Analysis of a Secreted Aspartic Peptidase Disruption Mutant of Glomerella cingulata

Peptidases have been implicated in the pathogenicity of fungi that cause disease in plants. Expression of the secreted aspartic peptidase gene (gcsap), of a Glomerella cingulata isolate pathogenic on apples, is induced during appressorium formation. To determine whether the secreted aspartic peptidase (GcSAP) is essential to pathogenicity, gcsap was disrupted using a vector containing a 637 bp fragment of genomic DNA that encodes the sequence spanning the two active site aspartic acid (Asp) residues. To ensure that the truncated gcsap gene products could not have residual peptidase activity the codons for the active site residues Asp¹¹² and Asp²⁹⁷ were both mutated to histidine residues. Both PCR and Southern analysis confirmed disruption of gcsap. Neither gcsap mRNA nor GcSAP activity was detected in the disruption mutant. Pathogenicity tests on fruit from three apple cultivars showed that GcSAP was not required for pathogenicity. The disruption mutant grew on medium containing protein as the sole source of nitrogen because G. cingulata secretes a previously undetected peptidase(s). A serine peptidase that had a pH optimum between pH 7.0 and 8.0 and a K _m of 0.25 mM for the synthetic substrate succinyl-Ala–Ala–Pro–Phe-p-nitroanilide was identified.     

Crystal and molecular structure of a double complex of manganese with phthalocyanato and pyridine ligands

A crystalline solid, formed by oxidation of phthalocyanatomanganese(II) in pyridine, has been identified as phthalocyanatopyridinemanganese(III)-micro-oxo-phthalocyanatopyridinemanganese(III)dipyridinate(C(74)H(42)Mn(2)N(18)O2C(5)H(5)N) byx-ray diffraction methods. This novel molecule consists of two manganese complexes joined by an essentially linear Mn-O-Mn bridge. Its structure may have some relation to oxidation processes in biological systems.     

Pathologic findings and association of Mycobacterium bovis infection with the bovine NRAMP1 gene in cattle from herds with naturally occurring tuberculosis

OBJECTIVE: To determine necropsy and Mycobacterium bovis culture results in cattle from herds with tuberculosis, the role of the bovine NRAMP1 gene in resistance and susceptibility to infection with M bovis, and the association between magnitude of the tuberculous lesions and various types of M bovis isolates. ANIMALS: 61 cattle from herds with tuberculosis in Texas and Mexico. PROCEDURE: 61 cattle were evaluated by necropsy; 59 had positive and 2 had negative caudal fold tuberculin intradermal test (CFT) results. Thirty-three cattle with positive CFT results were genotyped to evaluate polymorphism of the 3' untranslated region of the bovine NRAMP1 gene, using single-stranded conformational analysis, 9 were resistant to M bovis with no tuberculous lesions and negative M bovis culture results, and 24 were susceptible with tuberculous lesions and positive M bovis culture results. Isolates of M bovis were analyzed by restriction fragment length polymorphism (RFLP) on the basis of IS6110 sequences and direct-repeat fingerprinting patterns. RESULTS: 21 (35.6%; 21/59) cattle with positive CFT results had tuberculous lesions or positive culture results; in addition, 1 of 2 cattle with negative CFT results had tuberculous lesions and positive culture results. Tuberculous lesions were most common in the thorax (35/63; 55.5%) and lymphoid tissues of the head (10/63; 15.9%). Tuberculous lesions varied from 1 to 11/animal; 8 of 21 (38.1%) had solitary lesions. Associations were not found between resistance or susceptibility to infection with M bovis and polymorphism in the NRAMP1 gene or between the magnitude of the lesions and various RFLP types of M bovis isolates. CONCLUSIONS AND CLINICAL RELEVANCE: The NRAMP1 gene does not determine resistance and susceptibility to infection with M bovis in cattle.     

Flunixin meglumine attenuation of endotoxin-induced damage to the cardiopulmonary vascular endothelium of the pony

Endotoxic shock was induced in 5 ponies by intraperitoneal injections of 20, 40, 60, 80, and 80 micrograms of Escherichia coli endotoxin (LPS)/kg of body weight at 0, 6, 12, 18, and 24 hours, respectively. At 24 hours, the ponies also were given 20 micrograms of LPS/kg via catheter in the left ventricle of the heart. A 2nd group of 4 ponies was given 1.1 mg of flunixin meglumine (FM)/kg, IV, at 6, 12, 18, and 24 hours just before the corresponding LPS injection. Two hours after the 24-hour LPS injection, the ponies in both groups were anesthetized, the lungs were perfused with fixative, and portions of the pulmonary arteries and veins and right and left ventricles were prepared for scanning and transmission electron microscopy. In ponies that were given only LPS, some areas of pulmonary vascular endothelium appeared normal when compared with untreated controls, but other areas had disoriented endothelial cells or had varying amounts of sloughing, which ranged from focal areas of a few cells to large areas of denuded endothelium. Ponies treated with FM before LPS had less severe and less extensive endothelial cell damage. In both groups, leukocytes were attached to areas of the vessel wall; endothelial cell damage was greater in these regions. Administration of FM before LPS administration attenuated the LPS-induced endothelial cell damage.     

