Diagnostic and prognostic value of glycosyltransferase mRNA in glioblastoma multiforme patients.

Glioblastoma multiforme (GBM) is the most common and aggressive primary human brain tumour in adults with an average survival of 11 months. The 2-year survival is less than 10%, and only a small proportion of patients are alive at 3 years. Despite improved treatment strategies and aggressive therapy, the prognosis of GBM has changed little in past decades. Thus, any test that can reliably and rapidly diagnose the tumour and predict patient survival could be a valuable tool. Herein we report the use of quantitative real-time polymerase chain reaction (PCR) to quantify five glycosyltransferase transcripts in gliomas. Our results indicate that measuring GM1 synthase (beta-1,3 galactosyltransferase) mRNA may provide a useful method for segregating GBMs from other types of gliomas. In these studies, 97% of gliomas (36/37 tumours) below a threshold value had a diagnosis of GBM compared with 49% (52/106 tumours) above the threshold. More importantly, the increased expression of GD3 synthase mRNA in combination with decreased GalNAcT message correlated with increased survival in 79 GBM patients (proportional hazards model controlling for age, P = 0.02). These data were further corroborated by a data analysis from one of our previous studies on gangliosides of 80 GBMs, in which increased amounts of GM3 and GD3 (which accumulate in the absence of GalNAcT) correlated with a longer survival (P < 0.01). Thus, measuring GalNAcT and GD3 transcripts may provide a rapid method to assess prognosis in GBM patients. In summary, the data indicate that measuring glycosyltransferase mRNA levels by real-time PCR may be clinically useful for determining both diagnosis and prognosis in GBM patients.

TRAIL-induced apoptosis in U-1242 MG glioma cells.

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induces apoptosis in U-1242 MG cells. To investigate the molecular events involved in this process, we studied the effects of TRAIL on the localization within membrane fractions of molecules critical to the extrinsic apoptotic pathway. We report here that death receptor-5 (DR5), tumor necrosis factor receptor-1 (TNF-R1), and Fas receptor (FasR) are all located in the caveolin-1-enriched membrane fractions, and TRAIL caused the translocation of DR5, FasR, and TNF-R1 to the caveolar fractions. Caspase-8 is mainly located outside of caveolae, but TRAIL caused it to redistribute to the caveolin-1-enriched fractions where it was cleaved. Within 6 hours, the cleaved caspase-8 appeared in the high-density, noncaveolin fractions. Using confocal microscopy, we found that DR5, caspase-8, and caveolin-1 became progressively concentrated in blebs of plasmalemma as they formed in response to TRAIL. Our results provide the first evidence for the caveolar localization of TNF-R1 and DR5 and the coordinated redistribution among membrane fractions of several death receptors in response to TRAIL. We propose that the coordinated movement of these molecules among membrane compartments is probably an important component of the mechanisms regulating and initiating the extrinsic apoptotic pathway in human glioma cells.

Endogenous GD3 ganglioside induces apoptosis in U-1242 MG glioma cells.

GD3 ganglioside induces apoptosis in several cell types, but the molecular events through which this occurs are largely unknown. We investigated the apoptotic effects of GD3 expression using U-1242 MG glioblastoma cells, as these cells synthesize almost exclusively GM3 and GM2 but not GD3. To express GD3 under the control of the TetOn system with minimum leakage, we modified the system by constructing a single tri-cistronic retrovirus vector containing three genes separated by two internal ribosome entry sites: (a) transcriptional silencer, tTS; (b) mutant of reverse transcriptional activator, rtTA2(S)-M2 (provided by H. Bujard, Heidelberg, Germany); and (c) enhanced green fluorescent protein (EGFP), as an indicator of the tri-cistronic gene expression. Using flow cytometry, we selected glioma cells (U1242MG-GD3 clone) that express high levels of GD3 in response to doxycycline. Expression of GD3 was associated with apoptosis as verified by annexin-V binding, TdT-mediated dUTPnick end-labelling assay (TUNEL), and EGFP degradation. GD3-induced apoptosis occurred via caspase-8 activation, as GD3 caused cleavage of caspase-8 and inhibition of caspase-8 activation by zlETD-fmk minimized GD3-induced apoptosis.

Methodology for enumeration of coliphages in foods.

