Cell proliferation in the prostate complex of the castrate mouse

Cell proliferation during 100 h of continuous androgen challenge was studied in the seminal vesicle and coagulating gland of Balb/c mice castrated 3 days or 14 days prior to the first daily injection of 250 mug testosterone propionate. Continuous labelling with [3H] thymidine indicated that the seminal vesicle was almost totally responsive to androgen, as early as 3 days after castration, whereas the androgen sensitivity of the coagulating gland increased from 30% at 3 days after castration to 85% at 14 days after castration. In both tissues the magnitude of the proliferative reaction could be related to the extent of cell loss prior to stimulation. The duration of the pre-replicative phase in the response of the seminal vesicle to androgen was 20-25 h both at 3 and 14 days after castration. In the coagulating gland the pre-replicative phase was 40 h at 3 days after castration and 20 h at 14 days after castration. The maximum uptake of [7alpha-3H] testosterone administered to mice 3 days after castration was significantly greater (P less than 0-01) in the seminal vesicle compared to the coagulating gland. At 14 days the seminal vesicle and coagulating gland exhibited a similar capacity for uptake. The in vivo metabolism of [7alpha-3H] testosterone was studied by thin layer chromatography 30 min and 120 min after administration. A high proportion of the radioactivity extracted from all the tissues was associated with highly polar steroids. At 3 days after castration, the seminal vesicle, 2 h after administration of radioactive testosterone, retained a much higher proportion of radioactivity associated with dihydrotestosterone than did the coagulating gland. The localization of steroid in mice 3 days after castration was studied by dry-mount autoradiography at intervals up to 2 h after the injection of [1,2,6,7(n)-3H]-testosterone. A heavier deposition of silver grains was observed over autoradiographs of the seminal vesicle. In the seminal vesicle the grains were primarily located over nuclear areas whereas in the coagulating gland the grains were diffusely distributed over both nuclear areas and over cytoplasmic areas.     

Microbial growth,communities and sensory characteristics of vacuum and modified atmosphere packaged lamb shoulders

Packaging fresh lamb in a vacuum (VAC) versus a 100% CO₂ modified atmosphere (MAP) may influence product shelf-life and the bacterial communities. While VAC is a common packing method and 100% CO₂ MAP is used in some countries, there is little information about how these different techniques affect the growth of spoilage bacteria and sensory attributes of lamb. The aim of this study was to assess changes in microbiological and organoleptic properties, and determine differences in microbial communities by terminal restriction fragment length polymorphism (TRFLP) and 454 pyrosequencing, in bone-in (BI) and bone-out (BO) MAP- and VAC-packed lamb shoulders stored at −0.3 °C over 12 wk. VAC and MAP lamb shoulders were acceptable in sensory test scores over 12 wk of storage at −0.3 °C, despite total viable count (TVC) and lactic acid bacteria (LAB) levels increasing to 8 log₁₀ CFU/cm² for VAC lamb and 4–6 log₁₀ CFU/cm² for MAP lamb. Similar to the sensory results, there were no significant differences in microbial communities between BI and BO product. However, types of bacteria were different between VAC and MAP packaging. Specifically, while VAC shoulder became dominated by Carnobacterium spp. in the middle of the storage period, the MAP shoulder microbial population remained similar from the start until later storage times.     

A single, mild, transient scrotal heat stress causes DNA damage, subfertility and impairs formation of blastocysts in mice

Infertility represents a major clinical problem and 50% of cases are attributable to the male partner. Testicular function is temperature dependent, and in both man and mouse the position of the testes in the scrotum ensures that they are kept at between 2 and 8 degrees C below core body temperature. We used a mouse model to investigate the impact of a single, transient, mild, scrotal heat stress (38, 40 or 42 degrees C for 30 min) on testicular function, sperm DNA integrity and embryo survival. We detected temperature-dependent changes in testicular architecture, number of apoptotic cells and a significant reduction in testis weight 7 and 14 days after heat stress at 42 degrees C. We report for the first time that DNA strand breaks (gamma-H2AX-positive foci) were present in spermatocytes recovered from testes subjected to 40 or 42 degrees C. Fertility of heat-stressed males was tested 23-28 d after treatment (sperm at this time would have been spermatocytes at time of heating). Paternal heat stress at 42 degrees C resulted in reduced pregnancy rate, placental weight and litter size; pregnancies from the 40 degrees C group had increased resorptions at e14.5. Abnormalities in embryonic development were detected at e3.5 and in vitro fertilisation with sperm recovered 16 h or 23 d after scrotal stress at 42 degrees C revealed a block in development between the 4-cell and blastocyst stages. This study has provided evidence of temperature-dependent effects on germ cell DNA integrity and highlighted the importance of an intact paternal genome for normal embryo development.     

