Perceptual errors increase with movement duration and may contribute to hypokinesia in Parkinson's disease.

People with Parkinson's disease (PD) perceive that their movement amplitude is greater than what they actually perform. The neural mechanisms underlying one's perception of movement are believed to involve the sensorimotor integration process (SIP). How PD affects the SIP is not well understood. A previous study interrogating the SIP showed healthy adults (HAs) overestimated their limb position in the direction of movement and the error and its variance (VOE) depended on movement duration. We asked if PDs showed errors in perceived limb position and if the dependence on movement duration was different from HAs. We used an existing computational model of the SIP to explore mechanisms for the error and VOE as a function of movement duration. Twenty PDs, off medication, and 20 age-matched HAs were asked to estimate the position of their hand after performing 50, slow, non-visually guided wrist flexion or extension movements for a random period of time (<4.0 s). Both groups overestimated the amount they moved; however, the PDs' error and VOE were larger (p<0.001). HAs showed increasing error/VOE for small movement durations that reduced/stabilized for longer movement durations. PDs however showed increasing error/VOE with increasing movement duration that did not significantly improve/stabilize. The model suggested that the basis for such perceptual deficits may be abnormal proprioceptive feedback and/or processing of an abnormal internal impression (prediction) that underestimates movement amplitude. Simulation results imply that the PD's SIP could no longer effectively access sensory (proprioceptive) feedback to correct errors in other components of the SIP due to the abnormal processing of sensory feedback. We suggest from this study that an impaired perception of movement amplitude and sensory processing deficits contribute to hypokinesia in PD.

Successful urgent transplantation of an adult kidney into a child with inferior vena cava thrombosis.

Poor venous drainage options following inferior vena cava (IVC) thrombosis have been considered to complicate or preclude renal transplantation of adult kidneys into pediatric patients. We describe urgent renal transplantation in a 5-year-old (15.3 kg) male with IVC thrombosis using an adult living donor. Preoperative magnetic resonance venography revealed a patent infrahepatic/suprarenal vena cava and portal system. In surgery, the right liver lobe was mobilized sufficiently to anastomose the graft renal vein to the native IVC at the confluence of the native left renal vein and proximal vena cava. Graft function has remained excellent with serum creatinine of 0.5 mg/dL at 36 months. IVC thrombosis need not preclude successful transplantation of adult-sized kidneys into children.

Transient-state reduction and steady-state kinetic studies of menaquinol oxidase from Bacillus subtilis, cytochrome aa3-600 nm. Spectroscopic characterization of the steady-state species.

Cytochrome aa3-600 or menaquinol oxidase, from Bacillus subtilis, is a member of the heme-copper oxidase family. Cytochrome aa3-600 contains cytochrome a, cytochrome a3, and CuB, and each is coordinated via histidine residues to subunit I. Subunit II of cytochrome aa3-600 lacks CuA, which is a common feature of the cytochrome c oxidase family members. Anaerobic reduction of cytochrome aa3-600 by the substrate analogue 2,3-dimethyl-1,4-naphthoquinone (DMN) resolves two distinct kinetic phases by stopped-flow, single-wavelength spectrometry. Global analysis of time-resolved, multiwavelength spectra shows that during these distinct phases cytochromes a and a3 are both reduced. Cyanide binding to cytochrome a3 enhances the fast phase rate, which in the presence of cyanide can be assigned to cytochrome a reduction, whereas cytochrome a3-cyanide reduction is slow. The steady-state activity of cytochrome aa3-600 exhibits saturation kinetics as a function of DMN concentration with a Km of 300 microM and a maximal turnover of 63.5 s(-1). Global kinetic analysis of steady-state spectra reveals a species that is characteristic of a partially reduced oxygen adduct of cytochrome a3-CuB, whereas cytochrome a remains oxidized. Electron paramagnetic resonance (EPR) spectroscopy of the oxidase in the steady state shows the expected signal from ferricytochrome a, and a new EPR signal at g = 2.01. A model of the catalytic cycle for cytochrome aa3-600 proposes initial electron delivery from DMN to cytochrome a, followed by rapid heme to heme electron transfer, and suggests possible origins of the radical signal in the steady-state form of the enzyme.

