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PubMed1996

Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath).

An active preparation of the membrane-associated methane monooxygenase (pMMO) from Methylococcus capsulatus Bath was isolated by ion-exchange and hydrophobic interaction chromatography using dodecyl beta-D-maltoside as the detergent. The active preparation consisted of three major polypeptides with molecular masses of 47,000, 27,000, and 25,000 Da. Two of the three polypeptides (those with molecular masses of 47,000 and 27,000 Da) were identified as the polypeptides induced when cells expressing the soluble MMO are switched to culture medium in which the pMMO is expressed. The 27,000-Da polypeptide was identified as the acetylene-binding protein. The active enzyme complex contained 2.5 iron atoms and 14.5 copper atoms per 99,000 Da. The electron paramagnetic resonance spectrum of the enzyme showed evidence for a type 2 copper center (g perpendicular = 2.057, g parallel = 2.24, and magnitude of A parallel = 172 G), a weak high-spin iron signal (g = 6.0), and a broad low-field (g = 12.5) signal. Treatment of the pMMO with nitric oxide produced the ferrous-nitric oxide derivative observed in the membrane fraction of cells expressing the pMMO. When duroquinol was used as a reductant, the specific activity of the purified enzyme was 11.1 nmol of propylene oxidized.min-1.mg of protein-1, which accounted for approximately 30% of the cell-free propylene oxidation activity. The activity was stimulated by ferric and cupric metal ions in addition to the cytochrome b-specific inhibitors myxothiazol and 2-heptyl-4-hydroxyquinoline-N-oxide.

Alkenes↗
PubMed2001

Membrane-associated quinoprotein formaldehyde dehydrogenase from Methylococcus capsulatus Bath.

A membrane-associated, dye-linked formaldehyde dehydrogenase (DL-FalDH) was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The enzyme was the major formaldehyde-oxidizing enzyme in cells cultured in high (above 1 micromol of Cu per mg of cell protein) copper medium and expressing the membrane-associated methane monooxygenase. Soluble NAD(P)(+)-linked formaldehyde oxidation was the major activity in cells cultured in low-copper medium and expressing the soluble methane monooxygenase (Tate and Dalton, Microbiology 145:159-167, 1999; Vorholt et al., J. Bacteriol. 180:5351-5356, 1998). The membrane-associated enzyme is a homotetramer with a subunit molecular mass of 49,500 Da. UV-visible absorption, electron paramagnetic resonance, and electrospray mass spectrometry suggest the redox cofactor of the DL-FalDH is pyrroloquinoline quinone (PQQ), with a PQQ-to-subunit stochiometry of approximately 1:1. The enzyme was specific for formaldehyde, oxidizing formaldehyde to formate, and utilized the cytochrome b(559/569) complex as the physiological electron acceptor.

Aldehyde Oxidoreductases↗
PubMed2001

Expression of individual copies of Methylococcus capsulatus bath particulate methane monooxygenase genes.

The expression of the two gene clusters encoding the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus Bath was assessed by analysis of transcripts and by use of chromosomal gene fusions. The results suggest that the two clusters are functionally redundant but that relative expression alters depending on the copper levels available for growth.

Artificial Gene Fusion↗
PubMed2003

The membrane-associated methane monooxygenase (pMMO) and pMMO-NADH:quinone oxidoreductase complex from Methylococcus capsulatus Bath.

Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol.min(-1).mg of protein(-1). Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 microM) to 95 microM. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio.

Cell Membrane↗
PubMed2003

Production of high-quality particulate methane monooxygenase in high yields from Methylococcus capsulatus (bath) with a hollow-fiber membrane bioreactor.

