Zinc chloride inhibition of Nitrosococcus mobilis.

Nitrosococcus mobilis, a halophilic nitrifier, plays an important role in global nitrogen cycling and in the removal of nitrogen from wastewater treatment plants. However, ammonia oxidation is sensitive to a wide variety of inhibitors, including the heavy metal, zinc. Using a metabolic-steady-state reactor, shotgun DNA microarrays, and quantitative polymerase chain reaction (qPCR), this research looked at the dynamic physiological and transcriptional responses of N. mobilis to 1 and 10 microM ZnCl2. By oxygen uptake rate measurements, zinc was determined to act directly on the ammonia monooxygenase (AMO) enzyme. The addition of excess copper prevented the inhibition of AMO by ZnCl2 suggesting that zinc and copper compete for placement in the metal active site in AMO. Shotgun DNA microarrays identified four previously unsequenced genes that were up- or down-regulated in response to 10 microM ZnCl2. Genes up-regulated in response to zinc inhibition include methionine synthase I, UbiA prenyltransferase and a recG-like helicase. RuBisCO was the lone down-regulated gene identified. qPCR was used to track the gene expression of the identified genes over the course of the 4-h experiment for both ZnCl2 concentrations. Because of their physiological importance, the expressions of AMO and hydroxylamine oxidoreductase (HAO) were also monitored via qPCR. The qPCR results showed general agreement with the shotgun DNA microarray results for metH, UbiA, recG and RuBisCO, and revealed that AMO and HAO expression levels were maintained or modestly up-regulated during ZnCl2 inhibition.

Cometabolism of chlorinated solvents by nitrifying bacteria: kinetics, substrate interactions, toxicity effects, and bacterial response.

Pure cultures of ammonia-oxidizing bacteria, Nitrosomonas europaea, were exposed to trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), chloroform (CF), 1,2-dichloroethane (1,2-DCA), or carbon tetrachloride (CT), in the presence of ammonia, in a quasi-steady-state bioreactor. Estimates of enzyme kinetics constants, solvent inactivation constants, and culture recovery constants were obtained by simultaneously fitting three model curves to experimental data using nonlinear optimization techniques and an enzyme kinetics model, referred to as the inhibition, inactivation, and recovery (IIR) model, that accounts for inhibition of ammonia oxidation by the solvent, enzyme inactivation by solvent product toxicity, and respondent synthesis of new enzyme (recovery). Results showed relative enzyme affinities for ammonia monooxygenase (AMO) of 1,1-DCE approximately TCE > CT > NH(3) > CF > 1,2-DCA. Relative maximum specific substrate transformation rates were NH(3) > 1,2-DCA > CF > TCE approximately 1,1-DCE > CT (=0). The TCE, CF, and 1,1-DCE inactivated the cells, with 1,1-DCE being about three times more potent than TCE or CF. Under the conditions of these experiments, inactivating injuries caused by TCE and 1,1-DCE appeared limited primarily to the AMO enzyme, but injuries caused by CF appeared to be more generalized. The CT was not oxidized by N. europaea while 1,2-DCA was oxidized quite readily and showed no inactivation effects. Recovery capabilities were demonstrated with all solvents except CF. A method for estimating protein yield, the relationship between the transformation capacity model and the IIR model, and a condition necessary for sustainable cometabolic treatment of inactivating substrates are presented. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 520-534, 1997.

Body weight, body condition score, and wither height of prepartum Holstein cows and birth weight and sex of calves by parity: a database and summary.

Measurements of body weight (BW), body condition score, wither height, calf birth weight, and calf sex were recorded by parity from 728 Holstein cows and heifers at the Purina Research Center during the 8-wk prepartum period. Data were compiled over 5 yr. Mean daily BW gain was 0.93 kg. Loss of BW at parturition was 11.1%. Mean body condition score was 3.35 (five-point scale where 1 = thin to 5 = obese) and did not change during the 8-wk period. However, body condition score was lower for second parity cows than for heifers or cows in third or greater parity. Wither height averaged 138 cm at first parity and increased 3 cm from first to second parity. During fifth and sixth parities, wither height peaked at an additional 2 to 3 cm. Birth weights for all calves averaged 41.4 kg. Male calves were 8.5% heavier than female calves. Calves born to cows in second or greater parity had increased BW by 7 to 8%. Mean BW of twin calves was 15% less than that of single calves.

A cometabilic kinetics model incorporating enzyme inhbition, inactivation, and recovery: I. Model development, analysis, and testing.

Cometabolic biodegradation prcesses are important for bioremediation of hazardous waste sites. However, these proceeses are not well understood and have not been modeled thoroughly. Traditional Michaelis-Menten kinetics models often are used, but toxic effects and bacterial responses to toxicity may cause changes in enzyme levels, rendering such models inappropriate. In this article, a conceptual and mathematical model of cometabolic enzyme kinetics i described. Model derivation is based on enzyme/growth-substrate/nongrowth-substrate interaction and incorporates enzyme inhibition (caused by the presence of a cometabolic compound), inactivation (resulting from toxicity of a cometabolic product), and recovery (associated with bacterial synthesis of new enbzyme in response to inactivation). The mathematical model consists of a system of two, nonlinear ordinary differential equations that can be solved implicitly using numerical methods, providing estimates of model parameters. Model analysis shows that growth substraate adn nongrowth substrate oxidation rates are related by a dimensionless constant. Reliability of tehy model solution prcedure is verifiedl by abnalyzing data ses, containing random error, from simulated experimentss with trichhloroethyylene (TCE) degradation by ammonia-oxidizing bacterialunder various conditions. Estimation of the recovery rate contant is deterimined to be sensitive to intial TCE concentration. Model assumptions are evaluated in a companion article using data from TCE degradation experiments with amoniaoxidizing bacteria. (c) 1995 John Wiley & Sons, Inc.