The effects of eluent composition, pH, and chaotropic agents on the recovery of T2, MS2, and indigenous coliphages from various foods were investigated. Additionally, methods of sample suspension and clarification were evaluated for coliphage recovery and application to various foods. Clarified sample suspensions were assayed for coliphages with a modified agar layer technique and appropriate Escherichia coli hosts. Centrifugation and polypropylene mesh filtration were more rapid and effective than glass wool filtration for clarification of sample suspensions and subsequent recovery of coliphages. Blending, stomaching, and shaking procedures were generally comparable for sample liquefaction and release of coliphages from foods. Complex basal eluents, EC medium and 1% casein, were generally more effective than a less complex eluent, phosphate buffer, for elution of coliphages from foods. For most foods, incorporation of sodium chloride or chaotropic agents, i.e., sodium trichloroacetate, urea, Tween 80, Triton X-100, and sodium nitrate, into basal eluents did not enhance recovery of coliphages. Indigenous coliphage recovery was not affected by sample suspension pH over a range of 6.0 to 9.0. With an optimal procedure, i.e., EC medium eluent, blending, and centrifugation, the recovery of T2 and MS2 ranged from 48 to 81% and from 58 to 100%, respectively, depending on the food type.

Characterization of Coliphages Recovered from Foods According to Temperature of Infectivity.

Coliphages recovered from 38 samples of ten different food products were characterized with regard to temperature of infectivity. High temperature (HT) phages were capable of reproduction at or above 30°C, mid temperature (MT) phages over a range of 20 to 42°C and low temperature (LT) phages at or below 20°C. The percentage of HT coliphages isolated with Escherichia coli C-3000 host were consistently higher than corresponding percentages with E. coli C host. Coliphages recovered from all products were primarily HT or MT coliphages.

Distribution of Coliphages in Various Foods.

The distribution of coliphages in various foods and the relationship between the incidences of coliphages and bacterial indicators were investigated. A total of 120 food samples comprising twelve products and including fresh meats, shellfish, vegetables and processed meats, were analyzed for indigenous coliphages using Escherichia coli hosts C, C-3000 and B. Bacterial analyses included enumeration of E. coli , fecal coliforms and coliforms, as well as aerobic plate counts and Salmonella analyses. Coliphages were detected (≥10 PFU/100 g) in 56% of samples and eleven of twelve products. Coliphages, E. coli , fecal coliforms and coliforms were recovered at a level of at least 30 organisms per 100 g in 43, 43, 68 and 81% of samples, with overall mean recoveries of 13, 19, 93 and 4300 organisms/100 g, respectively. Highest and lowest recoveries of coliphages and E. coli were from fresh meats and vacuum-packaged processed meats, respectively. Significant nonparametric correlations between coliphages, E. coli , fecal coliforms and coliforms were found among all food samples.

Comparison of selective media for assay of coliphages in sewage effluent and lake water.

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.

Potential for Growth of Nonproteolytic Types of Clostridium botulinum in Pasteurized Restructured Meat Products: A Review 1.

Type E and nonproteolytic type B strains of Clostridium botulinum can grow and produce toxin at temperatures below 5°C. Recent publications describing the greater heat resistance of nonproteolytic type B C. botulinum spores than type E spores are discussed in relation to suitable proess lethalities required for a safe pasteurized product. The incidences of botulism in Europe caused by nonproteolytic type B spores were compared to the lack of such incidences in the U.S. and to published procedures for isolating the causative agent for botulism. The incidence of C. botulinum spores in meat products in the U.S. also is reviewed.

Application of Bioluminescence to Rapid Determination of Microbial Levels in Ground Beef.

The relationship between microbial ATP measurements and aerobic plate counts (APC's at 35, 20 and 7°C) was investigated for 75 ground beef samples. Samples (n = 27) were obtained from several local retail markets in one experiment, and ground beef samples obtained from a single, processing facility were sampled throughout 15 d of storage at 1°C for a total of 48 samples in another experiment. Bioluminescent assay time for a given sample was less than 1 h. The correlation coefficient (r) between log10 microbial ATP and log10 APC (20°C) per g was 0.86 and 0.99 for retail and single source samples, respectively. Differences between actual log10 APC (20°C)/g and corresponding values predicted by linear regression equations were ≤log10 0.5 for 25 of 27 retail samples and 48 of 48 single source samples. Variation was noted in values of ATP per bacterial cell and relative bioluminescent quenching (ATP per relative light unit, RLU) for most retail samples and for single source samples having low APC (20°C) levels (≤log10 7.0).

Recovery of Coliphages from Chicken, Pork Sausage and Delicatessen Meats.

Coliphages were recovered from 18 of 18 fresh chicken and pork sausage samples as well as from 2 of 6 processed delicatessen meat samples employing a rapid technique using EC medium as both an eluent and as a modified phage assay plating medium. Coliphage recoveries ranged from approximately 3.3 to 4.4 log10, 1.2 to 3.5 log10 and zero to 2.7 log10 plaque forming units per 100 g in fresh chicken, fresh pork sausage and roast turkey breast, respectively. High coliphage levels generally reflected high fecal coliform counts, particularly for fresh meat samples. These data indicate that coliphages can be readily enumerated in foods within 16 h.