In order to obtain particulate methane monooxygenase (pMMO)-enriched membranes from Methylococcus capsulatus (Bath) with high activity and in high yields, we devised a method to process cell growth in a fermentor adapted with a hollow-fiber bioreactor that allows easy control and quantitative adjustment of the copper ion concentration in NMS medium over the time course of cell culture. This technical improvement in the method for culturing bacterial cells allowed us to study the effects of copper ion concentration in the growth medium on the copper content in the membranes, as well as the specific activity of the enzyme. The optimal copper concentration in the growth medium was found to be 30 to 35 micro M. Under these conditions, the pMMO is highly expressed, accounting for 80% of the total cytoplasmic membrane proteins and having a specific activity as high as 88.9 nmol of propylene oxide/min/mg of protein with NADH as the reductant. The copper stoichiometry is approximately 13 atoms per pMMO molecule. Analysis of other metal contents provided no evidence of zinc, and only traces of iron were present in the pMMO-enriched membranes. Further purification by membrane solubilization in dodecyl beta-D-maltoside followed by fractionation of the protein-detergent complexes according to molecular size by gel filtration chromatography resulted in a good yield of the pMMO-detergent complex and a high level of homogeneity. The pMMO-detergent complex isolated in this way had a molecular mass of 220 kDa and consisted of an alphabetagamma protein monomer encapsulated in a micelle consisting of ca. 240 detergent molecules. The enzyme is a copper protein containing 13.6 mol of copper/mol of pMMO and essentially no iron (ratio of copper to iron, 80:1). Both the detergent-solubilized membranes and the purified pMMO-detergent complex exhibited reasonable, if not excellent, specific activity. Finally, our ability to control the level of expression of the pMMO allowed us to clarify the sensitivity of the enzyme to NADH and duroquinol, the two common reductants used to assay the enzyme.

Amino Acid Sequence↗
PubMed2000

Infection of hickman catheter by Pseudomonas (formerly flavimonas) oryzihabitans traced to a synthetic bath sponge.

Pseudomonas (formerly Flavimonas) oryzihabitans is an uncommon pathogen that may cause catheter-associated infections. Although it has occasionally been isolated from the environment, the source of human infection has not previously been documented. We describe an AIDS patient who developed Pseudomonas oryzihabitans bacteremia due to colonization of a Hickman catheter. The patient reported having strictly followed the recommendations for catheter hygiene. The only flaw detected was the use of a synthetic bath sponge in the shower. The sponge was cultured and yielded P. oryzihabitans among other nonfermentative, gram-negative bacilli. To determine the prevalence of P. oryzihabitans in sponges, we cultured 15 samples from unrelated households. The microorganism was isolated from 3 of the 15 samples. Molecular typing by arbitrarily primed PCR (AP-PCR) was performed with the environmental and clinical isolates. Three different profiles were obtained for the six isolates analyzed from the patient's sponge. The strain from the AIDS patient was identical to one of those from his sponge and was different from all the remaining strains. The AP-PCR typing results were subsequently confirmed by pulsed-field gel electrophoresis. It can be concluded that sponges are occasionally colonized by P. oryzihabitans. For the first time a probable source of an indwelling catheter contamination with this bacterium has been found. Patients carrying these devices should avoid using sponge-like materials, as these are suitable environments for nonfermentative, gram-negative bacilli.

Adult↗
PubMed1986

In-bath filming during extracorporeal shock wave lithotripsy.

Extracorporeal shock wave lithotripsy (ESWL) is now the preferred method for treating most renal calculi. We designed a cassette and grid holder and a technique for filming in the water bath. The excellent film quality permits initial localization of small or faint calculi and confirmation of satisfactory fragmentation during ESWL. The technique facilitates patient treatment and throughput and should reduce the repeat treatment rate.

Humans↗
PubMed1996

Relationship between volume of bathing medium and ectoenzyme activity in monolayers of cultured BPAEC.

It is unclear whether all or a fraction of the capillary plasma volume (Vcp) serves as the reaction volume (Vr) for pulmonary capillary endothelial ectoenzymes, in vivo. Cultured endothelial cell (EC) monolayers provide a convenient model for studying EC-bound enzyme-Vr relationships. Because the Michaelis-Menten parameter [maximum velocity of enzyme reaction (Vmax) = E x kcat/Vr, where E is enzyme mass and kcat is the constant of product formation] is inversely proportional to Vr, we hypothesized that increasing the volume of medium (Vm) bathing EC monolayers would proportionally reduce the calculated Vmax (or Vmax/K(m), where K(m) is the Michaelis constant) values of an ectoenzyme reacting with a substrate only if, and as long as, Vm = Vr. To test this hypothesis, studies were performed in bovine pulmonary arterial EC grown to confluence. Activities of angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT) were assayed in Earle's balanced salts solution utilizing [3H]benzoyl-Phe-Ala-Pro ([3H]BPAP) and 5'-[14C]AMP as substrates, respectively. Under first-order reaction conditions and at constant substrate concentrations ([BPAP] = 15 nM, [AMP] = 1 microM), Vmax/K(m) ratios of ACE and NCT declined to 20% of their original values, as Vm increased from 0.6 to 2 ml. ACE activity was also studied at constant substrate mass (BPAP = 7 pmol) under first-order reaction conditions. Again, enzyme activity (Vmax/K(m)) declined proportionally to increasing Vm. Under zero-order reaction conditions ([BPAP] = 250 microM), ACE activity (Vmax) was similarly related to Vm. Linear regression analyses revealed that ACE or NCT would recognize up to at least 3 ml Vm, a volume vastly exceeding that of Vcp in a section of the capillary bed composed of an equivalent number of ECs, thus suggesting that Vcp could serve as the reaction volume for pulmonary capillary EC ectoenzymes in vivo.