Control of vancomycin-resistant enterococcus in health care facilities in a region.

BACKGROUND: In late 1996, vancomycin-resistant enterococci were first detected in the Siouxland region of Iowa, Nebraska, and South Dakota. A task force was created, and in 1997 the assistance of the Centers for Disease Control and Prevention was sought in assessing the prevalence of vancomycin-resistant enterococci in the region's facilities and implementing recommendations for screening, infection control, and education at all 32 health care facilities in the region. METHODS: The infection-control intervention was evaluated in October 1998 and October 1999. We performed point-prevalence surveys, conducted a case-control study of gastrointestinal colonization with vancomycin-resistant enterococci, and compared infection-control practices and screening policies for vancomycin-resistant enterococci at the acute care and long-term care facilities in the Siouxland region. RESULTS: Perianal-swab samples were obtained from 1954 of 2196 eligible patients (89 percent) in 1998 and 1820 of 2049 eligible patients (89 percent) in 1999. The overall prevalence of vancomycin-resistant enterococci at 30 facilities that participated in all three years of the study decreased from 2.2 percent in 1997 to 1.4 percent in 1998 and to 0.5 percent in 1999 (P<0.001 by chi-square test for trend). The number of facilities that had had at least one patient with vancomycin-resistant enterococci declined from 15 in 1997 to 10 in 1998 to only 5 in 1999. At both acute care and long-term care facilities, the risk factors for colonization with vancomycin-resistant enterococci were prior hospitalization and treatment with antimicrobial agents. Most of the long-term care facilities screened for vancomycin-resistant enterococci (26 of 28 in 1998 [93 percent] and 23 of 25 in 1999 [92 percent]) and had infection-control policies to prevent the transmission of vancomycin-resistant enterococci (22 of 25 [88 percent] in 1999). All four acute care facilities had screening and infection-control policies for vancomycin-resistant enterococci in 1998 and 1999. CONCLUSIONS: An active infection-control intervention, which includes the obtaining of surveillance cultures and the isolation of infected patients, can reduce or eliminate the transmission of vancomycin-resistant enterococci in the health care facilities of a region.

Assignment of CDC weak oxidizer group 2 (WO-2) to the genus Pandoraea and characterization of three new Pandoraea genomospecies.

CDC weak oxidizer group 2 (WO-2) consists of nine phenotypically similar human clinical isolates received by the Centers for Disease Control and Prevention between 1989 and 1998. Four of the isolates were from blood, three were from sputum, and one each was from bronchial fluid and maxillary sinus. All are aerobic nonfermentative, motile gram-negative rods with one to eight polar flagella per cell. All grew at 25 and 35 degrees C and were positive for catalase, urease (usually delayed 3 to 7 days), citrate, alkalinization of litmus milk, oxidization of glycerol (weakly), and growth on MacConkey agar and in nutrient broth without NaCl. All except one strain were oxidase positive with the Kovács method, and all except one isolate weakly oxidized D-glucose. All were negative for oxidation of D-xylose, D-mannitol, lactose, sucrose, maltose, and 20 other carbohydrates, esculin hydrolysis, indole production, arginine dihydrolase, and lysine and ornithine decarboxylase. Only two of nine isolates reduced nitrate. Broth microdilution susceptibilities were determined for all strains against 13 antimicrobial agents. Most of the strains were resistant to ampicillin, extended-spectrum cephalosporins, and aminoglycosides, including gentamicin, tobramycin, and amikacin, but they varied in their susceptibility to fluoroquinolones. High-performance liquid chromatographic and mass spectrometric analyses of the WO-2 group identified ubiquinone-8 as the major quinone component. The percent G+C of the WO-2 strains ranged from 65.2 to 70.7% (thermal denaturation method). All shared a common cellular fatty acid (CFA) profile, which was characterized by relatively large amounts (7 to 22%) of 16:1omega7c, 16:0, 17:0cyc, 18:1omega7c, and 19:0cyc(11-12); small amounts (1 to 3%) of 12:0 and 14:0; and eight hydroxy acids, 2-OH-12:0 (4%), 2-OH-14:0 (trace), 3-OH-14:0 (12%), 2-OH-16:1 (1%), 2-OH-16:0 (3%), 3-OH-16:0 (4%), 2-OH-18:1 (2%), and 2-OH-19:0cyc (3%). This profile is similar to the CFA profile of Pandoraea, a recently described genus associated with respiratory infections in cystic fibrosis patients (T. Coenye et al., Int. J. Syst. Evol. Microbiol., 50:887-899, 2000). Sequencing of the 16S rRNA gene (1,300 bp) for all nine strains indicated a high level (> or =98.8%) of homogeneity with Pandoraea spp. type strains. DNA-DNA hybridization analysis (hydroxyapatite method; 70 degrees C) confirmed the identity of WO-2 with the genus Pandoraea and assigned three strains to Pandoraea apista and three to Pandoraea pnomenusa, and identified three additional new genomospecies containing one strain each (ATCC BAA-108, ATCC BAA-109, ATCC BAA-110). This study also shows that Pandoraea isolates may be encountered in blood cultures from patients without cystic fibrosis.

