Clathrin assembly involves a light chain-binding region.

Two regions on the clathrin heavy chain that are involved in triskelion interactions during assembly have been localized on the triskelion structure. These regions were previously identified with anti-heavy chain monoclonal antibodies X19 and X35, which disrupt clathrin assembly (Blank, G. S., and F. M. Brodsky, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:2087-2095). Antibody-binding sites were determined based on their reactivity with truncated triskelions, and were mapped to an 8-kD region in the middle of the proximal portion of the triskelion arm (X19) and a 6-kD region at the triskelion elbow (X35). The elbow site implicated in triskelion assembly was also shown to be included within a heavy chain region involved in binding the light chains and to constitute part of the light chain-binding site. We postulate that this region of the heavy chain binds to the interaction site identified on the light chains that has homology to intermediate filament proteins (Brodsky, F. M., C. J. Galloway, G. S. Blank, A. P. Jackson, H.-F. Seow, K. Drickamer, and P. Parham, 1987, Nature (Lond.), 326:203-205). These findings suggest the existence of a heavy chain site, near the triskelion elbow, which is involved in both intramolecular and intermolecular interactions during clathrin assembly.

Effects of diet type and enzyme addition on growth performance and gut health of broiler chickens during subclinical Clostridium perfringens challenge.

The effects of diet type (corn- vs. wheat-based) and multicarbohydrase addition on growth performance, digesta pH and viscosity, intestinal populations of Clostridium perfringens and lactic acid bacteria, and gut lesion score (from 0 to 4, where 0 = no gross lesions, 4 = severe extensive necrosis) of broiler chickens during oral challenge with C. perfringens (none or 10(8) cfu/bird on d 13) were studied in a 39-d experiment. A total of 1,216 male Ross-308 chickens was assigned to 8 dietary treatments in a randomized complete block design providing 8 replicate pens per treatment. Diets were formulated to meet the NRC protein requirement but were suboptimal in energy level. When compared with birds fed corn-based diets, chickens fed wheat-based diets had inferior (P < 0.01) final BW (2.49 vs. 2.59 kg) and feed conversion ratio (FCR; 1.83 vs. 1.78). Pathogen challenge significantly (P < 0.05) impaired growth performance and increased C. perfringens numbers and average lesion score. Increased (P < 0.01) C. perfringens counts (2.4 vs. 1.5 log(10) cfu/g of digesta) and intestinal lesion score (0.9 vs. 0.4) were observed for challenged birds fed wheat-based diets. No difference in digesta pH and lactic acid bacteria numbers were found among the treatments. Enzyme addition to both the corn- and wheat-based diets increased bird final BW (2.57 vs. 2.51 kg; P < 0.01), decreased overall FCR (1.78 vs. 1.83; P < 0.01), and, in those consuming wheat-based diets, reduced digesta viscosity (from 4.1 to 2.7 mPa.s; P < 0.01). Enzyme supplementation assisted the challenged birds in maintaining their optimal growth performance by improving (P < 0.05) average daily gain (59.5 vs. 56.9 g) in those consuming corn-based diets and FCR (1.83 vs. 1.90) in those consuming wheat-based diets to values similar to those observed in control birds (59.7 g/d and 1.84, respectively). In conclusion, enzyme addition improved growth performance and mitigated the negative effects of C. perfringens challenge.

Effects of superheated steam on Geobacillus stearothermophilus spore viability.

AIMS: To examine the effect of processing with superheated steam (SS) on Geobacillus stearothermophilus ATCC 10149 spores. METHODS AND RESULTS: Two inoculum levels of spores of G. stearothermophilus were mixed with sterile sand and exposed to SS at 105-175 degrees C. The decimal reduction time (D-value) and the thermal resistance constant (z-value) were calculated. The effect of cooling of spores between periods of exposure to SS was also examined. A mean z-value of 25.4 degrees C was calculated for both inoculum levels for SS processing temperatures between 130 degrees C and 175 degrees C. CONCLUSIONS: Spore response to SS treatment depends on inoculum size. SS treatment may be effective for reduction in viability of thermally resistant bacterial spores provided treatments are separated by intermittent cooling periods. SIGNIFICANCE AND IMPACT OF THE STUDY: There is a need for technologies that require short thermal processing times to eliminate bacterial spores in foods. The SS processing technique has the potential to reduce microbial load and to modify food texture with less energy in comparison to commonly used hot air treatment. This work provides information on the effect of SS processing parameters on the viability of G. stearothermophilus spores.

Suitability of pea starch and calcium alginate as antimicrobial coatings on chicken skin.

