis media with effusion.

Cleft palate in children is very frequently associated with otitis media with effusion. In this prospective study of 150 cleft palate children aged between 2 and 18 months, the prevalence of otitis media with effusion at myringotomy before palate repair was 92 percent. Otoscopic and tympanometric follow-up assessments for 140 children indicate that there is minimal improvement in middle ear status after palate repair. The condition is persistent in 70 percent of children up to 4 years of age. Furthermore, there is no evidence that age at repair or type of cleft is influential. These findings have implications for otologic management of cleft palate children. Early routine unilateral ventilation to ensure adequate hearing but minimize the morbidity of ventilation tubes is advocated.