ALBERT

Notes: We describe a rapid non-radioactive DNA typing of the serological types DR1-DRw10 using polymerase chain reaction (PCR)-amplified DNA and 15 sequence-specific oligonucleotides (SSO) which are labelled enzymatically at their 3’end with one digoxigenin (DIG). The hybridized SSOs were detected using anti-DIG alkaline phosphatase and Fab fragments and visualization was obtained with the chemilumine-scent substate 3-(2‘-spiroadamantan)-4-(3′-phosphoryloxy)-phenyl-1,2-dioxetan (AMPPD). The results were identical with those of the previously used 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/4-nitrobluetetrazolium chloride (NBT) system. The use of AMPPD is more rapid and allows the repeated rehybridization of the membrane-bound DNA.

Notes: Cell lines and a clone established from the C57BL/6 (H-2b) Lewis lung (3LL) tumour were previously characterized with respect to tumour growth and metastatic spread in vivo, and to the expression of a 3LL tumour-specific antigen (3LL TA) using a monoclonal antibody raised in syngeneic mice immunized with 3LL cells. No correlation was observed between the presence of 3LL TA and the prevention of metastatic spread which suggests that the immune recognition of this tumour antigen requires the presence of a self H-2 molecule absent from these tumour cells. Indeed, radioimmunoassay (RIA) and cytofluorometric analysis using specific monoclonal antibodies have shown that the H-2Kb molecule was not expressed at the cell surface of all 3LL cell lines and clones, while the H-2Db molecule was present at normal levels. This defect, which was not the consequence of a lack Of (32m expression, was accompanied by an absence or a marked reduction of the H-2K mRNA level (which has been reversed in the M4 cell line by in vitro gamma interferon treatment), while the H-2D class I gene was normally transcribed. Another defective transcription was also observed for a gene in the Tla region (gene 37). This low‘37’phenotype was corrected by in vitro treatment of the M4 cell line with gamma interferon, which indicates that this class I gene of the Qa/Tla region has an interferon response sequence in the promoter.

Notes: Using H-2 recombinant congenic strains of mice, genetic analysis of resistance to murine hepatitis virus type 3 (MHV3)-induced paralysis was performed. It appeared that both H-2K and H-2D, two class I gene regions of the mouse major histocompatibility complex (MHC), can play independent significant roles in the establishment of such resistance.

Notes: The HLA-DQw2 specificity, homogeneous in serology, is strongly associated to two HLA-DR specificities: DR3 and DR7. These alleles are found mainly on DQw2 bearing extended haplotypes with strong linkage disequilibrium. We describe, with BamHI, HindIII and RsaI, two restriction fragments length polymorphisms (RFLP) for the A gene of DQw2. These two subtypes correlated with the DR3 and DR7 specificities. Interestingly, by non-equilibrium pH gradient electrophoresis (NEPHGE), two DQα chains were also found, respectively correlated with the same DR specificities. In addition, Hindi polymorphism allowed us to distinguish several patterns of B genes for (DR7) DQw2 haplotypes but without any detectable association with another HLA marker. However, only one DQ(3 chain was found by NEPHGE in the (DR7) DQw2 haplotype. Furthermore, MncII discriminated the B genes of the two extended haplotypes: (B8, DR3) DQw2 and (B18, DR3) DQw2. The same result was found by NEPHGE: two DQβ chains were described, corresponding to the same extended haplotypes. The use of exon-specific DQB probes showed that the genomic polymorphism in DOw2 haplotypes is located, at least, at the 3’end of the gene. These data add new characteristics to the different DQw2 extended haplotypes.

Notes: Differences between the mouse Ly5a and Ly5b alleles can be distinguished on the basis of polymerase chain reaction (PCR)-restriction enzyme analysis and differential monoclonal antibody reactivities. To more precisely map the Ly5 gene on the mouse chromosome 1, analytical DNA and protein tests were performed on recombinant inbred strains of mice prepared from SJL/J (Ly5a) and B ALB/cke (Ly5b) progenitor strains. Each recombinant inbred strain was characterized to determine whether it carried the Ly5a or Ly5b allele. Both assays, DNA-PCR and protein-immunofluorescence, yielded identical results for each strain examined. Placement of the Ly5 gene with respect to other characterized markers of mouse chromosome 1 for these recombinant inbred mouse strains shows a gene order of Idh-1.Ity:Pep3:/Ly5, CfhJ.

Notes: Polymerase chain reaction/oligonucleotide typing was used to identify HLA-Cw*0601 (Cw6) in patients with psoriasis and psoriatic arthritis. The assignment of HLA-Cw*0601 was established by the concordant presence of codons for alanine (position 73), lysine (position 80) and tryptophan (position 97). The frequencies of all three codons were increased in the patient groups.

Notes: Associations between a large number of diseases and markers within the major histocompatibility complex (MHC) have been described. In particular, susceptibility to several autoimmune disorders, including type I diabetes mellitus and rheumatoid arthritis, is linked to genes within the MHC and strong population associations are demonstrable between certain HLA class II alleles and these conditions. Genetic mapping of HLA susceptibility loci has traditionally relied on the use of phenotypic markers defined by alloantisera, cellular typing reagents and biochemical analysis of histocompatibility antigens. Polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) typing combines the ability to define the finest of HLA specificities, by analysis of the corresponding DN A sequences, with the possibility of studying large populations of normal and affected individuals. The applications of this technology to characterizing precisely the MHC loci associated with susceptibility to autoimmune diseases such as rheumatoid arthritis, type I diabetes mellitus, coeliac disease and pemphigus vulgaris are reviewed here.