The effect of incorporating trisodium phosphate (TSP) in pea starch (PS) and acidified sodium chlorite (ASC) in calcium alginate upon the antimicrobial activity of TSP and ASC was studied against a 3-strain cocktail of Salmonella inoculated on chicken skin. The influence of polymer coating concentration on skin pH, coating-skin adhesion, and coating absorption upon antimicrobial performance were investigated. Aqueous solutions of 0.5 to 4.8% (wt/vol) PS were prepared with 10% (wt/vol) TSP (PS + TSP coating), and alginate + ASC coatings contained 1% (wt/vol) calcium chloride in 1,200 ppm of ASC mixed with an aqueous solution of 0.5, 1.0, or 1.5% (wt/vol) sodium alginate. Coating drops (10 microL) were placed on chicken skin thighs, and the angle formed by the tangent of the liquid surface at the skin interface (contact angle) was measured using a digital camera to assess coating-skin adhesion. Excised skins were mounted in a ring holder, and 5 mL of the coatings was applied to the skin. Weight changes in the skins that were related to coating absorptiveness were recorded. The TSP dissolved in 3.5% PS and ASC in 1% alginate reduced Salmonella by 1.6 log cfu/g and 1.4 log cfu/g, respectively, within 24 h. These reductions were significantly greater than those caused by TSP or ASC alone in water for up to 120 h. In coatings, TSP and ASC caused significant elevation or reduction of skin surface pH for up to 120 h, respectively. The TSP destabilized PS with 88% of the coating having dripped from the skin 1 h later. Coatings with 0.5% PS were absorbed quickly by the skin and had high skin adhesion, whereas those with >3.5% PS had low skin adhesion and slow absorption. Alginate coatings with or without ASC were stable, and about 50% of the coating weight was retained at 120 h. The latter coatings appeared to have low absorptiveness because the skin gained approximately 1.0% of its weight within 60 min following application. These findings indicate that effects of the agents in coatings on skin pH, the extent of coating adhesion, and absorption may contribute to overall antimicrobial behaviors.

Selective, efficient modulation of activated CD4+ αßT cells by the novel humanized antibody GZ-αßTCR targeting human αßTCR.

Allograft rejection and immunosuppression are two major issues in transplantation medicine. The specific targeting of alloreactive T cells, the initiators and promoters of allograft rejection, would be a promising strategy to reduce unwanted T-cell responses and side effects of lifelong immunosuppression. The novel humanized monoclonal antibody GZ-αßTCR, specific for the human αßT-cell receptor, was tested in vitro and in vivo for its specificity and efficacy to modulate the αßT-cell compartment. GZ-αßTCR moderately induced apoptosis in resting αßT cells in vitro, an effect considerably amplified in activated T cells. A single dose of GZ-αßTCR significantly reduced human CD45(+)CD3(+) T cells in vivo, with a preferential modulation of CD4(+) αßT cells. Importantly, naive T cells, the T-cell subset from which alloreactivity emanates, were significantly reduced. Simultaneously, a significant, compensatory increase of γδ T cells was observed in vitro and in vivo in both humanized mouse models examined. GZ-αßTCR did not induce cytokines and was well tolerated. Thus, specificity and high efficacy make GZ-αßTCR a powerful tool to selectively eliminate putatively detrimental T-cell subsets, a major goal in transplantation medicine. At the same time, GZ-αßTCR spares γδ and natural killer cells, thus leaving the recipient's immune system competent for cell-mediated immunoregulation and cell-mediated immunity.

An assessment of behavioral aging in the Mongolian gerbil.

Male Mongolian gerbils (Meriones unguiculatus) 14-54 months old (n = 77) were evaluated in a battery of psychomotor (open field, locomotor, and runwheel activity, rotorod performance) and learning (one-way active avoidance in a straight runway and in 14-unit T-maze performance) tests. Body weight and seizure activity were also monitored. According to linear regression analysis, runwheel activity decreased with age; and the number of errors in the 14-unit T-maze increased as a function of age (ps < 0.05). None of the other behavioral measures or body weight were significantly correlated with age. This gerbil strain (Tumblebrook Farms; West Brookfield, MA) tended to be very prone to seizures with 64% of the gerbils experiencing at least one seizure while being tested. Seizures tended to occur when the gerbil was exposed to a novel situation (e.g., initial weighing, placement on the rotorod). An age-related decline in some aspects of psychomotor and learning performance was observed, suggesting the gerbil as an additional mammalian model of aging. The high incidence of seizure activity presented a complicating and confounding variable to the interpretation of the results of the behavioral tests used in the present study. Interventions to control seizure activity (e.g., systematic, controlled breeding; adaptation to apparati) in this model will likely increase its viability as a mammalian model of aging.

Microwave plasmas for low-temperature dry sterilization.