Effect of Subjective Condition of Beef Quarters on the Microbiology and Storage Stability of Vacuum-Packaged Clods and Ground Beef Patties.

The effect of subjective condition of beef quarters, as determined by United States Department of Agriculture (USDA) personnel, on the microbial and sensory quality of vacuum-packaged clods and ground beef during refrigerated or frozen storage was investigated. In addition, the effect of reconditioning or trimming beef quarters considered to be in off-condition before fabrication into clods or ground beef patties was studied. The microbial quality of beef quarters was directly related to the subjective condition classification in that aerobic plate counts (APC's) of beef quarters and derived products were greater with increasing off-condition on the basis of condition "1" being "excellent" and condition "5" being "unfit for consumption." No significant differences (P<0.05) were observed in APC's between clods from quarters in various conditions initially or following 14 or 28 d of vacuum packaged storage at 1 to 2°C. Patties derived from quarters in condition "1" had significantly lower (P<0.05) APC's than those of patties from quarters in condition "4" or "5". The microbial quality of ground beef patties prepared from quarters in conditions "3" and "4" was not significantly affected by trimming of the quarters before fabrication. Few consistent differences in the sensory quality of ground beef patties were noted as a result of condition classification or trimming of quarters from which they were fabricated. These data indicate that reconditioning or trimming of "off-condition" beef quarters cannot be relied upon to improve the microbial quality of derived products such as ground beef.

Survival of Salmonella infantis and Staphylococcus aureus on smoked broiler halves.

Effects of storage at 5 ad -18 C for up to 28 days and high and low inoculation levels on survival of Salmonella infantis and Staphylococcus aureus on smoked broiler halves were investigated. S. infantis and Staph. aureus counts were significantly reduced during storage. The reduction in counts of S. infantis could not be attributed solely to either temperature or inoculation levels. Storage at 5 C significantly (P less than .05) reduced Staph. aureus counts on smoked broiler halves as compared to storage at -18 C. For Staph. aureus, low inoculation levels (10(3) to 10(4) organisms/ml) resulted in significantly higher mortality rates than did high inoculation levels (10(7) to 10(8) organisms/ml). At both storage conditions and inoculation levels, Staph. aureus was able to maintain higher populations than S. infantis.

Changes in the microbiological and shelf-life characteristics of beef tongues and livers following transcontinental and transoceanic shipment.

The effects of transcontinental (interstate) transport and transoceanic shipment were determined on microbiological and shelf-life characteristics of beef tongues and livers. These variety meats were evaluated for both microbiological and shelf-life characteristics following slaughter in Guymon, Oklahoma, USA. The samples were then frozen and sent by refrigerated truck (4·5°C) to storage facilities in Jacksonville, Florida, USA. Further microbiological and shelf-life evaluations took place prior to overseas shipment at the University of Florida and following overseas shipment at The Institute CIVO-Technology, TNO, Zeist, The Netherlands. Aerobic plate counts (APCs) at 35°C for beef tongues showed a significant (P < 0·0.5) decrease following overseas shipment. Similar results were noted for beef livers. For both organs, the surface thawing in Florida, required for sampling, did not appear to affect the final bacterial counts. The 20°C APCs for beef tongues revealed a significant (P < 0·0.5) decrease following interstate transport but not transoceanic shipment. The 20°C APCs for beef livers did not differ significantly during the entire transportation period. The colour differences noted during transport of the product were probably the result of freezing and not of the actual shipping and storage conditions.

Microbiological Characteristics of Beef Tongues and Livers as Affected by Temperature-Abuse and Packaging Systems.

Effects of various handling, packaging, temperature-abuse and storage conditions were determined on the microbiological characteristics of beef livers and tongues. These organs were evaluated: (a) initially following slaughter, (b) immediately following the frozen storage period of 2-4 weeks at -29°C and (c) following a simulated shipping-temperature abuse of 24 h at 22-28°C followed by 13 days of storage at -1 ± 0.5°C. Initial counts (log/cm2) of coliforms, coagulase-positive Staphylococcus aureus and Clostridium perfringens ranged from 0.19-1.37. Generally, neither freezing nor temperature-abuse had a significant effect on these microorganisms. Vacuum-packaged beef tongues and livers, generally, had lower bacterial counts than did either naked or polyvinyl chloride film-wrapped products. Generally, it was observed that abusive storage temperatures, in conjunction with the naked and film-wrapped packaging systems, appear to present potential microbial spoilage problems when compared with vacuum packaging.

Identification of Bacteria Isolated from Fresh and Temperature Abused Variety Meats 1.