Notes: A method for predicting the depth of penetration of bacteria into various beef tissues was developed. Blue Lake, an insoluble dye, was used to simulate bacteria on the meat surface because movement of the Blue Lake into the meat tissue during spray washing could be easily visualized. The beef tissue surfaces studied were: (1) exterior lean, (2) exterior fat, (3) interior body cavity (peritoneum) and (4) cut tissue. The model indicated that many bacteria not removed during washing are driven into the beef tissue by the washing process. Interior body cavity tissue was most resistant to penetration followed by exterior lean and fat tissues, which were about equal in resistance. Cut surfaces were the most susceptible to Blue Lake penetration.

Notes: The potential for controlling the growth of Listeria monocytogenes in refrigerated foods using Carnobacterium piscicola LK5, a bacteriocin-producing strain originally isolated from raw ground beef, was studied using co-culture techniques. Eight foods, including UHT milk, canned “all-beef”dog food (cooked meat), raw ground beef, irradiation-sterilized raw ground beef, chicken roll, pasteurized crabmeat, canned creamed corn, and frankfurters, were inoculated with 103 cfu/g L. monocytogenes Scott A, with and without 104cfu/g LK5, and incubated at 5 and 19C. Samples were removed periodically and assayed for total aerobic plate count using Brain Heart Infusion Agar and L. monocytogenes using Vogel-Johnson Agar or Modified Vogel Johnson Agar. The growth of L. monocytogenes was suppressed in milk, dog food, crabmeat, creamed corn, and frankfurters stored at 5C. The microorganism was less inhibitory at 19C. In sterile raw ground beef, LK5 inactivated the pathogen at 5C and prevented its growth at 19C. No activity attributable to LK5 was observed in refrigerated nonsterile ground beef or chicken roll; however, these products did not support the psychrotrophic growth of the pathogen even in the absence of LK5. LK5 was most effective in products where the background microflora was reduced by either thermal processing or irradiation treatment. The results indicate that C. piscicola LK5 has potential as a means for preventing the growth of L. monocytogenes in a variety of refrigerated food products.

Notes: The efficacy of an enzyme-linked immunosorbent assay for botulinal toxin using polyclonal antibodies was evaluated in relation to the standard mouse assay. Qualitative tests for toxin in a meat system inoculated with Clostridium botulinum and pure cultures of various aerobic and anaerobic bacteria showed that both the mouse neurotoxin and enzyme-linked immunosorbent assays detected toxin in the samples. However the ratios of mouse:ELISA activity of culture supernatant toxins of C. botulinum showed wide disparity among strains of types A and B. Trypsin treatment resulted in a slight loss of ELISA activity but the mouse response increased.

Notes: This study on sanitizing beef surfaces was designed to evaluate effects of mixtures of acetic, lactic, citric and ascorbic acids with individual solutions of acetic and lactic acids. Acetic acid (3%), lactic acid (3%), MA1 (2% acetic, 1% lactic, 0.25% citric and 0.1% L-ascorbic acids) and MA2 (2% lactic, 1% acetic, 0.25% citric and 0.1% L-ascorbic acids) solutions were applied to beef core samples of muscle inoculated with bacteria. Experimental variables were type, concentration and temperature of acid solutions and type of microorganisms. Overall, an increase in either acid concentration or treatment temperature decreased numbers of residual viable bacteria. Lactic acid solution was the most effective against S. typhimurium with a reduction of 2 log10 at 70°C. For enterobacteria, acetic, lactic and MA2 solutions at 70°C resulted in a 1.5 log10 reduction. MA2 was the most effective acid solution at both 45 and 70°C, whereas, lactic acid and the MA2 mixture did not differ in effectiveness at 20°C.

Notes: Daminozide residues were determined in ppm or ppb on ‘Starking', ‘Red King', ‘Rome’and ‘Golden Delicious’apples after repeated long term exposure (21 years) and on ‘Top Red’apples after short term (1 year) exposure. Application rates varied from 2.2 to 9.0 kg/ha for short term exposure and 2.2 to 4.5 kg/ha for long term exposure. Two and three years after cessation of the daminozide spray program no daminozide residues were found in ‘Top Red’regardless of the rate applied or analytical procedure used. Daminozide residues (〈1 ppm) were present in ‘Starking', ‘Red King', ‘Rome’and ‘Golden Delicious’one year after cessation of the spray program. The cultivar ‘Rome’had consistently higher daminozide residue levels followed by ‘Golden Delicious', ‘Red King’and ‘Starking'.

Notes: Book reviews in this article: MANUAL OF CLINICAL MICROBIOLOGY, 5th edition. A. Balow (editor-in-chief).

