Increase of glycocalyx and altered lectin agglutination profiles of Pasteurella haemolytica A1 after incubation in bovine subcutaneous tissue chambers in vivo or in ruminant serum in vitro.

Pasteurella haemolytica serotype A1 (bovine strain OK) was incubated for 2 and 6 h in bovine subcutaneous tissue chambers in vivo, and ovine strain 82-25 and bovine strain L011 were incubated in vitro for 2 h in heat-inactivated ovine or bovine serum from which gamma globulin had been depleted by protein G affinity chromatography to assess changes in morphology and lectin agglutination profiles (strains 82-25 and L101 only). Cells, removed from chambers after 2 h, were covered with an extensive, dense glycocalyx extending approximately 0.5 microm from the cell surface. In many cells, the glycocalyx was separated from the cell surface by a clear, electron-transparent area. Cells, removed at 6 h, were covered with a sparse glycocalyx of fine fibers 0.2 to 0.3 microm from the cell surface. Strains 82-25 and L101, incubated for 2 h in heat-inactivated ovine or bovine serum or in heat-inactivated ovine or bovine serum depleted of gamma globulin by protein G affinity chromatography, were also covered with a glycocalyx. The glycocalyx did not bind protein A-colloidal gold and therefore did not contain aggregates of accumulated antibody. Strains 82-25 and L101 were incubated individually for 2 h in 10 mM sodium phosphate buffer (pH 7.2) containing 0.14 M NaCl, 0.5 mM CaCl2, and 0.15 mM MgCl2 or with this buffer and either 25% heat-inactivated, gamma globulin-depleted ovine serum or 25% heat-inactivated, gamma globulin-depleted bovine serum. Agglutination profiles were then determined with 17 lectins in 10 mM HEPES-buffered saline (pH 8.4) with 0.1 mM CaCl2 and 0.08% sodium azide. Profiles did not vary with 10 of 17 lectins. However, profiles did vary with peanut agglutinin, Phaseolus vulgaris leucoagglutinin, Sophora japonica agglutinin, Maackia amurensis lectin II, Narcissus pseudonarcissus (daffodil) lectin, Griffonia simplicifolia lectin I, and Pisum sativum agglutinin. Altered profiles indicate a change in the bacterial cell surface, possibly by adsorption or alteration of surface carbohydrate moieties by serum constituents.     

The protective effect of ipratropium bromide aerosol against bronchospasm induced by hyperventilation and the inhalation of allergen, methacholine and histamine

The ability of the anticholinergic agent, ipratropium bromide, Atrovent, (40 mcg from a metered dose inhaler) to prevent bronchoconstriction produced by four different provocation tests was compared with placebo in 12 asthmatic patients. The provocation tests used were hyperventilation and inhalations of histamine, methacholine and an allergen to which the subject was known to be sensitive. The order in which each patient received the tests was determined according to a Latin-square design and remained the same on both test days. Pretreatment with ipratropium bromide and placebo was allocated randomly and administered in double-blind fashion. Ipratropium bromide provided significant protection at the 5% level against all four types of challenge. The average number of breaths required to produce a fall of at least 20% in FEV1 (forced expiratory volume in one second) was doubled for both histamine and allergen and increased by a factor of six for methacholine. The fall in FEV1 induced by hyperventilation was approximately halved. No side effects were noted with ipratropium bromide.     

Cytokeratin 5/6 in normal human breast: lack of evidence for a stem cell phenotype

In recent studies, B?cker and colleagues described a population of cells in paraffin wax sections of normal human breast that express cytokeratins (CK) 5/6 without expression of CK8/18 or smooth muscle actin (SMA). They proposed that these represent stem cells that give rise to differentiated luminal and myoepithelial cells. The data have been used to generate a model for breast cancer progression and classification with associated implications for management of pre-invasive disease. In this study, the expression of CK5/6, CK8/18, and SMA was investigated using multiple immunofluorescence on matched pairs of paraffin wax-embedded and frozen breast specimens. The staining patterns reported previously in antigen-retrieved paraffin wax-embedded sections were confirmed but no CK5/6-only cells were found in frozen sections of normal breast. There were cells with low levels of CK8/18 expression in frozen sections that may correspond to the CK8/18 'negative' cells seen in paraffin wax sections. This study brings into question the previously described profile of breast 'stem cells' based on CK5/6 staining and hence the breast cancer progression model and classification based on this phenotype.     