The use of microwave plasmas for dry sterilization has been investigated. The dry-sterilization process is a process similar to plasma etching. Bacteria and viruses can be killed by chemical reactions which disintegrate their bodies and remove them from the surface to be sterilized. The removal of bacteria or viruses from material surfaces is caused by the reaction of activated oxygen species in the plasma with hydrocarbon bonds of the cell wall of the bacteria or the capsid of the viruses. Preliminary experiments indicate that the low-temperature dry sterilization method is easy to use, requires much less time than other methods for sterilization, and is also non-toxic. It is feasible for use in the field of sterilization in dental and medical clinics.

Expanded bed protein A affinity chromatography of a recombinant humanized monoclonal antibody: process development, operation, and comparison with a packed bed method.

We show that expanded bed protein A affinity chromatography using Streamline rProtein A media is an efficient method for purifying a recombinant humanized monoclonal antibody from unclarified Chinese hamster ovary cell culture fluid and that it provides purification performance comparable to using a packed bed. We determined that the dynamic capacity of the expanded bed media is related to flow rate (measured in column volumes per hour) by a power function, which allows a high capacity at a low flow rate. At 250 cm h-1 with a 25 cm bed height (10 column volumes h-1), the dynamic capacity is 30 g l-1. The yield and purity (measured by the amount of host cell proteins, DNA, SDS-PAGE, and turbidity) of the antibody purified by expanded bed is comparable to the yield and purity obtained on a standard packed bed method using Prosep A media.

Real-time control of antibody loading during protein A affinity chromatography using an on-line assay.

We show that an on-line chromatographic assay can reliably control antibody loading in real-time during protein A affinity chromatography purification of a recombinant antibody from clarified Chinese hamster ovary cell culture fluid. The on-line assay directly sampled preparative column effluent and provided real-time measurement of antibody breakthrough during loading. The on-line assay used protein A immobilized on perfusion chromatography media, equilibrated with phosphate-buffered saline at pH 7.2 and eluted with phosphate-buffered saline at pH 2.2. The assay reliably ended loading at 1% breakthrough with minimal yield loss. Reproducible yield and purity were obtained over 23 consecutive cycles. Yield remained constant while breakthrough capacity and the antibody concentration in the load changed.

Membrane ion-exchange chromatography for process-scale antibody purification.

The large-scale production of recombinant monoclonal antibodies demands economical purification processes with high throughputs. The potential for ion-exchange membrane adsorbers to replace traditional ion-exchange columns was evaluated. Breakthrough capacities of commercially available cation-exchange membranes were determined as a function of flow-rate and layer number. Due to economic and process restrictions, cation-exchange membranes may not currently be advantageous for process-scale antibody purification in a bind and elute mode. However, anion-exchange membranes in a flow-through mode may provide a reasonable alternative to columns for the removal of low levels of impurities such as DNA, host cell protein, and virus.

Non-flammable preparative reversed-phase liquid chromatography of recombinant human insulin-like growth factor-I.

Acetonitrile is used as an eluent for reversed-phase chromatography. However, because it is a flammable solvent, using acetonitrile on a large scale requires expensive equipment and facilities specially designed for flammable solvents. Using a non-flammable solvent as an eluent eliminates this expense. A method was developed to purify recombinant human insulin-like growth factor I by reversed-phase high-performance liquid chromatography using gradient elution with hexylene glycol, a non-flammable replacement for acetonitrile. The separation produced equivalent yield, purity and throughput as reversed-phase chromatography using elution with acetonitrile.

Real-time control of purified product collection during chromatography of recombinant human insulin-like growth factor-I using an on-line assay.

During preparative reversed-phase chromatography of recombinant human insulin-like growth factor-I (IGF), the separation of IGF from IGF aggregates cannot be determined using UV absorbance. An on-line reversed-phase chromatographic assay was developed that provides a quantitative measurement of IGF and IGF aggregates every 4 min, allowing real-time control of purified IGF collection. Process control using the on-line assay is a reliable and accurate method to collect purified IGF.

Comparison of resistance of fungal spores to gamma and electron beam radiation.

The irradiation sensitivity of fungal spores to either gamma or electron beam irradiation was evaluated in distilled water. The D10 (the dose required to reduce the initial population by 90%) gamma values ranged from 0.236 to 0.416 kGy and from 0.209 to 0.319 kGy for Penicillium and Aspergillus species, respectively. The D10 electron beam values ranged from 0.194 to 0.341 and from 0.198 to 0.243 kGy for Penicillium and Aspergillus species, respectively. Of the aspergilli species evaluated, only half exhibited significantly (P < 0.05) greater sensitivity to the electron beam treatment compared to gamma irradiation. Four of the six penicillia species evaluated also exhibited significantly (P < 0.05) higher sensitivities to electron beam treatment.

