Modeling of enzymatic reactions in vesicles: the case of alpha-chymotrypsin.

The kinetic behavior of the alpha-chymotrypsin-catalyzed hydrolysis of the two p-nitroanilide substrates succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA) and benzoyl-L-Tyr-p-nitroanilide (Bz-Tyr-pNA) was modeled and simulated for two different systems, namely for an aqueous solution and for a vesicle system, which was composed of phospholipid vesicles containing entrapped alpha-chymotrypsin. In the case of the vesicles, the substrate was added to the bulk, exovesicular aqueous phase. The experimentally determined time-dependence of product (p-nitroaniline) formation was modeled by considering the kinetic behavior of the enzyme and-in the case of vesicles-the substrate permeability across the bilayer membrane. In aqueous solution-without vesicles-the kinetic constants kcat and KS (respectively KM) were determined from fitting the model to experimental data of batch product concentration-time curves. The results were in good agreement with the corresponding values obtained from initial velocity measurements. For the vesicle system, using the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), simulation showed that the substrate permeation across the bilayer was rate limiting. Using experimental data, we could obtain the substrate permeability coefficient for Bz-Tyr-pNA by parametric fitting as 2. 45 x 10(-7) cm/s.

Transient-state behavior of a biofilter removing mixtures of vapors of MEK and MIBK from air.

In the work reported here, selected aspects of the dynamic behavior of biofilters for waste air treatment have been investigated. Emphasis was placed on transient state elimination of mixtures of methyl ethyl ketone (MEK) and methyl isobutyl ketone (MIBK) vapors and on explanation of the observed phenomena. The initial startup, the response of the biofilter to step changes in the pollutant loadings, responses to pollutant pulses, restarting after starvation, and the influence of step changes in gaseous phase oxygen partial pressure are presented and discussed.

The value of open lung biopsy in immunocompetent patients with community-acquired pneumonia requiring hospitalization.

OBJECTIVE: To determine the role of open lung biopsy in immunocompetent patients with community-acquired pneumonia who require hospitalization. DESIGN: A group of 1,118 patients with severe community-acquired pneumonia that required hospitalization were enrolled in the study. Of the patients, 26 underwent open lung biopsy. Another 18 of these patients were immunocompromised and were excluded from this segment of the study. SETTING: Tertiary care 800-bed hospital from November 1981 to May 1989. RESULTS: Progressive diffuse pulmonary infiltrates and negative conventional cultures were the indications for biopsy in most of these patients. Eighteen (69 percent) were immunocompromised. The eight immunocompetent patients underwent a retrospective review of their course in hospital. Three patients died. The diagnostic yield from open lung biopsy was 25 percent. A specific histologic diagnosis was made in one patient--lipoid pneumonia. The pulmonary histologic finding were nonspecific in the remaining patients, but in four, in combination with the clinical data, gave useful information and resulted in therapy change. Culture of a pulmonary tissue yielded cytomegalovirus in one other patient. Serologic testing had a low yield in this group with three patients having a positive result. CONCLUSIONS: Open lung biopsy is rarely necessary in immunocompetent patients with community-acquired pneumonia. In a small group of patients where it is necessary, however, both positive and negative results are important in directing therapy.

The complementary role of hepatobiliary scintigraphy and abdominal sonography in a patient with a hepatic cyst and suspected acute cholecystitis.

A 51-year-old woman was admitted with suspected acute cholecystitis. A large hepatic cyst was found incidentally by abdominal sonography, which displaced an "abnormal" gallbladder. Hepatobiliary scintigraphy visualized the gallbladder and excluded acute cholecystitis, but required the anatomic information from sonography to verify the abnormal location of the gallbladder. Additionally, scintigraphy showed the cyst not to communicate with the biliary tract. Pathologic findings revealed mild chronic cholecystitis and a simple mesothelial hepatic cyst. This case illustrates the complementary role of hepatobiliary scintigraphy and abdominal sonography in patients with distorted hepatobiliary anatomy and suspected acute cholecystitis.

A simulation model for the continuous production of acetoin and butanediol using Bacillus subtilis with integrated pervaporation separation.