Notes: Vibrio metschnikovii, a potentially pathogenic marine bacterium, was recovered from a wide variety of finfish and shellfish purchased at fish markets and supermarkets. Because limited information is available on the occurrence of this organism in food products and human disease, the observations may be relevant to public health considerations of seafood consumption.

Notes: An appreciable percentage of electric power is generated by oil-fired power plants. The ash resulting from such plants is typically very high in elements such as vanadium, nickel, aluminum and molybdenum. Oil bottom ash and fly ash when disposed or fly ash fallout from such power plants could result in absorption of such metals by agricultural crops. Swiss chard (Beta vulgaris L.) was grown in potted soils amended with oil bottom ash and fly ash at 2% and 1% (w/w), respectively. The crop absorbed significantly higher concentrations of each of the metals as compared to those in the control crop grown on soil alone. Higher levels of the ashes in soil resulted in phytotoxicity to the swiss chard.

Notes: The “Wisconsin process'’(a combination of lactic acid starter culture and sucrose) has previously been shown to be effective in preventing botulinum toxigenesis in reduced nitrite bacon. This technology has been applied to other low-acid refrigerated foods which receive less than a 12D thermal process. A combination of Pediococcus acidilactici and dextrose was effective in preventing botulinum toxigenesis in chicken salad. When the chicken salad was temperature abused, the P. acidilactici catabolized available dextrose to lactic acid. Extracellular accumulation of the lactic acid caused a decrease in the pH of the product. Pathogen challenge tests verified that the rate and extent of lactic acid accumulation in the chicken salad during temperature abuse was sufficient to preclude botulinum toxigenesis. This technology has the potential to be of use in a variety of low-acid refrigerated foods.

Notes: Sterile slices of cooked uncured turkey loaf were inoculated with 106 CFU of either Salmonella typhimurium, Listeria monocytogenes, Escherichia coli, Enterococcus faecalis, Citrobacter freundii, Klebsiella pneumoniae, or Enterobacter cloacae. Inoculated samples were vacuum-packaged and stored at 3 ± 1°C. Microorganisms were enumerated at 0, 3, 6, 9, 12, and 15 days on nonselective media. K. pneumoniae exhibited the least cold-tolerance with a log10 1.70 decrease in numbers. The coliforms E. cloacae, E. coli, and C. freundii had a survival pattern similar to that of S. typhimurium, with population decreases of log10 0.65, 0.82, 1.13, and 0.79, respectively. E. faecalis and L. monocytogenes were significantly more cold-resistant, with a decrease of log10 0.20 and no significant change in numbers, respectively. Survival of E. faecalis was not significantly (p 〈 0.01) different than that of L. monocytogenes, suggesting the use of enterococci as indicators of L. monocytogenes contamination of processed meats.

Notes: Three methods of threshold estimation are compared for the detection of 1,8-cineole added to Concord grape juice presented in a semi-ascending paired difference design (Lundahl et al. 1986). Method A tests the significance of obtaining a nonzero response, method B determines the transition point of a 2-phase linear model fitted to the data, method C fits a logistic model to the data and defines threshold as the point of maximum curvature, Cmax, of the curve. Method A yields interval estimates of thresholds, the midpoints of which systematically decrease with increasing sample size. Methods B and C yield point estimates of thresholds which do not systematically decrease with increasing sample size, are a feature of the dose-response curve, and are similar in value to each other.

Notes: Two studies are reported on the effect ofingestion on the relative-to-ideal ratings of salt in soup. In the first study there was a reduction in the measure of ideal concentration from pre- to post-ingestion, with subjects consuming the soups ad libitum. However, a measure of ideal concentration derived from several small samples presented in each session agreed well with the final post-ingestion ideal. A measure of most preferred concentration of salt based on total soup consumption was unrelated to the other measures.In the second experiment, the post-ingestion decrease in most preferred salt concentration was replicated when subjects consumed a fixed quantity of soup. The post-ingestion ideal was similar to that derived from rating either several small samples in one session or one single small sample per session.

Notes: A quality control chart technique was tested for feasibility in measuring panelist performance in product profile development. The descriptive sensory score sheet evolved by Filipino trained panelists for fresh-frozen young coconut meat and water at “malakanin”(7–8 months old coconut fruit) stage was utilized for this study. A comparison of panelist performance in terms of their congruence, within themselves and between each other, in identifying the intensity or presence of an attribute was made. Results indicated that a similar measurement of significant differences could be obtained using both the analysis of variance (ANOVA) approach and the control chart (c.c.) technique. Considering the volume of data to be handled, the c.c. technique appeared to be more manageable than ANOVA. Besides, the c.c. was found by the panelists and the researchers to be more interesting to analyze, easier to apply, to interpret and to use for improvement follow-through compared to the ANOVA.

Notes: Eighteen commercial cat litters were characterized for the following physical characteristics: water holding capacity, drying time, pH, cost, density, residual water, and particle size. Using sensory analysis, eight of the litters were evaluated for dustiness, clumpability and odor control before and after use and storage. The cat litters differed substantially in both physical and sensory properties. The crystal litters were noted for their small uniform size, high water holding capacity and clumpability. The recycled litter was most dusty and ammoniacal; whereas, the granule litter had an innately offensive odor. The alfalfa litter had superior odor control, but neither clumped nor dried well. The clay litters could be differentiated by pH, residual water, water holding capacity and could be classified as either perfumed and nonperfumed. Some of the perfumed litters became intensely perfumed when used and stored; whereas, the other perfumed litters behaved like the nonperfumed litters and became more offensive.