Classification of Proteus vulgaris biogroup 3 with recognition of Proteus hauseri sp. nov., nom. rev. and unnamed Proteus genomospecies 4, 5 and 6.

Strains traditionally identified as Proteus vulgaris formed three biogroups. Biogroup 1, characterized by negative reactions for indole production, salicin fermentation and aesculin hydrolysis, is now known as Proteus penneri. Biogroup 2, characterized by positive reactions for indole, salicin and aesculin, was shown by DNA hybridization (hydroxyapatite method) to be a genetic species separate from biogroup 1 and from biogroup 3 which is positive for indole production and negative for salicin and aesculin. In this study, 52 strains were examined, of which 36 strains were Proteus vulgaris biogroup 3, which included the current type strain of the species P. vulgaris (ATCC 29905T), and compared to seven strains of Proteus vulgaris biogroup 2 and nine type strains of other species in the genera Proteus, Providencia and Morganella. By DNA hybridization, these 36 strains were separated into four distinct groups, designated as Proteus genomospecies 3, 4, 5 and 6. DNAs within each separate Proteus genomospecies were 74-99% related to each other in 60 degrees C hybridization reactions with < or = 4.5% divergence between related sequences. Proteus genomospecies 3 contained the former P. vulgaris type strain and one other strain and was negative in reactions for salicin fermentation, aesculin hydrolysis and deoxyribonuclease, unlike the reactions associated with strains considered as typical P. vulgaris which are positive in reactions for salicin, aesculin and DNase. Genomospecies 3 can be distinguished from Proteus genomospecies 4, 5 and 6 because it is negative for Jordan's tartrate. Proteus genomospecies 4, containing five strains, was differentiated from Proteus penneri, genomospecies 3 and 6 and most, but not all, strains of genomospecies 5, by its ability to ferment L-rhamnose. Proteus genomospecies 5 and 6, containing 18 and 11 strains, respectively, could not be separated from each other by traditional biochemical tests, by carbon source utilization tests or SDS-PAGE of whole-cell proteins. In an earlier publication, a request was made to the Judicial Commission that the former type strain of P. vulgaris (ATCC 13315) be replaced by P. vulgaris biogroup 2 strain ATCC 29905T, a strain considered more biochemically typical of P. vulgaris strains. This would have the effect of assigning the name P. vulgaris to P. vulgaris biogroup 2. Since this request has been acceded to, the name Proteus hauseri is herein proposed for Proteus vulgaris genomospecies 3. Its type strain is ATCC 700826T. Proteus genomospecies 4, 5 and 6 will remain unnamed until better phenotypic differentiation can be accomplished. All Proteus genomospecies were similar in their antimicrobial susceptibility patterns. Nineteen strains were isolated from urine, four from faeces, two from wounds, nine from other human sources and two from animals.