The potential for producing acetoin and butanediol with a Bacillus subtilis strain was investigated with continuous culture using molasses as carbon substrate. The steady-state results were influenced by both oxygen and undetermined limiting compounds. Employing the known metabolic pathways, four overall stoichiometry relations were used with an energetic assumption on the energy requirements for biomass formation to establish a linear relations were used with an energetic assumption on the energy requirements for biomass formation to establish a linear relation between the overall rates, whose parameters were determined by linear regression. This provided a relationship for the product formation rate. The chemostat culture data were described with a growth kinetics model, which included limitation by molasses and oxygen as well as diauxic effects and product inhibition. The biokinetics model was combined with an experimentally verified model for the membrane Pervaporation. From this combined model were determined the influence of the membrane characteristics (enrichment factors and membrane area) and the dilution rate on the performance of the integrated process. Simulations revealed that an increase of the enrichment factor, possible by membrane improvement, would have counteracting influences, owing to decreased product inhibition but with lower biomass concentration.

Operation of a three-phase biofilm fluidized sand bed reactor for aerobic wastewater treatment.

A biofilm fluidized sand bed column reactor (14 L) has been operated in the three-phase mode on a soluble glucose-yeast hydrolysate substrate in which the biofilm-sand phase (1-2.5 L) was suspended by direct aeration of the bed. Within two weeks a tight biofilm was formed whose activity resulted in a 90% reduction, with loads of 10.7 kg TC/m(3)day. The residence time was 1 h. The biofilm remained intact during operation with high residence times (up to 23 h) over three weeks. Oxygen transfer coefficients varied with aeration rate and sand quantity between 0.02 and 0.04 s(-1) during non growth conditions; they decreased with increasing amounts of clean sand and were higher and relatively independent of the sand fraction with biofilm-covered sand. Aeration rates used in the 14 L reactor were 23-40 L/min (2.4-4.1 cm/s) and were sufficient to suspend 78-92% f the biofilm-covered sand. Clean sand was 50-75% suspended. Oxygen uptake rates varied between 15.4 and 23.1 mol/m(3) h.

Packed- and fluidized-bed biofilm reactor performance for anaerobic wastewater treatment.

Anaerobic degradation performance of a laboratory-scale packed-bed reactor (PBR) was compared with two fluidized-bed biofilm reactors (FBRs) on molasses and whey feeds. The reactors were operated under constant pH (7) and temperature (35 degrees C) conditions and were well mixed with high recirculation rates. The measured variables were chemical oxygen demand (COD), individual organic acids, gas composition, and gas rates. As carrier, sand of 0.3-0.5 mm diameter was used in the FBR, and porous clay spheres of 6 mm diameter were used in the PBR. Startup of the PBR was achieved with 1-5 day residence times. Start-up of the FBR was only successful if liquid residence times were held low at 2-3 h. COD degradations of 86% with molasses (90% was biodegradable) were reached in both the FBR and PBR at 6 h residence time and loadings of 10 g COD/L day. At higher loadings the FBR gave the best performance; even at 40-45 g COD/L day, with 6 h residence times, 70% COD was degraded. The PBR could not be operated above 20 g COD/L day without clogging. A comparison of the reaction rates show that the PBR and FBR per formed similarly at low concentrations in the reactors up to 1 g COD/L, while above 3 g COD/L the rates were 17.4 g COD/L day for the PBR and 38.4 g COD/L day for the FBR. This difference is probably due to diffusion limitations and a less active biomass content of the PBR compared with the fluidized bed.The results of dynamic step change experiments, in which residence times and feed concentrations were changed hanged at constant loading, demonstrated the rapid response of the reactors. Thus, the response times for an increase in gas rate or an increase in organic acids due to an increase in feed concentration were less than 1 day and could be explained by substrate limitation. Other slower responses were observed in which the reactor culture adapted over periods of 5-10 days; these were apparently growth related. An increase in loading of over 100% always resulted in large increases inorganic acids, especially acetic and propionic, as well as large increases in the CO(2) gas content. In general, the CO(2) content of the gas was very low, due to the large amount of dissolved CO(2) that exited with the liquid phase at low residence times. The performance of the FBR with whey was comparable to its performance with molasses, and switching of molasses to whey feed resulted in immediate good performance without adaptation.

Modeling dynamic experiments on the anaerobic degradation of molasses wastewater.