Notes: This study dealt with the determination of the most preferred level of sugar in plain yoghurt. Twenty-four consumers participated in 13 lab meals. The results of a rapid test, based on ideal relative-rating were compared to the results of a consumption test, based on the amount of test product consumed, in order to assess if this rapid test predict actual consumption behavior. Linear regression of the ideal-relative intensity ratings on logarithms of sucrose concentrations was used to estimate, for each subject, ideal-point (i.e., sucrose level corresponding to a null distance from ideal) and slope (which accounts for tolerance) with a good reproducibility. The consumption tests revealed behavior which could not be related to the level of sugar for 30% of the subjects. Moreover, the most preferred level of sugar obtained with the rapid test was not always the most consumed. However, the two mean values of ideal level are close: 6.3% with ideal-relative rating, and 7.5% with measurement of eaten quantity. Thus our experimentation underlines the interest of ideal-relative rating as a good predictor of consumption behavior.

Notes: Different types of panelist by treatment interaction are explored to determine how they influence the outcomes of discrimination tests. The study compares the situations where panelists are considered as fixed or random effects over the range of most testing conditions for small panels (5–15 panelists) that replicate their judgements. Magnitude interaction and nonperceivers or nondiscriminators have minor effects on test outcomes. Cross-over interaction increases the chances for a type II error, especially when panelists are considered as random effects. False discrimination increases the chances for a type I error when panelists are considered as fixed effects. Applications of methods to reduce the chances for these errors in the testing for differences among treatments are discussed.

Notes: In this paper we discuss methods that can be used to evaluate the performance of sensory panels. In particular we concentrate on detection of variation within and among panelists. A systematic presentation of some simple, graphically oriented tools will be given. Interpretation aspects of the tools will be emphasized. The methods will be illustrated by computations from a sensory experiment based on 4 sausage products. The example demonstrates how the tools can be used to evaluate the reproducibility of the assessors and also how they can be used to detect different types of individual differences among the assessors.

Notes: Visual masking techniques are frequently used to prevent panelists from discriminating among samples on the basis of color differences, only. The purpose of the study was to determine if these conditions alter the responses of the judges. Raspberry gelatins sweetened with 9, 10, 14 and 16% sucrose, respectively, and lemonades sweetened with 8, 10, 12 and 14% sucrose, respectively, were rated for perceived sweetness intensity using four different visual masking conditions (red lights, red glasses, dyes and blindfolds) and a white light and clear glasses control condition. Most of the visual masking techniques did not significantly affect the sweetness scores for the gelatins or lemonades. The one exception was the dye condition for the lemonades where the color (turquoise) probably affected the results.

Notes: A panel of untrained judges was asked to assess spreadability of selected solid edible fat samples (butter, margarine, low fat products) of different temperature with regard to hedonic preferences. Instrumental measurements were performed by cone penetration with constant load. The statistical analysis of the ranked sensory data showed significant spreadability optima in the apparent yield value range of approximately 30–60 kPa. Differences between the selected fats were not observed.

Notes: Subjects rated the intensities of 42 different attributes for 26 different bread products. Principal component analysis was used to categorize the breads based on their attribute ratings. The first two principal components were interpreted as a richness dimension and a nutrition/fiber dimension. The influence of these dimensions on sensory-specific satiety in bread products was investigated.Subjects tasted and rated their liking of small portions of a set of bread products representative of the extremes of the richness dimension and the nutrition/fiber dimension before and after eating a 400 calorie serving of one of the breads. Measurements of hunger were taken before and after the meal.Bread appeared resistant to sensory-specific satiety. The richness of the breads did not affect the amount of sensory-specific satiety. The high nutrition/fiber breads decreased hunger and tended to decrease liking more than the low nutrition/fiber breads.

Notes: Aroma concentrates from Alphonso and Totapuri mango pulps were prepared and the aroma stripped pulps were concentrated two folds. Ready to serve beverages were prepared from fresh mango pulps and pulp concentrates by adding different levels of aroma concentrates. Sensory evaluation of beverages showed that in the case of Alphonso mango addition of 50%, 75% and 100% aroma concentrate gave comparable quality while with Totapuri only 50% level showed comparable quality.The aroma stripped pulp concentrates were canned as such and after mixing with 50% aroma concentrates and stored at 25 × 5°C. Aroma concentrates were bottled and stored at 2°C. At different intervals of storage, beverages were prepared and evaluated. The results showed that throughout the storage period, beverages prepared from mango concentrates to which 50% aroma was added before canning was superior to those prepared from aroma stripped canned pulp concentrates to which aroma was added at the time of preparation of beverage indicating the effect of mango pulp on aroma stability.