Characterization of YpmQ, an accessory protein required for the expression of cytochrome c oxidase in Bacillus subtilis.

A search of the Bacillus subtilis genome identifies a potential homolog, ypmQ, of the inner mitochondrial membrane protein Sco1 from yeast. Sco1 has been found to aid the delivery of copper to cytochrome c oxidase. B. subtilis expresses two members of the cytochrome oxidase family, a cytochrome c oxidase that has two copper centers, Cu(A) and Cu(B), and a menaquinol oxidase that has only Cu(B). Deletion of ypmQ in B. subtilis depresses expression of cytochrome c oxidase but not menaquinol oxidase. Levels of cytochrome c oxidase recover when copper is added to the growth medium of the DeltaypmQ strain or when ypmQ is expressed from a plasmid. Neither treatment affects the amount or activity of menaquinol oxidase. YpmQ in which two conserved cysteines are replaced by serines and a conserved histidine is replaced by alanine do not complement the deletion of ypmQ even though these mutant forms are found in the membrane extract at a level similar to the wild type protein. We propose that the two cysteines and the histidine are critical for the function of YpmQ and suggest they are involved in copper exchange between YpmQ and the Cu(A) site of cytochrome c oxidase.

Characterization of a structural model of membrane bound cytochrome c-550 from Bacillus subtilis.

A structural model of Bacillus subtilis cytochrome c-550 has been built based upon hydropathy analysis, sequence alignment, homology modeling, and energy minimization. The model has a single transmembrane alpha-helix and a water-soluble domain folded around covalently attached heme C. Physical measurements on purified, recombinant cytochrome c-550 have been made to test aspects of the model. Excitation at either 280 or 295 nm yields fluorescence with an emission maximum at 334 nm and a quantum yield of 25% relative to n-acetyltryptophanamide. The model places one (i.e., W115) of the two tryptophans of cytochrome c-550 in the heme domain and the second (i.e., W3) in the transmembrane domain. The indole ring of W115 is within 5 A of the heme macrocycle and is expected to be highly quenched via resonance energy transfer to the heme. In contrast, W3 is at the start of the putative transmembrane helix and could be located a considerable distance from the heme. Förster theory assigns a distance of 42 A from W3 to the heme. This distance is important in adjusting the relative positions of the membrane-spanning and heme-binding domains. Circular dichroism measurements in the ultraviolet region indicate increased alpha-helical content of B. subtilis cytochrome c compared to mitochondrial cytochrome c in support of an alpha-helical transmembrane domain. The ionic strength dependence of redox kinetics for cytochrome c-550 indicates an overall negative charge that is consistent with a calculated pI of 5.4. However, the charge distribution specified by the model indicates a surface for electron exchange that is different from the classical front face used by mitochondrial cytochrome c.

A simplified method for testing Bordetella pertussis for resistance to erythromycin and other antimicrobial agents.

Present methods of antimicrobial susceptibility testing of Bordetella pertussis are time consuming and require specialized media that are not commercially available. We tested 52 isolates of B. pertussis for resistance to erythromycin, trimethoprim-sulfamethoxazole, chloramphenicol, and rifampin by agar dilution with Bordet-Gengou agar (BGA) containing 20% horse blood (reference method), Etest using BGA and Regan-Lowe agar without cephalexin (RL-C), and disk diffusion using BGA and RL-C. The organisms tested included four erythromycin-resistant isolates of B. pertussis from a single patient, a second erythromycin-resistant strain of B. pertussis from an unrelated patient in another state, and 47 nasopharyngeal surveillance isolates of B. pertussis from children in the western United States. The results of agar dilution testing using direct inoculation of the organisms suspended in Mueller-Hinton broth were within +/-1 dilution of those obtained after overnight passage of the inoculum in Stainer-Scholte medium, which is the traditional method of testing B. pertussis. The Etest method produced MICs similar to those of the agar dilution reference method for three of the four antimicrobial agents tested; the trimethoprim-sulfamethoxazole results were lower with Etest, particularly when the direct suspension method was used. Most of the Etest MICs, except for that of erythromycin, were on scale. Disk diffusion testing using RL-C medium was helpful in identifying the erythromycin-resistant strains, which produced no zone of inhibition around the disk; susceptible isolates produced zones of at least 42 mm. Thus, the antimicrobial susceptibility testing of B. pertussis can be simplified by using the Etest or disk diffusion on RL-C to screen for erythromycin-resistant isolates of B. pertussis.

