Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.     

Microbiological Deterioration of Frozen Parfried Potatoes upon Holding After Thawing

Frozen parfried potatoes were thawed and stored at 55, 45, and 34 F (12.8, 7.2, 1.1 C). Significant changes in flavor and texture did not occur at these temperatures until the total bacterial count exceeded 100 million per gram. These sensory changes were produced after 4, 8, and 20 days of storage at 55, 45, and 34 F, respectively. Detectable color change appeared sooner and probably was not of microbial origin. It is unlikely that any health hazard exists under the range of conditions studied. Nevertheless, it seems undesirable to market food with such a high bacterial count. At half the storage periods given above, the count did not exceed 100,000 per gram.     

Microbiological Standards and Handling Codes for Chilled and Frozen Foods. A Review

The usefulness of microbiological standards for frozen foods is now a controversy in the trade and scientific literature. Most reviewers have given arguments both for and against, and have concluded that they should be applied with great caution. Such standards have the advantage of putting questions of safety on a convenient numerical basis. Canadian workers have reported that promulgation of standards has invariably raised the hygienic level of the products controlled.

Bacteriological standards have often been associated with the question of safety to the consumer. Everyone recognizes that food poisoning bacteria are a potential danger in any food. But many have argued that the history of food poisoning outbreaks from frozen foods is excellent and that there is no need for standards; on the other hand, proponents of standards have pointed to the incomplete investigation and reporting of outbreaks, and have argued that there may be more outbreaks than we realize. They have pointed to laboratory studies that have shown grossly mishandled precooked frozen foods to be truly dangerous. Some have proposed that pathogens should be absent from foods; but others have questioned that a microbiological standard can accomplish this end. Some pathogens, such as Salmonella or Staphylococcus have been shown to be so ubiquitous that their presence in some commercial foods is unavoidable. Also, sampling and analytical methods have been described as inadequate to guarantee that pathogens present will be detected. Some have argued that control at the source is a better way—through inspections of the plant operation, by enforcement of handling codes, or by processing procedures such as pasteurization, which would be more certain to result in a pathogen-free food.

A most important part of any of the proposed standards is a “total count” of viable aerobic bacteria. English workers have found that foods causing poisoning outbreaks usually had total viable counts above 10 million per gram. On the other hand, these same workers found Salmonella on meats with very low total viable count. The assumption by many that low total count indicates safety has been shown to be not always true. Furthermore, high counts of nonpathogenic organisms, such as psychrophilic saprophytes would have no public health significance.

The relation between bacterial level and quality is open to less controversy. Some authorities have pointed to bacterial level as a measure of sanitation, adequacy of refrigeration, or speed of handling. Others have indicated that to determine which of these factors caused a high count would be impossible with only a total count on the product as a guide. Some investigators have said a high count affects flavor adversely before actual spoilage is evident, and this may be a factor in competition on today's market. It is well established that initial bacterial level will affect the shelf-life of a chilled product. Methods of analysis are more nearly adequate for counts than for pathogens, but they need improvement, and should be clearly specified as part of any bacteriological standard. Foods with high count could sometimes be brought into compliance merely by storing them for a sufficient period frozen, or by heating them slightly. This has been cited by some authors as a disadvantage of bacteriological standards.

The enterococci and the coliform group (except Escherichia coli) have been shown to be ubiquitous and therefore should not be used alone to indicate fecal contamination. Although E. coli has greater significance, its source should be determined each time it is found.

Various reviewers have expressed the need for caution in the application of standards. The principal precautionary arguments we have found are as follows:

1) A single set of microbiological standards should not be applied to foods as a miscellaneous group, such as “frozen foods” or “precooked foods.”

2) Microbiological standards should be applied first to the more hazardous types of foods on an individual basis, after sufficient data are accumulated on expected bacterial levels, with consideration of variations in composition, processing procedures, and time of frozen storage.

3) When standards are chosen, there should be a definite relation between the standard and the hazard against which it is meant to protect the public.

4) Methods of sampling and analysis should be carefully studied for reliability and reproducibility among laboratories, and chosen methods should be specified in detail as part of the standard.

5) Tolerances should be included in the standard to account for inaccuracies of sampling and analysis.

6) At first, the standard should be applied on a tentative basis to allow for voluntary compliance before becoming a strictly enforced regulation.

7) Microbiological standards will be expensive to enforce.

8) If standards are unwisely chosen they will not stand in courts of law.

     

Deep-level diagnostic value of the rDNA-ITS region

The similarity of certain reported angiosperm rDNA internal transcribed spacer (ITS) region sequences to those of green algae prompted our analysis of the deep-level phylogenetic signal in the highly conserved but short 5.8S and hypervariable ITS2 sequences. We found that 5.8S sequences yield phylogenetic trees similar to but less well supported than those generated by a ca. 10-fold longer alignment from rDNA-18S sequences, as well as independent evidence. We attribute this result to our finding that, compared to 18S, the 5.8S has a higher proportion of sites subject to vary and greater among-site substitution rate homogeneity. We also determined that our phylogenetic results are not likely affected by intramolecular compensatory mutation to maintain RNA secondary structure nor by evident systematic biases in base composition. Despite historical homology, there appears to be no ITS2 primary sequence similarity shared sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that groups, however, share sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that ITS region sequences can diagnose organismal origins and phylogenetic relationships at many phylogenetic levels and provide a useful paradigm for molecular evolutionary study.      

Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro

ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.