Detection of OXA-4 beta-lactamase in Pseudomonas aeruginosa isolates by genetic methods

In Pseudomonas aeruginosa, resistance to cefclidin is usually associated with resistance to another third-generation cephalosporin, ceftazidime. In this study we analysed 22 isolates of P. aeruginosa, collected at Showa University Fujigaoka Hospital between 1992 and 1993, which were resistant to cefclidin but susceptible to ceftazidime. All polymerase chain reaction (PCR) products amplified by a primer pair covering the full-length gene of OXA-4 (also OXA-1) precursor beta-lactamase were 0.84 kb in length. The isoelectric points of the beta-lactamases produced by these isolates were typical of the OXA-4 type of beta-lactamase (pl 7.5) rather than the OXA-1 type (pl 7.4). All PCR products at 216 bp were amplified by the primer pair covering the A928-->T point mutation, which corresponds to the Asp48-->Val amino acid substitution of OXA-1 beta-lactamase to form OXA-4 beta-lactamase. These single-strand conformation polymorphism (SSCP) patterns are typical of the OXA-4 gene, rather than the OXA-1 gene, demonstrating that these enzymes can be classified by SSCP analyses based on the PCR method. Although OXA-4 beta-lactamase is generally plasmid-mediated, the chromosomal DNA of these isolates, but not their plasmids, hybridized with the OXA-4 gene amplified by the PCR method. Based on these results, we suspected that the plasmids encoding OXA-4 beta-lactamase had been spontaneously cured, or that the gene had been deleted from the plasmid. The distribution of P. aeruginosa producing OXA-4 beta-lactamase amongst hospital wards and clinical specimens demonstrated that the OXA-4 enzyme in this collection period was representative of hospital P. aeruginosa.     

Covalent association of C3b with C4b within C5 convertase of the classical complement pathway

The C5 convertase of the classical complement pathway is a complex enzyme consisting of three complement fragments, C4b, C2a, and C3b. Previous studies have elucidated functional roles of each subunit (4, 6, 7), but little is known about how the subunits associate with each other. In this investigation, we studied the nature of the classical C5 convertase that was assembled on sheep erythrocytes. We found that one of the nascent C3b molecules that had been generated by the C3 convertase directly bound covalently to C4b. C3b bound to the alpha' chain of C4b through an ester bond, which could be cleaved by treatment with hydroxylamine. The ester bond was rather unstable, with a half-life of 7.9 h at pH 7.4 and 37 degrees C. Formation of the C4b-C3b dimer is quite efficient; e.g., 54% of the cell-bound C3b was associated with C4b when 25,000 molecules of C4b and 12,000 molecules of C3b were present per cell. Kinetic analysis also showed the efficient formation of the C4b-C3b dimer; the rate of dimer formation was similar to or even faster than that of cell-bound monomeric C3b molecules. These results indicate that C4b is a highly reactive acceptor molecule for nascent C3b. High-affinity C5-binding sites with an association constant of 2.1 X 10(8) L/M were demonstrated on C4b-C3b dimer-bearing sheep erythrocytes, EAC43 cells. The number of high-affinity C5-binding sites coincided with the number of C4b-C3b dimers, but not with the total number of cell-bound C3b molecules. Anti-C4 antibodies caused 80% inhibition of the binding of C5 to EAC43 cells. These results suggest that only C4b-associated C3b serves as a high-affinity C5 binding site. EAC14 cells had a small amount of high-affinity C5 binding sites with an association constant of 8.1 X 10(7) L/M, 100 molecules of bound C4b being necessary for 1 binding site. In accordance with the hypothesis that C4b-associated C4b might also serve as a high-affinity C5-binding site, a small amount of C4b-C4b dimer was detected on EAC14 cells by SDS-PAGE analysis. Taken together, these observations indicate that the high-affinity binding of C5 is probably divalent, in that C5 recognizes both protomers in the dimers. The high-affinity binding may allow selective binding of C5 to the convertase in spite of surrounding monomeric C3b molecules.     

Antenatal diagnosis and treatment of a case of fetal goitrous hypothyroidism associated with high-output cardiac failure.

A case of fetal goitrous hypothyroidism associated with high-output cardiac failure is presented. At 32 weeks of gestation, the antenatal diagnosis of goiter was made based on ultrasound examination, and the fetal thyroid function was examined by amniocentesis and cordocentesis. Color and pulsed Doppler examinations demonstrated a high vascular flow pattern in the goiter and marked elevation of the maximum velocity in the common carotid artery at the level of the neck. It was suspected that arteriovenous shunting through the large goiter resulted in high-output cardiac failure with cardiomegaly and pleural effusion. The fetus was treated by injection of levothyroxine sodium into the amniotic fluid at 33 weeks of gestation and the goiter thereafter decreased in size, with subsequent improvement of the high-output cardiac failure. The maximum velocity in the common carotid artery fell rapidly before the shrinkage of the fetal goiter and in parallel with the fetal level of thyroid stimulating hormone.     

Effects of new quinolones on Mycoplasma pneumoniae-infected hamsters.