5'-Nucleotidase↗
PubMed1990

Role of EPSPs in initiation of spontaneous synchronized burst firing in rat hippocampal neurons bathed in high potassium.

1. Spontaneous discharges that resemble interictal spikes arise in area CA3 b/c of rat hippocampal slices bathed in 8.5 mM [K+]o. Excitatory postsynaptic potentials (EPSPs) also appear at irregular intervals in these cells. The role of local synaptic excitation in burst initiation was examined with intracellular and extracellular recordings from CA3 pyramidal neurons. 2. Most (70%) EPSPs were small (less than 2 mV in amplitude), suggesting that they were the product of quantal release or were evoked by a single presynaptic action potential in another cell. It is unlikely that most EPSPs were evoked by a presynaptic burst of action potentials. Indeed, intrinsic burst firing was not prominent in CA3 b/c pyramidal cells perfused in 8.5 mM [K+]o. 3. The likelihood of occurrence and the amplitude of EPSPs were higher in the 50-ms interval just before the onset of each burst than during a similar interval 250 ms before the burst. This likely reflects increased firing probability of CA3 neurons as they emerge from the afterhyperpolarization (AHP) and conductance shunt associated with the previous burst. 4. Perfusion with 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a potent quisqualate receptor antagonist, decreased the frequency of EPSPs in CA3 b/c neurons from 3.6 +/- 0.9 to 0.9 +/- 0.3 (SE) Hz. Likewise, CNQX reversibly reduced the amplitude of evoked EPSPs in CA3 b/c cells. 5. Spontaneous burst firing in 8.5 mM [K+]o was abolished in 11 of 31 slices perfused with 2 microM CNQX.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2005

CdS nanoparticles grown in a polymer matrix by chemical bath deposition.

Nanocrystallites of CdS have been grown by chemical bath deposition within the pores of poly(vinyl alcohol) (PVA) on glass and Si substrates. The CdS-PVA composite films are transparent in the visible region. XRD and TEM diffraction patterns confirmed the nanocrystalline CdS phase formation. TEM study of the film revealed the manifestation of nano CdS phase formation and the average particles size was varied in the range 5-12 nm. UV-vis spectrophotometric measurement showed high transparency (nearly 80% in the wavelength range 550-900 nm) of the films with a direct allowed band gap lying in the range 2.64-3.25 eV. Particle sizes have also been calculated from the shift of band gap with respect to that of bulk value and were found to be in the range 3.3-6.44 nm. The high dielectric constant (lies in the range 120-250 at high frequency) of PVA/CdS nanocomposite compared to that of pure PVA (-28) has been observed. The dielectric constant decreases with increase of dispersibility of the CdS nanoparticles within PVA. The nanocrystalline PVA/CdS thin films have also showed field emission properties with a turn-on field of approximately 6.6 V/microm, whereas only PVA thin film and bulk CdS on PVA have shown no field emission.

Cadmium Compounds↗
PubMed2005

Optical studies on chemical bath deposited nanocrystalline CdS thin films.

Nanocrystalline cadmium sulphide thin films were prepared by the chemical bath (CB) deposition using a mixed aqueous solution of cadmium chloride, thiourea, and ammonium chloride. The XRD patterns showed that the CdS films were of hexagonal phase with preferred (002) orientation. From ellipsometric measurements, the film thickness was found to be in order of 66 nm and the optical band gap was estimated to be 2.4 eV.