Isolation of Providencia heimbachae from human feces.

Providencia heimbachae was first described in 1986. It has been isolated from penguin feces and an aborted bovine fetus. To date, there has been no reported isolation of this organism from human specimens. We now report the isolation of P. heimbachae from the stool of a 23-year-old woman with idiopathic diarrhea. The identity of the human strain was determined biochemically and by DNA relatedness to the type strain of P. heimbachae.

Regional dissemination of vancomycin-resistant enterococci resulting from interfacility transfer of colonized patients.

During early 1997, the Siouxland District Health Department (SDHD; Sioux City, IA) reported an increased incidence of vancomycin-resistant enterococcal (VRE) isolates at area health care facilities. To determine the prevalence and risk factors for colonization with VRE strains at 32 health care facilities in the SDHD region, a prevalence survey and case-control study were performed. Of 2266 patients and residents, 1934 (85%) participated, and 40 (2.1%) were positive for (gastrointestinal) VRE colonization. The prevalence of VRE isolates was significantly higher in acute care facilities (ACFs) than in long-term care facilities (LTCFs) (10/152 [6.6%] vs. 30/1782 [1.7%]; odds ratio [OR], 4.1; 95% confidence interval [CI], 1.8-9.0). LTCF case patients were significantly more likely than controls to have been inpatients at any ACF (19/30 vs. 12/66; OR, 8.0; 95% CI, 2.7-23.8). Of 40 VRE isolates, 34 (85%) were a related strain. The predominant strain was present in all 12 LTCFs that had at least 1 case patient in each facility. Soon after the introduction of VRE isolates into this region, dissemination to multiple LTCFs resulted from resident transfer from ACFs to LTCFs.

Lithium and valproate differentially regulate brain regional expression of phosphorylated CREB and c-Fos.

Previous studies in our laboratory have shown that the mood stabilizers, lithium and valproate (VPA), regulate the transcription factors, cyclic AMP responsive element binding protein (CREB), c-Fos and c-Jun, differentially in cultured human neuroblastoma SH-SY5Y cells. Here, we confirm these findings in rat brain and further study the brain-regional effects of these drugs using immunohistochemistry. We found that although chronic treatment with LiCl or VPA did not change the expression of c-Fos and c-Jun, acute treatment with either drugs increased c-Fos expression but not c-Jun expression in CA1 and CA3 regions of hippocampus. Chronic treatment with LiCl, but not VPA, decreased CREB phosphorylation in rat cerebral cortex and hippocampus. These results suggest that lithium and VPA may act on different pathways to bring about their long-term prophylactic effects on bipolar disorder (BD). The regulation of CREB phosphorylation may be relevant to lithium effect. VPA, which is also effective in BD, may be linked to other pathways.

Staphylococcus aureus with reduced susceptibility to vancomycin isolated from a patient with fatal bacteremia.

A Staphylococcus aureus isolate with reduced susceptibility to vancomycin was obtained from a dialysis patient with a fatal case of bacteremia. Comparison of the isolate with two methicillin-resistant S. aureus (MRSA) isolated obtained from the same patient 4 months earlier suggests that the S. aureus with reduced susceptibility to vancomycin emerged from the MRSA strain with which the patient was infected. Atypical phenotypic characteristics, including weak or negative latex-agglutination test results, weak or negative-slide coagulase test results, heterogeneous morphologic features, slow rate of growth, and vancomycin susceptibility (by disk diffusion test) were observed.

