The norA gene cloned from chromosomal DNA of quinolone-resistant Staphylococcus aureus TK2566 conferred relatively high resistance to hydrophilic quinolones such as norfloxacin, enoxacin, ofloxacin, and ciprofloxacin, but only low or no resistance at all to hydrophobic ones such as nalidixic acid, oxolinic acid, and sparfloxacin in S. aureus and Escherichia coli. The The increase in methicillin-resistant Staphylococcus aureus is a serious problem because only a few effective agents are clinically available. Some quinolones have been used for the treatment of methicillin-resistant S. aureus infections, but the emergence of quinolone resistance has been reported elsewhere (32). Unlike the mechanism underlying the quinolone resistance of gram-negative bacteria such as Escherichia coli (2,7,9,11,12,15,27,31,(36)(37)(38)(39) and Pseudomonas aeruginosa (4,13,16,29,30,36,40)

The affinity of [ 3 H]benzylpenicillin for penicillin-binding protein (PBP) 3A was reduced in 25 clinical isolates of ␤-lactamase-negative ampicillin (AMP)-resistant (BLNAR)Haemophilus influenzae for which the AMP MIC was >1.0 g/ml. The affinities of PBP 3B and PBP 4 were also reduced in some strains. The sequences of the ftsI gene encoding the transpeptidase domain of PBP 3A and/or PBP 3B and of the dacB gene encoding PBP 4 were determined for these strains and compared to those of AMP-susceptible Rd strains. The BLNAR strains were classified into three groups on the basis of deduced amino acid substitutions in the ftsI gene, which is thought to be involved in septal peptidoglycan synthesis. His-517, near the conserved Lys-ThrGly (KTG) motif, was substituted for Arg-517 in group I strains (n ‫؍‬ 9), and Lys-526 was substituted for Asn-526 in group II strains (n ‫؍‬ 12). In group III strains (n ‫؍‬ 4), three residues (Met-377, Ser-385, and Leu-389), positioned near the conserved Ser-Ser-Asn (SSN) motif, were replaced with Ile, Thr, and Phe, respectively, in addition to the replacement with Lys-526. The MICs of cephem antibiotics with relatively high affinities for PBP 3A and PBP 3B were higher than those of AMP and meropenem for group III strains. The MICs of ␤-lactams for H. influenzae transformants into which the ftsI gene from BLNAR strains was introduced were as high as those for the donors, and PBP 3A and PBP 3B showed decreased affinities for ␤-lactams. There was no clear relationship between 7-bp deletions in the dacB gene and AMP susceptibility. Even though mutations in another gene(s) may be involved in ␤-lactam resistance, these data indicate that mutations in the ftsI gene are the most important for development of resistance to ␤-lactams in BLNAR strains.Haemophilus influenzae is one of the important pathogens causing respiratory tract infection (RTI), acute otitis media (AOM), pneumonia, and purulent meningitis. Ampicillin (AMP) resistance in this organism is due to two well-known mechanisms. One is resistance mediated by the production of TEM-1 (20) and ROB-1 (13) ␤-lactamases, and the other is decreasing affinity of AMP for penicillin-binding proteins (PBPs) involved in peptidoglycan synthesis (14)(15)(16)19). Strains with resistance due to the latter mechanism are termed ␤-lactamase-negative AMP-resistant (BLNAR) H. influenzae.In surveillance studies conducted in the United States the incidence of ␤-lactamase-producing AMP-resistant (BLPAR) H. influenzae has gradually increased (6, 7, 10) and accounted for 36.4% of all isolates (5) in 1994 and 1995. In contrast, BLNAR isolates remain extremely uncommon in the United States.In Japan, according to nation wide surveillance studies conducted in 1997 and 1998, the proportion of clinical isolates supposed to be BLNAR has rapidly increased to 28.8% in parallel with the increased prevalence of penicillin (PEN)-resistant Streptcoccus pneumoniae. The characteristic of resistant H. influenzae isolates is that the AMP MIC is Ն1 g/ml, whereas the MIC for susce...

No abstract

A novel penicillin-binding protein, PBP-2' (Mr about 75,000), is known to be induced in excessively large amount by most beta-lactam compounds in cells of a clinically isolated strain of Staphylococcus aureus, TK784, that is highly resistant to beta-lactams and also most other antibiotics. This protein has very low affinities to most beta-lactam compounds and has been supposed to be the cause of the resistance of the cells to beta-lactams. A 14-kilobase DNA fragment was isolated from the cells that carried the gene encoding this penicillin-binding protein and also a genetically linked marker that is responsible for the resistance to tobramycin. This DNA was cloned on plasmid pACYC184 and was shown to cause both production of PBP-2' and resistance to tobramycin in Escherichia coli cells. However, the formation of PBP-2' in E. coli was only moderate and was independent of normal inducer beta-lactams. The PBP-2' formed in the E. coli cells showed slow kinetics of binding to beta-lactams similar to that of PBP-2' formed in the original S. aureus cells and gave a similar pattern of peptides to the latter when digested with the proteolytic V8 enzyme of S. aureus.