Differential reactivity of bovine lymphocytes to species of Brucella

The reactivity of bovine lymphocytes to 4 species of Brucella was tested in thymidine-uptake assays, using long-term cultured lymphocytes and freshly obtained blood mononuclear cells. Lymphocytes were taken from cows that had been challenge exposed with a virulent strain of B abortus at midgestation. The cows were classified retrospectively as being naturally resistant or susceptible to brucellosis. Lymphocytes taken from these cows had 3 patterns of reactivity with species of Brucella: pattern 1 was defined by reactivity with 4 species (B abortus, B canis, B suis, and B melitensis); pattern 2 was defined by reactivity with all these species, except B melitensis; pattern 3 was defined by reactivity with B abortus and B canis, but not with B suis or B melitensis. There was a statistically significant correlation between susceptibility to brucellosis and expression of lymphocyte cross-reactivity with B suis (P less than 0.01) and with B melitensis (P less than 0.001).     

Survival of smooth, rough and transposon mutant strains of Brucella abortus in bovine mammary macrophages

Transposon mutants offer a unique way to evaluate the role of lipopolysaccharide (LPS) by producing a theoretical single-gene difference between the original strain and the transposon mutant strain. Comparative survival of Brucella abortus smooth strain 2308, rough RB51, smooth strain 19, and two transposon mutant strains (rough strain 2308::Tn5 Lac Z [m106] and rough strain 19::Tn5 Lac Z [m3], was tested in restrictive bovine mammary macrophages that were able to effectively reduce the percentage of intracellular bacterial survival and permissive bovine mammary macrophages that were unable to control the intracellular replication of B. abortus. The theoretical single-gene difference between strain 19 and strain 19::Tn5 lac Z [m3] and between smooth virulent strain 2308 and rough transposon mutant 2308::Tn5 lacZ [m106] is likely related to differences in LPS content or structure. Significant (P less than 0.05) reduction in the survival of rough strain 19::Tn5 Lac Z [m3] with no significant reduction in the rough transposon mutant strain 2308::Tn5 lacZ [m106] indicated that at least one factor other than LPS contributes to the intracellular survival of B. abortus in bovine macrophages.     

Hemodynamics, plasma eicosanoid concentrations, and plasma biochemical changes in calves given multiple injections of Escherichia coli endotoxin

Twelve male neonatal calves (39 to 50 kg) were allotted to 3 groups of 4 calves each. All calves were anesthetized with halothane, and then Escherichia coli endotoxin was given intravenously (3 times) and intraperitoneally (3 times) during a 6-hour period. Group-1 calves were untreated, group-2 calves were pretreated with a low dose of flunixin meglumine (1.1 mg/kg of body weight), and group-3 calves were pretreated with a high dose of flunixin meglumine (4.4 mg/kg). In calves of group 1, the mean systemic arterial blood pressure (MABP) and cardiac output (CO) decreased, but pulmonary arterial pressure increased after the initial intravenous and intraperitoneal injections of endotoxin. In calves of this group, these changes were accompanied by increased plasma thromboxane B2 (TxB2) concentration. During this period, increased plasma TxB2 concentration or hemodynamic changes were not detected in calves of groups 2 and 3. Only calves of group 1 had altered hemodynamics early in the experiment; however, after 6 hours, calves of all 3 groups had similarly decreased CO and MABP. In calves of the untreated group, plasma 6-keto-prostaglandin (PG)F1 alpha concentration increased steadily from the beginning of the experiment until 3 hours later. The CO and MABP were low at the time when serum 6-keto-PGF1 alpha concentration was high; however, these 2 measurements also were low in treated calves who did not have correspondingly high plasma 6-keto-PGF1 alpha concentration. Regional blood flow analysis did not reveal correlations between prostanoid concentrations and altered blood flow to selected tissues.(ABSTRACT TRUNCATED AT 250 WORDS)