Expression, purification, and characterization of recombinant forms of membrane-bound cytochrome c-550nm from Bacillus subtilis.

Bacillus subtilis expresses a cytochrome c-550nm that participates in respiratory electron transfer and is an integral membrane protein. Analysis of the B. subtilis cytochrome c-550nm amino acid sequence predicts a single N-terminal transmembrane helix attached to a water-soluble heme binding domain [C. von Wachenfeldt and L. Hederstedt (1990) J. Biol. Chem. 265, 13939-13948]. We have purified cytochrome c-550nm from wild-type B. subtilis and B. subtilis transformed with the shuttle vector pHP13 containing the gene for B. subtilis cytochrome c-550nm (cccA). In B. subtilis transformed with pHP13/cccA there is better than eightfold more membrane-bound cytochrome c-550nm than in wild-type B. subtilis. The overexpressed cytochrome c-550nm can be purified by chromatography on hydroxylapatite and Q-Sepharose media. A six-histidine tag has been added to the C-terminus of cytochrome c-550nm from B. subtilis as a further aid for purification. This strain produces cytochrome c-550nm to a level fourfold greater than wild type and allows for one-step purification using metal affinity chromatography. UV-Vis spectroscopy detects no change in the heme C spectrum due to the addition of six histidines. Neither form of B. subtilis cytochrome c-550nm is stable in its reduced state in aerated buffer, unless EDTA is added. The two forms, wild-type and his-tagged, of cytochromes c have similar midpoint redox potentials of 195 and 185 mV, respectively, and are equally good substrates for B. subtilis cytochrome c oxidase. We conclude that the addition of the histidine tag eases the purification of cytochrome c-550nm from B. subtilis plasma membranes and that the additional metal binding site does not compromise the stability or functional properties of the protein.

Electron transfer kinetics during the reduction and turnover of the cytochrome caa3 complex from Bacillus subtilis.

The cytochrome caa3 complex from Bacillus subtilis is a member of the cytochrome oxidase superfamily of respiratory enzyme complexes. The key difference in the cytochrome caa3 complex lies in the addition of a domain, homologous with mitochondrial cytochrome c, that is fused to the C-terminal end of its subunit II. Measurements of steady-state and transient reduction kinetics have been carried out on the cytochrome caa3 complex. Reduction of the cyanide-bound enzyme with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) supports a sequence of electron transfer in which cytochromec is reduced initially, and this is followed by rapid internal electron transfer from cytochrome c to CuA and from CuA to cytochrome a. Steady-state kinetics with exogenous cytochrome c as the substrate demonstrates the capability of the cytochrome caa3 complex to act as a cytochrome c oxidase. The cytochrome c from B. subtilis is the most efficient cytochrome c of those tested. Steady-state kinetics with ascorbate-TMPD as the reductant, in the absence of exogenous cytochrome c, reveals a biphasic pattern even though only a single, covalent cytochrome c interaction site is present. The two-phase kinetics are characterized by a low activity phase associated with a high apparent affinity for TMPD and a high activity phase with a low affinity for TMPD. This pattern is observed over a wide range of ionic strengths and enzyme concentrations, and with both purified and membrane extract forms of cytochrome caa3. It is proposed that the biphasic steady-state kinetics of this oxidase, and other members of the cytochrome oxidase superfamily, do not result directly from different interactions with cytochrome c but are due to a change in the redox kinetics within the centers of the conventional oxidase unit itself. Our results will be related to models that account for the biphasic steady-state kinetics exhibited by cytochrome oxidase.

Characterization of staphylococci with reduced susceptibilities to vancomycin and other glycopeptides.