Notes: These studies examined the influence of learned flavor cues on daily food intake in 28, normal-weight adults. Subjects were trained to associate distinctly flavored high or low calorie lunches with their postingestive effects. Following training, the flavors in the lunches were switched. Twenty-five percent of subjects (responders) adjusted their daily food intake based on their acquired sensory experience and were initially misled by the changes in the flavor cues; i.e., following the flavor switch, responders consumed additional or fewer calories based on the expected rather than actual energy intake of the meals. Energy intake gradually returned to baseline levels. The remaining subjects (nonresponders) were not misled by the flavor switch and maintained their customary level of intake. Responses were more pronounced following conditioning with the high calorie as compared to the low-calorie lunch. These data suggest that sensory cues may play an important role in the control of long-term food intake in individuals who are sensitive to such training.

Notes: A base syrup formulation containing nonvolatile flavor compounds was used to systematically evaluate the sensory contributions of selected volatile compounds (Furaneol, 2,5–dimethyl-4-hydroxy-3(2H)-furanone; sugar furanone, 4,5-dimethyl-3-hydroxy-2(5H)-furanone; guaiacol, 2-methoxy phenol; vanillin, 4-hydroxy-3-methoxy-benzaldehyde) to maple syrup flavor. Descriptive sensory panels showed that all compounds contributed to maple flavor intensity, but sugar furanone was an exceptionally important determinant. Medium and dark amber syrups contained generally higher concentrations of the selected flavor compounds than the light amber syrup. 5′-Inosine monophosphate was tentatively identified in maple syrup by HPLC analysis, and was found to contribute important taste characteristics to maple syrup. Consumer studies showed that more flavorful, darker grade A syrups were preferred over light amber grade A syrup.

Notes: Clostridium perfringens Type A is one of the most common causes of food-borne disease in the USA. Diarrheal and cramping symptoms associated with this illness are caused by a protein enterotoxin (abbreviated as CPE). Recently, diagnosis of C. perfringens food poisoning has been improved by the development of direct assays to detect CPE in feces or CPE production by food or fecal isolates of C. perfringens. The enterotoxin has a membrane-active action which appears to involve four early steps: (1) binding of CPE to a mammalian receptor, (2) insertion of CPE into membranes, (3) formation of a complex between CPE and mammalian membrane proteins and (4) onset of toxin-induced ion and water permeability changes. CPE has a unique amino acid sequence, and recent studies have indicated that the receptor-binding region of enterotoxin is localized at the extreme C-terminus. Further studies of the CPE structure versus function relationship may facilitate development of vaccines for human or veterinary usage.

Notes: Shelf-life and quality of fresh fishery products can be extended by the use of a modified atmosphere (MA) and high barrier film packaging coupled with refrigerated storage. MAs with elevated levels of carbon dioxide inhibit or slow the growth of various aerobic spoilage bacteria of fishery products by extending the lag phase. However, at the same time, MAs provide conditions for the growth of Grampositive bacteria and food pathogens within the package. The extension of the storage life of the refrigerated MA products may enable the slower-growing Gram-positive bacteria to reach high populations. The shelf-life of fishery products packaged under MAs rich in carbon dioxide coupled with storage at 8.0°C or below can be extended more than 100%. Major safety concerns regarding the risk of foodborne botulism can result from MA packaging of fresh fishery products that contain the spores of nonproteolytic C. botulinum and are subsequently temperature-abused. Minimizing the risk of foodborne botulism by including inhibitory factors such as antimicrobial agents before packaging fishery products under MAs and strict adherence to refrigerated storage temperatures are discussed.

Notes: The N-nitrosoamino acids (NNA) N-nitrosoproline (NPRO) and N-nitrososarcosine (NSAR) are precursors of the carcinogenic N-nitrosamines. Methodology using gas chromatography for their determination and data on their contents in 52 food products are presented. Food products consisted of smoked meats (12), smoked (9) and boiled (11) sausage, cheese (10), fish (7) and beer (3). Results showed that NNA formed during processing, particularly in meat, where levels of NPRO were as high as 353μg/kg. The compounds accumulated in the adipose tissue and surface portion of the product. Smoking caused increased formation of NPRO, while the use of liquid smoke minimized it. NPRO in meat products in natural casings was higher than in synthetic ones, and frying of boiled sausage and hams further enchanced NPRO formation. Like meat, smoked cheese and fish showed consistently higher NNA levels than the non-smoked products. These data indicate means of reducing NNA contents in foods by modification of processing techniques.

Notes: Toxoplasmosis, a disease of mammals and birds, is caused by the obligate intracellular protozoan parasite, Toxoplasma gondii. It is believed that approximately half of the human population of the U.S. is infected and that 500 million of the world population demonstrate a positive serological reaction to the parasite. Only a small percentage of the infected individuals show symptoms: however, in immunocompromised persons, the disease can be quite severe and even fatal. The infectious agent is generally foodborne and is due to the ingestion of raw or undercooked meats derived from infected animals. In this review, several aspects of the T. gondii organism, including its survival, its distribution in the environment and animals, its presence and survival in foods, role of virulence factors, and its transmission to humans, are reviewed. In addition, human toxoplasmosis, its treatment and prevention, and the economic aspects of the disease are discussed.