Mutation analysis of PEX7 in 60 probands with rhizomelic chondrodysplasia punctata and functional correlations of genotype with phenotype

PEX7 encodes the cytosolic receptor for the set of peroxisomal matrix enzymes targeted to the organelle by the peroxisome targeting signal 2 (PTS2). Mutations in PEX7 cause rhizomelic chondrodysplasia punctata (RCDP), a distinct peroxisome biogenesis disorder. In previous work we described three novel PEX7 mutant alleles, including one, L292X, with a high frequency due to a founder effect. We have now extended our analysis to 60 RCDP probands and identified a total of 24 PEX7 alleles, accounting for 95% of the mutant PEX7 genes in our sample. Of these, 50% are L292X, 13% are IVS9+1G>C, and the remainder are mostly private. IVS9+1G>C occurs on at least three different haplotypes and thus appears to result from recurrent mutation. The phenotypic spectrum of RCDP is broader than commonly recognized and includes minimally affected individuals at the mild end of the spectrum. To relate PEX7 genotype and phenotype, we evaluated the consequence of the disease mutation on PEX7 RNA by Northern analysis and RT/PCR. We evaluated the function of the encoded Pex7 protein (Pex7p) by expressing selected alleles in fibroblasts from RCDP patients and assaying their ability to restore import of a PTS2 marker protein. We find that residual activity of mutant Pex7p and reduced amounts of normal Pex7p are associated with milder and variant phenotypes.     

Multiple sets of visual cortical neurons projecting transitorily through the corpus callosum

In areas 17 and 18 of adult cats only a few neurons send bifurcating axons to more than one contralateral area or to areas in both hemispheres; most neurons seem to project to only one area. We now found that such a selective connectivity is already present at birth, i.e. several weeks before transitory callosal axons are eliminated. This is true even for those portions of cortex which will lose access to the corpus callosum: in particular different neurons project transitorily from medial area 17 to different contralateral areas. Thus cortical neurons may be induced to project to specific targets by a mechanism operating long before (and possibly independent of) the axon elimination. The latter may, however, be responsible for the final topographic organization of the connections.     

Pasteurella haemolytica Leukotoxin-Induced Increase in Phospholipase A2 Activity in Bovine Neutrophils

Exposure of bovine neutrophils to Pasteurella haemolytica leukotoxin (LKT) stimulates the production of leukotriene B₄ (LTB₄), which is believed to be an important chemotactic agent in the development of acute fibrinopurulent pneumonic infection in cattle. The involvement of phospholipase A₂ (PLA₂) in LKT-induced synthesis of LTB₄ was studied by using bovine neutrophils labeled with ³H-arachidonate ([³H]AA). Incubation of isolated neutrophils with [³H]AA resulted in incorporation of radioactivity in the PLA₂ substrates phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Exposure of radiolabeled neutrophils to LKT caused concentration- and time-dependent release of radioactivity and redistribution of radioactivity in neutrophil membranes consistent with utilization of phosphoglyceride substrate and release of free fatty acid and eicosanoid products. These LKT-induced effects could be inhibited by pretreatment with arachidonyl trifluoromethyl ketone, an inhibitor of type IV cytoplasmic PLA₂, and were dependent on extracellular calcium. These results support the conclusion that LKT-induced synthesis of LTB₄ involves a calcium-mediated increase in PLA₂ activity.     

Timed sequential analysis of creatine kinase in the diagnosis of myocardial infarction in patients over 65 years of age.