During the last several years a series of staphylococcal isolates that demonstrated reduced susceptibility to vancomycin or other glycopeptides have been reported. We selected 12 isolates of staphylococci for which the vancomycin MICs were > or =4 microg/ml or for which the teicoplanin MICs were > or =8 microg/ml and 24 control strains for which the vancomycin MICs were < or =2 microg/ml or for which the teicoplanin MICs were < or =4 microg/ml to determine the ability of commercial susceptibility testing procedures and vancomycin agar screening methods to detect isolates with reduced glycopeptide susceptibility. By PCR analysis, none of the isolates with decreased glycopeptide susceptibility contained known vancomycin resistance genes. Broth microdilution tests held a full 24 h were best at detecting strains with reduced glycopeptide susceptibility. Disk diffusion did not differentiate the strains inhibited by 8 microg of vancomycin per ml from more susceptible isolates. Most of the isolates with reduced glycopeptide susceptibility were recognized by MicroScan conventional panels and Etest vancomycin strips. Sensititre panels read visually were more variable, although with some of the panels MICs of 8 microg/ml were noted for these isolates. Vitek results were 4 microg/ml for all strains for which the vancomycin MICs were > or =4 microg/ml. Vancomycin MICs on Rapid MicroScan panels were not predictive, giving MICs of either < or =2 or > or =16 microg/ml for these isolates. Commercial brain heart infusion vancomycin agar screening plates containing 6 microg of vancomycin per ml consistently differentiated those strains inhibited by 8 microg/ml from more susceptible strains. Vancomycin-containing media prepared in-house showed occasional growth of susceptible strains, Staphylococcus aureus ATCC 29213, and on occasion, Enterococcus faecalis ATCC 29212. Thus, strains of staphylococci with reduced susceptibility to glycopeptides, such as vancomycin, are best detected in the laboratory by nonautomated quantitative tests incubated for a full 24 h. Furthermore, it appears that commercial vancomycin agar screening plates can be used to detect these isolates.

Spectral and cyanide binding properties of the cytochrome aa3 (600 nm) complex from Bacillus subtilis.

The cytochrome aa3 (600 nm) complex, or menaquinol oxidase, from Bacillus subtilis is a member of the cytochrome oxidase superfamily of respiratory membrane protein complexes. We have characterized some spectral properties of this enzyme and its reaction with cyanide. The magnetic circular dichroism (MCD) spectrum of the oxidized enzyme has a single band at 1560 nm in the near-infrared region assigned to bis-histidine-ligated, low-spin ferricytochrome a. The other heme, cytochrome a3, is presumably high-spin in the oxidized enzyme, as isolated. The absence of a trough in the MCD spectrum at 790 nm, observed previously with mammalian cytochrome c oxidase and assigned to CuA (Greenwood et al., Biochem. J. 215, 303-316, 1983), is consistent with the absence of this center from the menaquinol oxidase. When the heme ligand cyanide is added to oxidized menaquinol oxidase, a new MCD band appears at 2010 nm, while the band at 1560 nm is unperturbed. The new band is assigned to low-spin ferricytochrome a3 bound with cyanide. The long-wavelength position of this cyanide-induced band is proposed to arise from the close interaction of cytochrome a3 with the copper atom, CuB. The kinetics of cyanide binding to oxidized cytochrome aa3(600 nm) reveal a spectrally simple, yet kinetically complex process. The reaction is biphasic with second-order rate constants of 45 and 0.61 M-1s-1 at 1 mM KCN, with each phase constituting about 50% of the overall reaction. When the enzyme is subjected to a cycle of anaerobic reduction and air oxidation, the subsequent reaction with cyanide occurs in a single phase at the faster rate. This behavior is ascribed to different conformations of the binuclear center exhibiting different reactivities with cyanide, and is in keeping with that previously established for the structurally more complex mitochondrial cytochrome c oxidase. However, the electronic spectral characteristics of some of the species involved in these reactions are different in the present bacterial case from those of reported eukaryotic systems.

First report of a human isolate of Erwinia persicinus.