The kinetics of anaerobic degradation of a molasses wastewater were measured under constant pH conditions in a laboratory scale packed bed reactor. In continuous and batch experiments the formation and degradation rates of the organic acids (butyric, propionic and acetic) have been followed. The influence of hydrogen gas on the acid degradation rates has been measured and, contrary to the literature and the thermo-dynamic calculations, no inhibition was detected, biofilm diffusional effects may be the reason. Two dynamic simulation models were tested, a heterogeneous model, which considered the biofilm diffusion-reaction phenomena and a quasihomogeneous model with the same kinetics. Except for hydrogen, the diffusion effects were found to be negligible. Otherwise both models gave essentially the same results and the time profiles of acids, hydrogen, carbon dioxide and methane agreed relatively well with dynamic startup experiments. Batch experiments showed the acid concentrations to be highly sensitive to the initial molasses concentration. This aspect was not included in the model but is being investigated further.

Mast cell protease release and mucosal ultrastructure during intestinal anaphylaxis in the rat.

Intestinal anaphylaxis is associated with disturbances in gut function that are antigen-specific and dependent on mast cell degranulation. Using an animal model of intestinal anaphylaxis, we have correlated alterations in water and electrolyte transport, associated with intraluminal challenge, with specific intestinal mucosal mast cell degranulation by following systemic as well as local release of rat mast cell protease II. This protease is specific for intestinal mucosal mast cells and is known to selectively attack type IV collagen, which is found in basement membranes. Intraluminal antigen challenge in sensitized animals dramatically increased serum and intraluminal levels of rat mast cell protease II. Serum levels continued to rise throughout the duration of antigen challenge. Although light microscopy of challenged intestine demonstrated little distortion of mucosal architecture, ultrastructural examination revealed significant disruption to the basement membrane and underlying collagenous matrix of the intestinal mucosa. Our findings indicate that during mucosal immunoglobulin E-mediated reactions, rat mast cell protease II is released and is associated with ultrastructural changes in the intestinal mucosa. The systemic appearance of this specific protease provides a serum marker of intestinal anaphylaxis.

The response of the intestinal epithelium in B10.A mice to infection with Trichinella spiralis.

Previous studies on intestinal trichinosis have dealt mainly with areas other than the intestinal epithelium. Since the epithelium is now known to be the parasite's habitat, its response to infection is important. Infection with Trichinella spiralis in immunologically slow-responding B10.A mice was associated with crypt hyperplasia and villus atrophy. With similar infection levels in both primary and challenge infections, there was no difference in the maximal degree of atrophy or hyperplasia between the 2 groups. However, challenged mice underwent these mucosal changes in about half the time. Expulsion of worms always occurred during regeneration of the intestinal epithelium suggesting that the host's defense mechanism of altering the kinetics of the epithelium was not the prime factor causing expulsion. Pulse labelling of enterocytes with [3H] thymidine showed that there was no significant increase in the relative size of the proliferation zone. This indicates that the crypt cell output was not altered by this parasite. Atrophy of the villus was analysed with respect to its 3-dimensional shape. There was a decrease in both height and width of the villus but not thickness. Thus, there is a real decrease in the size of the enterocyte population per villus. Histochemical staining of the enterocyte brush border by an alkaline phosphatase method showed that (1) hyperplastic crypts have an enlarged maturation zone and (2) the villus epithelium is composed entirely of mature cells. The distribution of the nematode population was compared to these changes in the intestine. Trichinella spiralis showed a marked anteriad (distal to proximal) migration prior to expulsion. Thus, utilizing a novel approach to study intestinal trichinosis, the response of the mucosal epithelium has been characterized.

Biological denitrification of drinking water using autotrophic organisms with H(2) in a fluidized-bed biofilm reactor.

Biological denitrification of drinking water was studied in a fluidized sand bed reactor using a mixed culture. Hydrogen gas was used as the reaction partner. The reaction kinetics were calculated with a double Monod saturation function. The K(s) value for hydrogen was below 0.1% of saturation. No appreciable biofilm diffusion effects were detected. Reactor performance was a function of the culture's past history. Batch experiments always exhibited an accumulation of NO(2) (-), but continuous experiments with a sufficiently long residence time always resulted in complete nitrogen removal. Rates of up to 23 mg N/L h, 25 mg N/g DW h, and 7.9 mg H(2)/L h were achieved. Residence times of 4.5 h would be required for complete denitrification of water containing 25 mg NO(3) (-)-N/L or approximately 1 h for every 5 mg/L.