A cometabolic kinetics model incoroporating enzyme inhibition, inactivation, and recovery: II. Trichloroethylene degradaation experiments.

A Cometabolism enzyme kinetics model has been presented which takes into account changes in bacterial activity associated with enzyme inhibitiion, inactivation, inactivation of enzyme resulting from product toxicty, and respondent synthesis of new enzyme. Although this process is inherently unsteady-state, the model assumes that cometabolic degradation of a compound exhibiting product toxicity can be modeled as pseudo-steady-staate under certain conditions. In its simplified from, the model also assumes that enzyme inactivation is directly propoertional to nongrawth substrate oxidation, and that recovery is directly proportionla to growth substrate oxidation. In part 1, model drivation, simplification, and analyses were described. In this articles, model assuptiions are tested by analyzing data from experiments exmining trichloroethylene (TCE) degradation by the ammoniaoxidizing baceterium Nitrosomonas europaea in a quasisteady-state bioreactor. Model solution results showed steady-state bioreactor. Model solution results showed TCE to be a competitive inhibitoer of ammonia oxidation, with TCE affinity for ammonia monooxygenase (AMO) being about four times greater than that of ammonia for the enzyme. Inhibition was independent fo TCE oxidation and occurred essentially instantly upon exposure to TCE. In contrast, inactivation of AMO occurred more gradually and was proportional to the rate and amount of TCE oxidized. Evaluation of other O(2)-dependent enzymes and electron transport proteins suggested that TCE-related damage was predominantly confined to AMO. In response to inhibition and/or inactivation, bacterial recovery was initiated, even in the presence of TCE, implying that membranes adn protein synthesis systems were functioning. Analysis of data and comparison of model results showed the inhibition/inactivation/recovery concept to provide a reasonable basis for understandign the effects fo TCE on AMO function and bacterial response. The model assumptions were verified except tht questions remain regarding the factores controlling recovery and its role in the long term. (c) 1995 John Wiley & Sons, Inc.

Inhibition, Inactivation, and Recovery of Ammonia-Oxidizing Activity in Cometabolism of Trichloroethylene by Nitrosomonas europaea.

The kinetics of the cometabolism of trichloroethylene (TCE) by the ammonia-oxidizing soil bacterium Nitrosomonas europaea in short-term (<10-min) incubations were investigated. Three individual effects of TCE cometabolism on this bacterium were characterized. First, we observed that TCE is a potent competitive inhibitor of ammonia oxidation by N. europaea. The K(infi) value for TCE (30 (mu)M) is similar to the K(infm) for ammonia (40 (mu)M). Second, we examined the toxicity associated with TCE cometabolism by N. europaea. Stationary-phase cells of N. europaea oxidized approximately 60 nmol of TCE per mg of protein before ammonia-oxidizing activity was completely inactivated by reactive intermediates generated during TCE oxidation. At the TCE concentrations used in these experiments, ammonia did not provide significant protection against inactivation. Third, we have determined the ability of cells to recover ammonia-oxidizing activity after exposure to TCE. Cells recovering from TCE inactivation were compared with cells recovering from the specific inactivation of ammonia-oxidizing activity by light. The recovery kinetics were indistinguishable when 40% or less of the activity was inactivated. However, at increased levels of inactivation, TCE-inactivated cells did not recover as rapidly as light-inactivated cells. The kinetics of recovery appear to be dependent on both the extent of inactivation of ammonia-oxidizing activity and the degree of specificity of the inactivating treatment.

External carotid artery reconstruction: its role in the treatment of cerebral ischemia.

Twenty-one patients who underwent 22 external carotid artery reconstructions for cerebral ischemia were reviewed. Ten patients had amaurosis fugax, 9 had hemispheric ischemia, and 2 were asymptomatic. With the exception of one patient, all had ipsilateral internal carotid artery occlusion and either external carotid stenosis or a cul-de-sac of the occluded internal carotid artery. Six patients had ipsilateral common carotid occlusions. The operative technique has been presented. There were no operative strokes or new neurologic deficits. All patients were relieved of symptoms. Patients were followed a mean of 32 months. During follow-up, 3 ipsilateral transient ischemic attacks occurred during the first 3 months. Four strokes occurred (one ipsilateral and three contralateral) from 4 to 33 months postoperatively. Ipsilateral stroke rate by life table analysis was 1 percent per year. These data suggest that external carotid artery reconstruction is a safe, effective, and durable therapeutic alternative for management of patients with cerebral ischemia and an ipsilateral occluded internal carotid artery. External carotid endarterectomy eliminates a potential source of emboli and increases cerebral perfusion. Because of the increase in cerebral blood flow, subsequent extracranial-to-intracranial bypass may be unnecessary.

The prognostic spectrum of left main stenosis.

Three-year survival for 163 consecutive medically treated patients with 50% or greater left main stenosis was 50%. Survival was significantly higher for patients with 50 to 70% left main stenosis (one and three-year survivals of 91% and 66%) than for patients with 70% or greater left main stenosis (one and three-year survivals of 72% and 41%). In fact, left main lesions of less than 70% were not associated with the increased risk usually attributed to patients with left main stenosis. A number of noninvasive and catheterization characteristics were significant predictors of survival for patients with 70% or greater left main stenosis. Noninvasive descriptors defined a low risk subgroup (one and three-year survivals of 97% and 74%) and a high risk subgroup (one- and three-year survivals of 59% and 25%). These observations have important implications both in assessing therapeutic interventions and in managing individual patients.