Erwinia persicinus was first described in 1990 after being isolated from a variety of fruits and vegetables, including bananas, cucumbers, and tomatoes. In 1994, it was shown to be the causative agent of necrosis of bean pods. We now report the first human isolate of E. persicinus. The strain was isolated from the urine of an 88-year-old woman who presented with a urinary tract infection. By the hydroxyapatite method, DNA from this strain was shown to be 94.5% related at 60 degrees C and 86% related at 75 degrees C to the type strain of E. persicinus. The biochemical profile of E. persicinus is most similar to those of Erwinia rhapontici, Pantoea agglomerans, and Enterobacter species. It is negative in tests for lysine, arginine, ornithine, dulcitol, and urea. It is motile and positive in tests for D-sorbitol and sucrose. It is susceptible to the expanded-spectrum cephalosporins, aminoglycosides, and fluoroquinolones, but it is resistant to ampicillin, ticarcillin, and cefazolin.

Risk factors for carriage of drug-resistant Streptococcus pneumoniae among children in Memphis, Tennessee.

OBJECTIVES: To determine risk factors for carriage of drug-resistant Streptococcus pneumoniae to understand better the factors promoting spread of these isolates. STUDY DESIGN: We obtained medical and demographic information and nasopharyngeal swab specimens from 216 children less than 6 years old with upper respiratory tract infections, seeking medical care at five Memphis, Tenn, study sites. We evaluated risk factors for carriage of penicillin-nonsusceptible S. pneumoniae (NSSP) among 100 children with S. pneumoniae isolates. Patterns of antimicrobial prescription were recorded for enrolled children. RESULTS: Independent risk factors for carriage of NSSP included an increased number of antimicrobial treatment courses during the previous 3 months and white race. Day care attendance approached statistical significance (p = 0.07). Most children with upper respiratory tract infection received a prescription for antimicrobial drugs. These prescriptions were more common for white children than for black children. CONCLUSIONS: Increased use of antimicrobial drugs enhances the risk of carriage of NSSP. This may contribute to the higher risk among white children of NSSP infection; however, after control for antimicrobial use, white children were still at an increased risk of infection with NSSP, possibly through greater exposure to resistant strains.

Stopped-flow, laser-flash photolysis studies on the reactions of CO and O2 with the cytochrome caa3 complex from Bacillus subtilis: conservation of electron transfer pathways from cytochrome c to O2.

The reaction of CO and O2 with fully reduced cytochrome caa3 from Bacillus subtilis has been studied by rapid reaction spectrophotometry. The fully reduced caa3 complex reacts with CO to give a spectrum that is characteristic of formation of ferrocytochrome a3-CO. This adduct is photosensitive, and its recombination rate is proportional to CO concentration with a bimolecular value of 1.2 x 10(5)M-1 s-1. When the CO compound of the reduced complex is exposed to O2, the rate of oxidation proceeds at 0.1 s-1, which is assigned as the CO off rate. These kinetic constants give an equilibrium dissociation constant for the CO complex of 0.83 microM. Photolysis of the CO adduct in the presence of O2 reveals three reaction phases over the first 3 ms and an additional phase on the second time scale. A kinetic model is proposed in which fully reduced oxidase first combines with O2 and then electron transfer commences from both cytochrome a and a3, followed rapidly by electron input from CuA and the cytochrome c domain. An equivalent kinetic model has been used to account for the reactivity of mammalian cytochrome c oxidase in its electrostatic complex with soluble cytochrome c [Hill, B. C., (1994) J. Biol. Chem. 269, 2419-2425]. However, unlike the mitochondrial complex, the reactivity of cytochrome c in the B. subtilis caa3 complex is unaffected by ionic strength. Thus the cytochrome c moiety in the B. subtilis caa3 complex seems to be fixed in a reactive orientation by its covalent association with the rest of the oxidase complex. The pathway of electron transfer from cytochrome c to O2 appears very well conserved from B. subtilis to the mammalian respiratory chain, making the B. subtilis protein a good model to probe intersite electron transfer within the cytochrome c-cytochrome oxidase complex.