AIM--To assess the value of timed sequential analysis of creatine kinase (CK) activity for the early diagnosis of acute myocardial infarction (AMI) in patients over 65 years of age. METHOD--Samples were collected on admission and eight to 12 hours later from 156 patients over 65 years of age. Routine cardiac enzyme activities were determined and serial electrocardiograms (ECGs) recorded. The predictive value of timed samples for CK activity, standard cardiac enzyme activities, and ECGs was compared with the final diagnosis on discharge. RESULTS--Forty one patients had a discharge diagnosis of AMI, 83 of angina pectoris, and the remaining 32 patients had other diagnoses. Electrocardiograms had a sensitivity of 55% and a specificity of 96%, giving a predictive value of 86% for a negative and 84% for a positive ECG. Standard cardiac enzymes had a predictive value of 99% for a negative result but only 68% for a positive result. The logarithm of the rate of change of CK activity had a predictive value of 97% for a negative result and 95% for a positive result. CONCLUSION--This study has shown that slope analysis of CK activity can be used for the early diagnosis of AMI in patients over 65 years of age, and that this was not affected by the presence of possible confounding diagnoses.     

Heterogeneity in spinal radial glia demonstrated by intermediate filament expression and HRP labelling

Summary Considerable evidence indicates that radial glial cells play an active role in guiding growing neurites during development of the vertebrate CNS. In this paper we describe subpopulations of radial glia in the spinal cord of the axolotl. Amphibians maintain radial glia throughout life, and subpopulations are described using anatomical criteria following filling of individual cells with horseradish peroxidase and immunocytochemical staining with a range of intermediate filament antibodies.Radial glial cells in specific regions of the spinal cord stain with a range of antibodies specific to human keratins 8 and 18, and to glial fibrillary acid protein (GFAP). Some of these antibodies show selective staining localized to specific regions of individual glial cell processes. Immunoblotting analysis indicates that two keratins are present in the axolotl CNS corresponding to the two earliest embryonic keratins of vertebrates, keratins 8 and 18. Comparisons of molecular weight indicate that these may correspond to keratins identified inXenopus laevis, the genes of which have been cloned. Axolotl GFAP is also identified in Western blots and may be present in two forms of differing molecular weight.These results are discussed in terms of the likely role of radial glial cells, and comparisons are drawn between the keratin and GFAP types seen, in the axolotl spinal cord and of those in other vertebrate groups.     

Identification of Neisseria meningitidis serogroups Y and W135 by siaD nucleotide sequence analysis

Rapid serogrouping of meningococci is essential for the effective public health management of cases of the disease and the contacts of infected patients. Here we describe an accurate nucleotide-sequencing method for the confirmation of serogroup Y and W135 meningococci by siaD gene analysis from cultures of Neisseria meningitidis.     

A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing

The lipopolysaccharide (LPS) O-antigen side chains of Klebsiella serotype O1 have been studied by using mutants selected by resistance to a Klebsiella bacteriophage designated O1-A. Two classes of LPS mutants were identified. The major group (90%) synthesized rough LPS. The remaining 10% of the mutants produced a novel LPS profile that lacked the highest-molecular-weight O-substituted molecules (HMW-LPS) but still produced lower-molecular-weight O-substituted species (LMW-LPS). By using antisera raised against mutant Klebsiella strains and antiserum specific for Pasteurella haemolytica serotype 4, it was demonstrated that HMW-LPS and LMW-LPS contain shared epitopes. HMW-LPS also contained an epitope absent in LMW-LPS. This unique epitope was recognized by a monoclonal antibody (O1-52.6) and appears to be responsible for the serological cross-reaction between the O antigens of Klebsiella O1 and Escherichia coli O19. This HMW-LPS epitope was present in eight other Klebsiella O1 isolates which were examined. Electron microscopy demonstrated that HMW-LPS excluded overlying capsular polysaccharide for a distance of 25 to 40 nm. The distance was reduced to 10 to 18 nm in strains which synthesized only LMW-LPS and to zero in rough LPS strains. The HMW-LPS of Klebsiella O1 was shown to be an important virulence determinant, since this molecule was responsible for the resistance of the bacterium to nonspecific, complement-mediated serum killing.