The microflora associated with fresh and temperature abused beef livers, kidneys, hearts, tongues and pork livers was identified. Variety meats were obtained from a packing plant and allocated to three packaging treatments, i.e., vacuum packaging, polyvinyl chloride film wrapping and no wrapping (unwrapped). Isolates were characterized from fresh variety meats following frozen storage for three weeks at -29 ± 2 C; and following simulated temperature abuse. Classification of 1555 isolates obtained from aerobic plate counts at 35, 20 and 7 C is provided. Fresh variety meats were found to be contaminated with a variety of bacteria commonly associated with fresh red meats immediately post-mortem, with Micrococcus sp. being the most frequently isolated gram-positive bacterium and Escherichia coli the predominating gram-negative isolate. Frozen variety meats before simulated temperature abuse reflected a higher proportion of gram-positive organisms and fewer Enterobacteriaceae than fresh variety meats. Abused variety meats yielded predominately Pseudomonas strains, except where vacuum packaging was used, in which case isolates were predominately Lactobacillus sp. and Micrococcus .

Microbiological Comparison of Hot-Boned and Conventionally Processed Beef Plate Cuts During Extended Storage 1.

The development of microflora on hot-boned and conventionally processed beef plate cuts was investigated from time of slaughter and/or fabrication throughout vacuum-packaged storage for 6 weeks at 0-1 C. Cuts from each processing treatment were analyzed immediately post-mortem and after 0.5, 1, 1.5, 2, 4, 7, 14, 21, 28 and 42 days of storage. Fabrication, packaging and chilling of beef plates were carefully controlled to minimize differences in chilling rates and contamination of hot and conventionally processed cuts. Microbial analyses included enumeration of mesophilic, psychrotrophic and total Enterobacteriaceae populations as well as taxonomic characterization of corresponding microbial isolates. Microbial counts of hot-boned cuts were generally higher than corresponding counts of conventionally processed cuts with significant differences (p<0.05) detected between mesophilic and psychrotrophic counts at most storage intervals between 14 and 42 days. Earlier predominance of organisms such as Lactobacillus spp. and Brochothrix thermosphacta on hot-boned vs. conventionally processed cuts was indicated by taxonomic determinations. Psychrotrophic Enterobacteriaceae , including Hafnia alvei and Yersinia enterocolitica -like organisms, were recovered in high numbers from a few samples after 28 and 42 days of storage regardless of processing technique. Differences in the development of microbial flora on hot and conventionally processed beef cuts could not be explained on the basis of differences in initial chill rates between treatments.

Microflora Recovered from Foods on Violet Red Bile Agar with and without Glucose and Incubated at Different Temperatures 1.

Counts and taxonomic distribution of typical colonies on violet red bile agar (VRB) and VRB with 1% glucose (VRBG) incubated at 45, 35, 20, 7 and 1 °C from 23 retail food samples were compared. Aerobic plate counts were also obtained at each incubation temperature. Samples included fresh meats, processed meats, frozen processed products and fresh vegetables. Overall mean VRBG counts were slightly higher than VRB counts at each incubation temperature although there was some variation according to sample type. No significant differences (p>0.05) between the two media were noted overall or for any food type. Highest counts for VRB and VRBG generally occurred at 20°C incubation followed by 35, 7, 45 and 1 °C; counts at 20 and 35°C were not significantly different (p>0.05). The taxonomic distribution of typical colonies varied according to incubation temperature and sample type but there were few differences between VRB and VRBG for a given sample and incubation temperature. At 45°C, Escherichia coli was the most frequently recovered organism from both media overall but Klebsiella pneumoniae , Erwinia herbicola or Enterobacter cloacae predominated in many samples. Serratia marcescens and Erwinia herbicola comprised the majority of isolates from both media at 35, 20, 7 and 1 °C with taxonomic diversity being greatest at 35 and 20°C.

Microbiological Analysis of Alligator ( Alligator mississippiensis ) Meat.

The two tail muscles, ilio caudalis and ischio caudalis, along with hide surface areas from four alligators, Alligator mississippiensis , were evaluated for microbial numbers and types. Microbial analyses of hide surfaces yielded a mean aerobic plate count (APC, 35 C) of approximately 4.45 logs/cm2 at 35 C, and total coliform counts were low. Salmonella was not recovered from hide or tissue samples. APCs of fresh meat samples were low (2.88 - 3.02 logs/g) regardless of muscle type, with no recovery of fecal coliforms or Escherichia coli . Microbial development in meat stored at 1.7 C for up to 15 days was comparable to that reported for fresh beef. Typical microorganisms recovered included Corynebacterium , Staphylococcus , Micrococcus , Flavobacterium , Pseudomonas , Acinetobacter , Arthrobacter and yeasts.