Notes: Ionizing radiation is shown capable of serving as an effective sanitizing treatment improving the sanitary quality of shellfish and providing an increased margin of safety for shellfish consumers. 60Co irradiation of the hard-shelled clam, Mercenaria mercenaria, and the oyster, Crassostrea virginica, significantly reduced virus carriage numbers without unduly affecting shellfish survival rates or desirable organoleptic qualities. A D10 value of 2 kGy was determined for depletion of hepatitis A virus in clams and oysters as measured by in situ hybridization fluorescent foci and cytopathology enumeration methods. A D10 value of 2.4 kGy was determined for depletion of rotavirus SA11 in clams and oysters as measured by a plaque forming unit enumeration method. Study results showed ionizing radiation capable of providing an extra, highly effective safeguard of shellfish sanitary quality when combined with traditional depuration treatment. Data drawn from other studies is introduced which shows D10 values as low as 1.0 kGy effectively eliminate Vibrio cholerae, and V. parahemolyticus, from shellfish.

Notes: The effects of sodium alginate (0.4 and 0.8%), calcium lactate (0.15 and 0.3%) and sodium lactate (60% solution; 1, 2, 3 and 4%) on growth and aminopeptidase activity of Pseudomonas fragi ATCC 4973 and Salmonella typhimurium in trypticase soy broth or autoclaved ground beef were studied for 5 days at 5°C. Sodium alginate and calcium lactate did not change colony counts of P. fragi or S. typhimurium; and had only slight effects on aminopeptidase activity. Sodium lactate (3 and 4%) showed inhibitory activity on growth and aminopeptidase activity of P. fragi. Sodium lactate also decreased aminopeptidase activity of S. typhimurium but had no major influence on colony counts. In general, aminopeptidase activity was inhibited more than cell multiplication by higher levels of alginate and sodium lactate.

Notes: Thirty-two foodborne bacterial isolates were tested as potential competitors of Listeria monocytogenes strain LM82 during enrichment because of their resistance to the selective agents in Listeria enrichment and isolation media. Competitive ability of each isolate was classified as weak, moderate, or strong by determining the ratio at which it masked identification of LM82 at an inoculation concentration of 10 colony forming units (CFU)/10 mL of Listeria enrichment broth. Of the competitive isolates identified, six were Enterococcus spp., two were Staphylococcus spp., and one was a Corynebacterium sp. Although several strains of Enterococcus faecium were examined, not all were competitive. Of six other bacterial strains associated with food fermentations and tested for competitiveness with LM82, one, a Gram-positive tetrad, was competitive. This study showed that although food microfloral strains that are able to survive in enrichment and isolation environments are fairly common, they do not necessarily compete with Listeria. Not all strains in a competitive species are necessarily competitive.

Notes: Salmonella typhimurium was inoculated into prepared dirt (moisture content 8.8%), sand (moisture content 0.06%) and phosphate buffer, and these menstrua were used to contaminate beef tissue. Fewer bacteria adhered to the tissue surface from the low moisture menstrua when compared to contamination by buffer. Determination of SR values, an estimate of the total population associated with the surface which was physically attached, indicated that more bacteria remained in the soil menstrum than adhered to the surface, when compared to buffer and sand menstrua. Contrary to expectations, bacteria which contaminated the surface from the low moisture menstrua were not more sensitive to organic acids than those which originated from the buffer.

Notes: A lactic acid bacterium (LK5) originally isolated from raw ground beef was characterized in relation to its ability to inhibit the growth of Listeria monocytogenes. The isolate, which was identified as Carnobacterium piscicola, inhibited the growth of 17 of 21 strains of Listeria (L. monocytogenes, L. innocua, L. ivanovii, L. welshimeri, and L. grayii). Its activity was not due to either acid or hydrogen peroxide production, but was related to the production of a heat stable bacteriocin. The isolate was most active against L. monocytogenes at refrigeration temperatures due to the combined effect of the pathogen's increased susceptibility, LK5's rapid growth rate, and enhanced bacteriocin production at low temperatures. Examination of the effect of inoculum ratios in co-cultures of C. piscicola LK5 and L. monocytogenes Scott A indicated that the lactic acid bacterium was active against L. monocytogenes even when the initial level of the pathogen was 100-fold greater. Evaluation of the impact of oxygen availability, initial pH, and sodium chloride content on the effectiveness of LK5 suggested that the isolate could be used to suppress the growth of Listeria in a variety of refrigerated foods.

Notes: Consumer food safety concerns and interest in pesticide related information was measured by personal interviews with 479 consumers in 14 counties in California. Although concern about pesticide safety was noted by almost half of the respondents, nutritional considerations had the greatest impact on food selection criteria. Of those who volunteered pesticide safety concerns, 15% indicated they sought organic produce and 13% said they avoided or reduced produce purchases. In addition to information about the human health effects of pesticide use, consumers expressed high interest in the environmental effects of pesticide use. Consumers currently use television and newspapers as information sources and prefer these to other sources.