The efficacies of the new quinolones temafloxacin, ofloxacin, and ciprofloxacin were investigated against Mycoplasma pneumoniae in an experimental hamster pneumonia model. Hamsters were infected intratracheally with M. pneumoniae and sacrificed 18 h after the final medication, and their lungs were aseptically removed, homogenized, and cultured quantitatively. The efficacies of these drugs were determined by the CFU of M. pneumoniae in lungs. Temafloxacin and ofloxacin, but not ciprofloxacin, were active when the oral administration of 200 mg/kg of body weight per day (once per day) for 5 days was initiated 24 h after infection. Although no effect on the elimination of M. pneumoniae was observed after the administration of these drugs at 200 mg/kg/day at 5 days after infection, the continuous administration for 15 days of temafloxacin, but not ofloxacin or ciprofloxacin, significantly reduced viable M. pneumoniae in the lungs. These results suggest that temafloxacin and ofloxacin are effective in the acute phase of infection and, moreover, that temafloxacin is effective in the late stage of infection during which progressive lung alterations and continuous increases in mycoplasmal growth occurred. The peak levels of temafloxacin in sera and lungs after oral administration were similar to those of ofloxacin and higher than those of ciprofloxacin. The areas under the curve of temafloxacin in the lung tissue, however, were higher than those of ofloxacin and ciprofloxacin. On the basis of these results, temafloxacin and ofloxacin might be promising antimicrobial agents for the treatment of mycoplasmal infection.     

Identification and quantification of a protein-bound ligand in uremic serum

A major protein-bound ligand in the serum of chronic hemodialysis patients was isolated from heat-deproteinized uremic serum by "high-performance" liquid chromatography (HPLC). The isolated compound was identified as 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid by use of liquid secondary-ion mass spectrometry and gas chromatography-mass spectrometry. On HPLC, an authentic sample of this compound showed a retention time identical to that of the protein-bound ligand peak. The concentration of the furancarboxylic acid in serum, as estimated by HPLC, is markedly greater in chronic hemodialysis patients than in normal subjects.     

Three‐dimensional structures of bacteriophage neck subunits are shared in Podoviridae,Siphoviridae and Myoviridae

Tailed bacteriophages (Caudovirales) are divided into three families: Myoviridae with long contractile tails, Siphoviridae with long noncontractile tails and Podoviridae with short noncontractile tails. All have an icosahedral head with a portal vertex connected to a neck structure followed by a tail. Bacteriophage Mu belongs to the Myoviridae family. Herein, the gp29 portal subunit and neck subunits gp35, gp36 and gp37 of the Mu phage were purified to elucidate their arrangement in the neck. Both gp29 and gp36 were monomeric in solution, like the corresponding subunits of Podoviridae P22 and Siphoviridae SPP1. X‐ray crystal structure of gp36 showed structural similarity to neck subunits of Siphoviridae and Podoviridae. The gp36 structure has a characteristic aromatic hydrophobic core, and the structure of the ring form of the Mu phage connector deduced from the Siphoviridae and Podoviridae connector showed that this feature builds the contact surface between gp36 subunits. Structural comparison with the neck of Siphoviridae and Podoviridae also implies direct interaction between gp36 and gp29. Because gp35 and gp36 form a stable complex, we predict that the head‐portal ring (gp29), the connector complex (gp36 and gp35), the tail terminator (gp37) and the tube (gp40) are arranged in the Mu phage neck in this order.     

Vascular endothelial growth factor receptor 1 (VEGFR1) tyrosine kinase signaling facilitates granulation tissue formation with recruitment of VEGFR1<Superscript>+</Superscript> cells from bone marrow

Vascular endothelial growth factor (VEGF)-A facilitates wound healing. VEGF-A binds to VEGF receptor 1 (VEGFR1) and VEGFR2 and induces wound healing through the receptor’s tyrosine kinase (TK) domain. During blood flow recovery and lung regeneration, expression of VEGFR1 is elevated. However, the precise mechanism of wound healing, especially granulation formation on VEGFR1, is not well understood. We hypothesized that VEGFR1-TK signaling induces wound healing by promoting granulation tissue formation. A surgical sponge implantation model was made by implanting a sponge disk into dorsal subcutaneous tissue of mice. Granulation formation was estimated from the weight of the sponge and the granulation area from the immunohistochemical analysis of collagen I. The expression of fibroblast markers was estimated from the expression of transforming growth factor-beta (TGF-β) and cellular fibroblast growth factor-2 (FGF-2) using real-time PCR (polymerase chain reaction) and from the immunohistochemical analysis of S100A4. VEGFR1 TK knockout (TK^?/?) mice exhibited suppressed granulation tissue formation compared to that in wild-type (WT) mice. Expression of FGF-2, TGF-β, and VEGF-A was significantly suppressed in VEGFR1 TK^?/? mice, and the accumulation of VEGFR1⁺ cells in granulation tissue was reduced in VEGFR1 TK^?/? mice compared to that in WT mice. The numbers of VEGFR1⁺ cells and S100A4⁺ cells derived from bone marrow (BM) were higher in WT mice transplanted with green fluorescent protein (GFP) transgenic WT BM than in VEGFR1 TK^?/? mice transplanted with GFP transgenic VEGFR1 TK^?/? BM. These results indicated that VEGFR1-TK signaling induced the accumulation of BM-derived VEGFR1⁺ cells expressing F4/80 and S100A4 and contributed to granulation formation around the surgically implanted sponge area in a mouse model.