Dementia is one of the most debilitating symptoms of Parkinson's disease. A recent longitudinal study suggests that up to 80% of patients with Parkinson's disease will eventually develop dementia. Despite its clinical importance, the development of dementia is still difficult to predict at early stages. We previously identified olfactory dysfunction as one of the most important indicators of cortical hypometabolism in Parkinson's disease. In this study, we investigated the possible associations between olfactory dysfunction and the risk of developing dementia within a 3-year observation period. Forty-four patients with Parkinson's disease without dementia underwent the odour stick identification test for Japanese, memory and visuoperceptual assessments, (18)F-fluorodeoxyglucose positron emission tomography scans and magnetic resonance imaging scans at baseline and 3 years later. A subgroup of patients with Parkinson's disease who exhibited severe hyposmia at baseline showed more pronounced cognitive decline at the follow-up survey. By the end of the study, 10 of 44 patients with Parkinson's disease had developed dementia, all of whom had severe hyposmia at baseline. The multivariate logistic analysis identified severe hyposmia and visuoperceptual impairment as independent risk factors for subsequent dementia within 3 years. The patients with severe hyposmia had an 18.7-fold increase in their risk of dementia for each 1 SD (2.8) decrease in the score of odour stick identification test for Japanese. We also found an association between severe hyposmia and a characteristic distribution of cerebral metabolic decline, which was identical to that of dementia associated with Parkinson's disease. Furthermore, volumetric magnetic resonance imaging analyses demonstrated close relationships between olfactory dysfunction and the atrophy of focal brain structures, including the amygdala and other limbic structures. Together, our findings suggest that brain regions related to olfactory function are closely associated with cognitive decline and that severe hyposmia is a prominent clinical feature that predicts the subsequent development of Parkinson's disease dementia.

The mechanism of methicillin resistance was investigated in methicillin-resistant staphylococci (MRS) and in variants which had lost methicillin resistance. Phase-contrast microscopy showed that cells swelled at low concentrations of 13-lactam antibiotics in both MRS and variants which had lost methicillin resistance. Cells of variants which had lost methicillin resistance were lysed easily when higher concentrations of antibiotic were used. In contrast, MRS cells remained swollen at even higher concentrations of antibiotics. Furthermore, bacterial growth was inhibited at antibiotic concentrations much lower than MICs for MRS. Examination of the penicillin-binding proteins (PBPs) in MRS revealed that a new PBP-2' (molecular weight, 74,000) was induced in large quantity by exposure to P-lactams. PBP-2' was produced constitutively in variants of MRS which had lost a penicillinase plasmid. The induction of PBP-2' by (-lactams was not detected in variants which had lost methicillin resistance. High concentrations of ,l-lactam were required for saturation of PBP-2'. The optimum antibiotic concentration for the induction of PBP-2' varied with the 3-lactam used as the inducer, and PBP-2' was produced in a larger amount at 32°C than at 370C. From these results, we suggest that the mechanism of methicillin resistance depends on the induction of PBP-2', which may function as a detour enzyme for PBP-2 or PBP-3 or may be a particular enzyme involved in peptidoglycan synthesis.Methicillin-resistant strains of Staphylococcus aureus were first reported in 1961 (19), soon after the introduction of methicillin into clinical use. Subsequently, it has been shown that these strains also are resistant to many penicillins and cephems other than methicillin (2,11,15). In addition, methicillin resistance (MR) has been found to be temperature dependent (1, 7, 12) and affected by pH (27), NaCl concentration (3,12), and inoculum size (12, 23). Although methicillip-resistant staphylococci (MRS) produce penicillinase (PCase), curing of the PCase plasmid does not reduce the level of MR (28).Four main penicillin binding proteins (PBPs) have been identified in S. aureus (20), and either one or both PBP-2 and PBP-3 have been suggested to be the lethal target(s) for P-lactam action (13). In MRS, production of altered PBPs with extremely low affinity for ,3-lactams (14,16,17) or an increase of an altered PBP-3 (6) has been described. However, the double-zone phenomenon occasionally detected on a disk diffusion susceptibility test of P-lactams, such as methicillin, nafcillin (24), or imipenem (4), for MRS cannot be explained by the decreased affinities of PBPs for ,-lactams. This phenomenon suggests the existence of another mechanism for MR.Although MRS had not been a major clinical problem in Japan, after the introduction of third-generation cephems in 1982 isolates of MRS increased. We isolated two variant types from our clinical isolates of MRS. One type retained MR, whereas PCase activity became negative, and the other type lost MR along with ...

BackgroundThe intracellular deposition of misfolded proteins is a common neuropathological hallmark of most neurodegenerative disorders. Increasing evidence suggests that these pathogenic proteins may spread to neighboring cells and induce the propagation of neurodegeneration.ResultsIn this study, we have demonstrated that α-synuclein (αSYN), a major constituent of intracellular inclusions in synucleinopathies, was taken up by neuronal and oligodendroglial cells in both a time- and concentration-dependent manner. Once incorporated, the extracellular αSYN was immediately assembled into high-molecular-weight oligomers and subsequently formed cytoplasmic inclusion bodies. Furthermore, αSYN uptake by neurons and cells of the oligodendroglial lineage was markedly decreased by the genetic suppression and pharmacological inhibition of the dynamin GTPases, suggesting the involvement of the endocytic pathway in this process.ConclusionsOur findings shed light on the mode of αSYN uptake by neuronal and oligodendroglial cells and identify therapeutic strategies aimed at reducing the propagation of protein misfolding.

A beta-lactam-sensitive strain of Staphylococcus aureus could be converted to methicillin resistance by the introduction of a plasmid carrying the 4.3-kilobase HindIII chromosomal DNA fragment which encoded the mecA gene from a methicillin-resistant S. aureus. Transformant cells produced methicillin-resistant S. aureus-specific penicillin-binding protein constitutively, and additional insertion of an inducible penicillinase plasmid caused production of the pencillin-binding protein to become inducible.