Notes: Thirteen spices were screened for growth inhibition effect against Listeria monocytogenes at 24°C using a concentration gradient plate method. Cloves, and oregano were the two most inhibitory spices with MIC values ranging from 0.5 to 0.7% (W/V). Inhibition was also detected with sage and rosemary (MIC of 0.7 to 1.0%) as well as nutmeg (MIC of 1.1–1.4%). Black pepper, chili, cinnamon, garlic, mustard, paprika, parsley and red pepper at a concentration up to 3.0% did not inhibit the growth of the organisms. The effect of cloves, oregano and sage on the growth and survival of L. monocytogenes Scott A was further tested in tryptic soy broth at both 4 and 24°C. At a concentration of 0.5 or 1.0%, cloves were bactericidal and oregano was bacteriostatic to this organism at both incubation temperatures. Sage at these two concentrations was bactericidal at 4°C and bacteriostatic at 24°C. When tested in sterile meat slurry, a 1.0% level of either cloves or oregano had little effect on the growth of L. monocytogenes Scott A at 4 or 24°C.

Notes: The corrosion inhibitor, cyclohexylamine (CHA), is a common additive for steam generation systems. If primary steam directly contacts food during blanching, heating, or cooking operations the possibility exists that contamination of the food by CHA may result. In this study, snap beans and green peppers were blanched with steam containing CHA. A gas chromatographic method was developed and used to show the presence of appreciable residues of CHA remaining in the post-blanched vegetables. The widespread replacement of primary steam generators with secondary systems for food blanching largely eliminates possible hazards to consumers. The known toxicologic properties of CHA are reviewed and the need for additional studies of possible food contamination or direct human exposure by other commonly used steam additives is emphasized.

Notes: The popularity of Cajun cuisine has promoted the consumption of Louisiana crayfish, Procambarus clarkii. Tail meat and hepatopancreatic tissues of crayfish captured from two locations in the Atchafalaya River Basin and four open ponds were analyzed separately for xenobiotic metal and mineral composition using an inductively coupled plasma emission spectrometer (ICP) and chlorinated hydrocarbons by gas chromatograph (GC) with electron capture detector. Less than 3 mg/kg of toxic xenobiotic metals were found in the tail meats and less than 5 mg/kg in the hepatopancreatic tissues. Mineral concentrations were similar to those reported for other crustacean species. Occasional trace amounts of DDD and DDE were found present in tissue samples.

Notes: Both fresh and frozen chicken meat were evaluated for microbiological status by screening for total bacterial counts and for the presence of pathogens like Enterobacteria, Bacillus cereus, coagulase positive Staphylococci and Salmonella spp. Most of the samples exhibited heavy bacterial contamination (1.2 × 105 - 2.6 × 106/g), mainly with Staphylococcus spp. (1.5 × 104 - 2.8 × 105/g). All the chicken samples also showed the presence of Salmonellae (3 × 101 - 2.1 × 102/g). Among the different serotypes observed in chickens. S. typhimurium was common in fresh as well as frozen chicken. Radicidation at 2 kGy at cryogenic conditions (−40°C) was efficient in eliminating the natural pathogenic contamination of the poultry. Salmonella spp. viz. S. seftenberg and S. typhimurium differed in radiation sensitivity, the D10 values in phosphate buffer (pH 7.2) being 0.25 kGy and 0.12 kGy, respectively. Chicken homogenate (10%) offered approximately 2-fold protection to these cells. Chicken samples artificially inoculated with a heavy inoculum (108 cells/g) of these 2 serotypes required higher gamma radiation doses of 4–5 kGy. The findings suggested that a dose of 2 kGy is adequate for normally contaminated chicken samples, but for the heavily contaminated chicken a dose of 4–5 kGy, depending upon the predominating Salmonella serotype present, is required.

Notes: Ackee fruit representing a range of maturities were analyzed for hypoglycin A (hyp A) content. Hyp A in the arilli (edible flesh) dropped from over 1000 ppm to undetectable (〈0.1 ppm) as the fruit matured (i.e., pod colored up, split and opened fully). At all stages the seed contained appreciable hyp A, ∼ 1000 ppm, and the membrane mirrored aril levels. These analyses support earlier observations that unopened or partially opened ackee should not be consumed, whereas fruit which open naturally to 〉 15 mm lobe separation pose little health hazard, provided that all seed and membrane portions are removed. Bright red, full sized ackee should never be forced open for human use.

Notes: Book reviewed in this article: Nitrite—Cured Meat—A Food Safety Issue in Perspective. R. G. CassensTechnical Handbook for the Chilled Foods Industry. Chilled Foods Association

Notes: A panel of trained sensory judges is a sensitive and valuable instrument. and a lot of time and work is invested in their selection and training. The importance of internal motivation and external stimulation to maintain the interest and wellbeing of these human instruments is supported by major food scientists and behavioral psychologists. Current stimulation procedures for sensory judges are listed. A new procedure is proposed, consisting of an annual celebration party on November 20 in recognition of sensory judges. Advantages of holding this day on an international level are discussed: mainly, promotion of fellowship among sensory judges from different groups and countries, and also its help in increasing cooperation in sensory evaluation. Actions to promote International Sensory Judges Day are listed. Suggestions for the actual celebration party are put forward: an open house luncheon, presentation of awards, greeting cards and letters from other sensory panels; and last but not least, a set of proven “sensory” games where everyone joins in.