Effective use of nisin to control Bacillus and Clostridium spoilage of a pasteurized mashed potato product

Heat-resistant spore-forming bacteria such as Bacillus and Clostridium can survive and grow in cooked potato products. This situation represents both a public health problem and an economic problem. The natural food preservative nisin is used in heat-treated foods to prevent the growth of such bacteria. A cocktail of Clostridium sporogenes and Clostridium tyrobutyricum spores was inoculated into cooked mashed potatoes, which were vacuum packed, pasteurized, and incubated at 8 and 25 degrees C. The shelf life of the mashed potatoes at 25 degrees C was extended by at least 58 days with the addition 6.25 microg of nisin per g. At 8 degrees C, in control samples not containing nisin, the natural contaminant Bacillus grew, but the inoculated Clostridium strains did not until the temperature was raised to 20 degrees C after 39 days. No bacterial growth occurred in nisin-containing samples. The shelf life of the mashed potatoes was extended by at least 30 days with 6.25 microg of nisin per g. In trials involving a cocktail of Bacillus cereus and Bacillus subtilis strains, 6.25 microg of nisin per g extended the shelf life of mashed potato samples that were not vacuum packed by at least 27 days at 8 degrees C. At 25 degrees C, 25 microg of nisin per g extended shelf life by a similar period. Shelf life extension was also observed at lower nisin levels. Microbiological analysis of the mashed potato ingredients showed that a high spore level was associated with the onion powder. It is emphasized that the preservative and the ingredients must be well mixed to ensure good nisin efficacy. Nisin remained at effective levels after pasteurization, and good retention was observed throughout the shelf life of the mashed potatoes.     

A hierarchical modeling approach for estimating national distributions of chemicals in public drinking water systems

Water quality studies often include the analytical challenge of incorporating censored data and quantifying error of estimation. Many analytical methods exist for estimating distribution parameters when censored data are present. This paper presents a Bayesian-based hierarchical model for estimating the national distribution of the mean concentrations of chemicals occurring in U.S. public drinking water systems using fluoride and thallium as examples. The data used are Safe Drinking Water Act compliance monitoring data (with a significant proportion of left-censored data). The model, which assumes log-normality, was evaluated using simulated data sets generated from a series of Weibull distributions to illustrate the robustness of the model. The hierarchical model is easily implemented using the Markov chain Monte Carlo simulation method. In addition, the Bayesian method is able to quantify the uncertainty in the estimated cumulative density function. The estimated fluoride and thallium national distributions are presented. Results from this study can be used to develop prior distributions for future U.S. drinking water regulatory studies of contaminant occurrence.     

Comparative evaluation of chloroethene dechlorination to ethene by Dehalococcoides-like microorganisms

Reductive dehalogenation of tetrachloroethene (PCE), trichloroethene (TCE), cis-1,2-dichloroethene (DCE), and vinyl chloride (VC) was examined in four cultures containing Dehalococcoides-like microorganisms. Dechlorination and growth kinetics were compared using a Monod growth-rate model for multiple electron acceptor usage with competition. Included were the Victoria mixed culture containing Dehalococcoides species strain VS (from Victoria, TX), the mixed culture KB-1/VC (from southern Ontario), the Pinellas mixed culture (from Pinellas, FL), and D. ethenogenes strain 195. All cultures, with the exception of D. ethenogenes strain 195, grew with VC as catabolic electron acceptor. A dilution method was developed that allows a valid comparison to be made of dehalogenating kinetics between different mixed cultures. Using this procedure, maximum growth rates on VC were found to be similar for strain VS and KB-1/VC (0.42-0.49 +/- 0.02 d(-1)) but slower for the Pinellas culture (0.28 +/- 0.01 d(-1)). The 16S rRNA gene sequences were determined to ensure that no cross contamination between cultures had occurred. Following enrichment of the VC dechlorinating microorganisms on VC, the cultures were amended with DCE, TCE, or PCE. The three mixed cultures failed to dechlorinate PCE or did so very slowly. However, the dilution technique indicated that all experienced growth on TCE and DCE as well as on VC. Maximum growth rates on DCE alone were quite similar (0.43-0.46 d(-1)), while the Pinellas culture grew faster on TCE alone (0.49 d(-1)) than did the other two mixed cultures (0.33-0.35 d(-1)). Half-velocity and inhibition constants for growth on TCE were also determined for the three mixed cultures; both constants were found to be essentially equal and the same for the different cultures, varying between only 8.6 and 10.5 microM. The ability of the strain VS, KB-1/VC, and Pinellas cultures to utilize TCE rapidly with conversion to ethene is quite different from that of any other reported microorganism. It was separately confirmed with more traditional cell-counting techniques that strain VS coupled TCE, as well as DCE and VC, utilization with growth. This is the first report of an organism obtaining energy for growth through every step in the reduction of TCE to ethene. Also, as suggested by the dilution technique, the dehalogenating organisms in the KB-1/VC and Pinellas cultures appear to obtain growth from TCE utilization as well. Such ability to grow while dehalogenating TCE to ethene will be an important advantage for their use in bioaugmentation.     