Ammonium Chloride↗
PubMed2004

Implementing intensive insulin therapy: development and audit of the Bath insulin protocol.

Intensive insulin therapy to control blood glucose has been found to reduce mortality among critically ill patients in a surgical intensive care unit, though a simple prescriptive insulin infusion protocol to achieve this has not been published previously. This study documents the development and routine use of a simple prescriptive intravenous insulin infusion protocol for critically ill patients and compares the results with previous practice. During development the protocol was optimized and practical issues of implementation addressed. The optimized protocol was then used for all ICU admissions, and a prospectively defined retrospective chart audit performed for the first month of use. Results were compared with a similar time period the previous year. In September 2002, 27 admissions were started on the protocol. Blood glucose for the time on the protocol had a median value of 6.2 (IQR 5.9-7.1) mmol/l compared with 9.2 (IQR 8.1-10.2) mmol/l for those on insulin in 2001. Blood glucose for the whole ICU stay for those on the protocol in 2002 had a median value of 6.6 (IQR 6.0-7.4) mmol/l compared with 8.6 (IQR 8.0-9.4) mmol/l in 2001. Blood glucose for all ICU patients in 2002 had a median value of 6.5 (IQR 6.0-7.3) mmol/l compared with 7.2 (IQR 6.3-8.3) mmol/l in 2001. Three blood glucose recordings were less than 2.2 mmol/l in September 2002. This study provides initial effectiveness and safety data for the Bath Insulin Protocol Further audits in a larger patient population are now needed.

Blood Glucose↗
PubMed1975

Potassium exchange between bathing solution and midgut of Hyalophora cecropia and time delay for potassium flux through the midgut.

Exchange between potassium of the isolated midgut of the American silkworm Hyalophora cecropia and the bathing solutions has been determined by different authors, but with contradictory results. Therefore the experiments were repeated with another technique and the exchange determined for varying time intervals. It was found that the exchange was fast-half of the midgut K was exchanged in 2-3 minutes, which confirms the earlier findings by Harvey & Zerahn (1969).

Animals↗
PubMed2003

Evaluation of recreational health risk in coastal waters based on enterococcus densities and bathing patterns.

We constructed a simulation model to compute the incidences of highly credible gastrointestinal illness (HCGI) in recreational bathers at two intermittently contaminated beaches of Orange County, California. Assumptions regarding spatial and temporal bathing patterns were used to determine exposure levels over a 31-month study period. Illness rates were calculated by applying previously reported relationships between enterococcus density and HCGI risk to the exposure data. Peak enterococcus concentrations occurred in late winter and early spring, but model results showed that most HCGI cases occurred during summer, attributable to elevated number of exposures. Approximately 99% of the 95,010 illness cases occurred when beaches were open. Model runs were insensitive to 0-10% swimming activity assumed during beach closure days. Comparable illness rates resulted under clustered and uniform bather distribution scenarios. HCGI attack rates were within federal guidelines of tolerable risk when averaged over the study period. However, tolerable risk thresholds were exceeded for 27 total days and periods of at least 6 consecutive days. Illness estimates were sensitive to the functional form and magnitude of the enterococcus density-HCGI relationships. The results of this study contribute to an understanding of recreational health risk in coastal waters.

California↗
PubMed2001

Excitatory ascending and descending motor responses in the guinea pig small intestine: a comparative study of longitudinal and circular muscles by a triple bath method.

A triple organ bath was developed to study the ascending and descending reflexes in a guinea pig small intestine model, allowing synchronous recording the motor activity of the longitudinal and circular muscle layers belonging to the oral and anal part of segment preparations. Field electrical stimulation (0.8 msec, 40 V, 5 Hz, 10 sec) applied either to the anal or oral part of the segments elicited both tetrodotoxin (1 microM)-sensitive contractile local motor responses of muscle layers belonging to the stimulated part and ascending and descending contractions of both muscle layers at a distance of 10 mm were observed when the electrical stimulation was applied to the middle part of the segments. Local responses of the circular muscle layer were considerably higher. The ascending motor responses of both muscle layer were expressed more than those of the circular one. It is concluded that locally induced nerve stimulation propagated via intrinsic ascending or descending neural pathways could be synchronously coactivated by one and the same stimulus. Prominent ascending motor responses and contractility of the longitudinal muscle layer in orally directed reflexes.

Anesthetics, Local↗