Survival of Escherichia coli O157:H7 in frozen foods: impact of the cold shock response.

The survival of Escherichia coli O157:H7 strains in both frozen foods and trypticase soy broth (TSB) was investigated following cold shocking at 10 degrees C for 1.5 h. Using both trypticase soy agar (TSA) and violet red bile agar (VRBA) as recovery media, it was demonstrated that survival levels between cold shocked (CS) and non-cold shocked (NS) E. coli in ground beef or pork were not significantly different (P < or = 0.05). In contrast, cold shocking E. coli in either milk, whole egg or sausage resulted in a significant(P < or = 0.05) enhancement in survival. For milk, survival levels of CS E. coli, by 28 days of frozen storage, were 1.89 and 1.66 log10 cfu/ml higher on TSA and VRBA, respectively, when compared to NS cells. In egg these values were 0.64 and 1.31, while in sausage, values of 0.74 and 1.19 were obtained. In TSB (pH 7) survival of CS E. coli for some strains was about 3 log10 cfu/ml higher when compared to NS cells. Acidification of TSB (pH 5), however, appeared to negate the protective effects of the cold shock treatment. In milk, increasing the differential between the growth and cold shock temperatures resulted in higher numbers of survivors. In this regard the growth-cold shock temperature protocol giving optimum protection was 37-10 degrees C. In contrast, growth of E. coli at 20 degrees C followed by cold shocking at 10 degrees C did not result in any significant freeze protection. In addition, increased protection due to cold shocking was correlated with the appearance of a novel protein appearing at pI 4.8 following isoelectric focusing analysis, thus demonstrating an alteration of protein synthesis.

Anti-CD18 antibodies improve cardiac function following cardiopulmonary bypass in dogs.

PURPOSE: Cardiopulmonary bypass is associated with activation of neutrophils, which may adhere to vascular endothelium causing lung, heart, and brain injury. We tested whether blocking neutrophil adherence would improve organ function following cardiopulmonary bypass in dogs. MATERIALS AND METHODS: All dogs received a standard anesthetic, and then one group (n = 6) received 2 hours of cardiopulmonary bypass followed by 4 hours of observation. A second group (n = 6) received a monoclonal antibody (6 mg/kg) to CD18, a neutrophil adherence factor, immediately before cardiopulmonary bypass. A third group (n = 6) did not receive cardiopulmonary bypass or antibody. RESULTS: Using flow cytometry we found that the antibody bound essentially all neutrophil CD18 sites. All three groups had similar gas exchange and hemodynamics. Lung and heart histology results were similar between groups. By echocardiography, five animals receiving cardiopulmonary bypass alone showed regional wall abnormalities, whereas only one receiving antibody showed wall motion abnormality (P < .05). Following cardiopulmonary bypass, intracellular myocardial pH was higher (P < .05) in the antibody-treated group compared with the group that had cardiopulmonary bypass alone (7.23 +/- 0.05 v 7.07 +/- 0.07 respectively). CONCLUSION: Monoclonal antibodies to CD18 can prevent the deterioration in cardiac function routinely observed following cardiopulmonary bypass.

Recovery of foodborne microorganisms from potentially lethal radiation damage.

A two-stage recovery protocol was examined for microorganisms following gamma irradiation in phosphate buffer at 0 degrees C. In the first stage, survivors were recovered on basal yeast extract agar and held at various temperatures suboptimal for their growth for 20 h (resuscitation protocol). In the second stage the survivors were incubated for an additional 24 h, but in this case at their optimum temperature for growth. Controls consisted of survivors which were not subjected to the resuscitation protocol (direct incubation at their optimum growth temperature). The ratio of survivors enumerated with and without the resuscitation protocol (control) at each specified temperature was used to formulate a recovery factor(RF). An RF was determined for each treatment dose. Results of this study indicated that the number of Escherichia coli, Salmonella serotype typhimurium and Brochothrix thermosphacta survivors increased following a resuscitation protocol (RF > 2.0). Overall, optimum resuscitation temperatures ranged from 14 to 22 degrees C. The extent of recovery also appeared dose dependent, with larger treatment doses giving rise to a higher RF. S. serotype typhimurium irradiated at 1.5 kGy exhibited the highest RF, 161, when resuscitated at 22 degrees C. Listeria monocytogenes, Yersinia enterocolitica, Staphylococcus aureus, Aeromonas hydrophila and Saccharomyces cerevisiae exhibited an RF < 2.0 regardless of resuscitation temperature. Results of this study indicated that the use of suboptimal holding temperatures as part of a recovery protocol may have advantages, especially with respect to the enumeration of E. coli and salmonellae survivors in irradiated foods such as poultry.