Isolation,cloning, and characterization of the 2S albumin: A new allergen from hazelnut

Scope: 2S albumins are the major allergens involved in severe food allergy to nuts, seeds, and legumes. We aimed to isolate, clone, and express 2S albumin from hazelnut and determine its allergenicity. Methods: 2S albumin from hazelnut extract was purified using size exclusion chromatography and RP‐HPLC. After N‐terminal sequencing, degenerated and poly‐d(T) primers were used to clone the 2S albumin sequence from hazelnut cDNA. After expression in Escherichia coli and affinity purification, IgE reactivity was evaluated by Immunoblot/ImmunoCAP (inhibition) analyses using sera of nut‐allergic patients. Results: N‐terminal sequencing of a ～10 kDa peak from size exclusion chromatography/RP‐HPLC gave two sequences highly homologous to pecan 2S albumin, an 11 amino acid (aa) N‐terminal and a 10aa internal peptide. The obtained clone (441 bp) encoded a 147aa hazelnut 2S albumin consisting of a putative signal peptide (22 aa), a linker peptide (20 aa), and the mature protein sequence (105 aa). The latter was successfully expressed in E. coli. Both recombinant and natural 2S albumin demonstrated similar IgE reactivity in Immunoblot/ImmunoCAP (inhibition) analyses. Conclusion: We confirmed the postulated role of hazelnut 2S albumin as an allergen. The availability of recombinant molecules will allow establishing the importance of hazelnut 2S albumin for hazelnut allergy.     

Preharvest internalization of Escherichia coli O157:H7 into lettuce leaves, as affected by insect and physical damage

Environmental pests may serve as reservoirs and vectors of zoonotic pathogens to leafy greens; however, it is unknown whether insect pests feeding on plant tissues could redistribute these pathogens present on the surface of leaves to internal sites. This study sought to differentiate the degree of tissue internalization of Escherichia coli O157:H7 when applied at different populations on the surface of lettuce and spinach leaves, and to ascertain whether lettuce-infesting insects or physical injury could influence the fate of either surface or internalized populations of this enteric pathogen. No internalization of E. coli O157:H7 occurred when lettuce leaves were inoculated with 4.4 log CFU per leaf, but it did occur when inoculated with 6.4 log CFU per leaf. Internalization was statistically greater when spinach leaves were inoculated on the abaxial (underside) than when inoculated on the adaxial (topside) side, and when the enteric pathogen was spread after surface inoculation. Brief exposure (～18 h) of lettuce leaves to insects (5 cabbage loopers, 10 thrips, or 10 aphids) prior to inoculation with E. coli O157:H7 resulted in significantly reduced internalized populations of the pathogen within these leaves after approximately 2 weeks, as compared with leaves not exposed to insects. Surface-contaminated leaves physically injured through file abrasions also had significantly reduced populations of both total and internalized E. coli O157:H7 as compared with nonabraded leaves 2 weeks after pathogen exposure.     

Introduction of sulphydryl groups into pea vicilin: Formation of intra-and inter-polypeptide disulphide bonds

Site directed mutagenesis was used to introduce two cysteine residues into the hydrophilic regions of the pea vichlin polypeptide previously expressed in Saccharomyces cerevisiae. The mutated polypeptide was expresed and the resultant protein was characterised by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. The presence of both intra- and inter-polypeptide sulphydryl bonds was indicated by bands of different mobility from those of the native polypeptide. The association of mutated vicili polypeptides into disulphide-bonded aggregates within the yeast was not a random process, trimers being the major aggregate produced. When the mutated vicilin was extracted from yeast and purified, random aggregates of the vicilin polypeptide were formed.