Notes: The differential sensitivity to sucrose or Aspartame in water was studied over the course of 90 min sessions, using a sequential method. The assessors had not eaten since 9 a.m. and the sessions began at 12 noon. Two factors were studied: water ingestion and solid sucrose ingestion every 5 min. The ingestion of water had no effect on discriminative sensitivity. However, the ingestion of solid sucrose had an effect; it maintained discriminant ability in contrast to the protocol without ingestion. The reason for this effect of sucrose is not known; it might be metabolic, hedonic or perhaps something else.

Notes: A consumer panel comprising 60 females evaluated three coffee whiteners and fresh milk for a number of sensory attributes including liking of flavor and appearance, overall liking, natural flavor and odor, creaminess and watery flavor. Fourier analysis was used to produce a plot which readily identified the whiteners with sensory profiles most similar to fresh milk. More importantly, the whitener whose sensory profile was significantly different from that of fresh milk was identified from the Fourier plot, something which could not be done by traditional univariate methods or even by an overall discriminant analysis.

Notes: Matrix matching techniques such as Generalized Procrustes Analysis (GPA) always produce a matrix of maximal agreement which can then be used to graphically represent samples in “consensus plots”. The degree to which the consensus plots produced by GPA on sensory data (such as that obtained from free choice profiling) actually give a picture of true consensus among panelists, as opposed to being merely artifacts of the analysis, has not been examined. Using a Monte Carlo approach, a statistical test is defined for qualifying this consensus. Examples of the application of the test to sensory profiling data of fruit flavors are given.

Notes: Australian and Hong Kong Chinese male and female consumers, aged 20 to 30 years, evaluated a range of 16 meat products using two questionnaires. One questionnaire comprised the 12 most frequently used terms by consumers to describe how, when and where they would most often consume each product. The alternate questionnaire for each individual comprised free choice vocabulary. Half of each consumer sample used the consensus questionnaire and the other half, the free choice questionnaire first, and indicated how often they would consume each meat product in the various contexts listed using graphic line scales of 100 mm anchored at either end by the words ‘never’ and ‘always’. After an interval of time, the alternate questionnaire was completed. It was found that similar conclusions can be drawn about major contextual dimensions either by principal component analysis of data from a consensus questionnaire or by generalized Procrustes analysis of data from free choice profiling.

Notes: Previous research on the flavor development of Cheddar cheese has largely concentrated on the chemical changes that occur, and less work has been published on the sensory aspects of cheese maturation. Cheddar cheese was produced under controlled conditions and a quantitative descriptive analysis procedure was developed and used to assess development of sensory properties during maturation. The method was successful and weekly changes were recorded during maturation. The sensory data also grouped cheeses according to commercial classifications such as Vintage, Mature and Medium, and showed how these were related to a reduced fat cheese. The quantitative descriptive analysis procedure could be used in conjunction with other chemical and physical methods to help in gaining a full understanding of the maturation process in Cheddar cheese.

Notes: The protein kinase C (PKC) family comprises calcium- and phospholipid-dependent kinases whose activity is stimulated by diacylglycerol and tumour-promoting phorbol esters such as 12-tetradecanoyl phorbol-13-acetate (TPA). In the Gram-negative bacterium Escherichia coli, functional similarity to PKC was demonstrated in crude extracts by calcium and phospholipid-dependent, TPA-stimulated phosphorylation of a small number of endogenous substrates. Activity was reduced by sphingosine, a known inhibitor of eukaryotic PKC. Structural similarity to PKC was demonstrated in crude and partially purified bacterial extracts by cross-reactivity with several monoclonal antibodies. This revealed isozyme-specific homology between a protein(s) of relative molecular mass 80–85000 in E. coli and the α-and γ-isozymes, but probably not the β-isozyme, of eukaryotic PKC.

Notes: Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4 and MAL6. Each MAL locus is complex consisting of at least three genes: a trans-acting activator, a maltose permease, and maltase. All the MAL loci show homology to each other both at the sequence level as determined by Southern transfer analysis and at the functional level as determined by complementation. We describe the organization of the MAL loci in yeast and the basic features of their regulation. The analysis of MAL has contributed to our understanding of the evolution of multigenic families. The global integration of carbohydrate metabolism, and gene regulation.

Notes: Both ATP and an electrochemical potential play roles in translocating proteins across the Inner membrane of Escherichia coli. Recent discoveries have dissected the overall transmembrane movement into separate subreactions with different energy requirements, identified a translocation ATPase, and reconstituted both energy-requiring steps of the reaction from purified components. A more refined understanding of the energetics of this fundamental process is beginning to provide answers about the basic issues of how proteins move across the hydrophobic membrane barrier.

Notes: Electrotransformation of Rhodococcus fascians by non-replicating plasmids containing a suitable resistance marker resulted in stable transformants by integration of these constructs at various sites in the genome, thereby generating different mutations. Tagged genes could be isolated in Escherichia coli owing to the presence of a CoIE1 replicon and an ampicillin resistance gene in the inserted sequences.Southern analysis and nucleotide sequencing revealed that recombination can occur at defined locations in the plasmid, while no site preference for target sequences could be detected. Low homology between the recombining sequences indicates illegitimate recombination. The specificity of the plasmid sites could be explained by assuming a linear recombination intermediate, generated by cleavage of the transformed plasmid.

Notes: A group of four genes of Erwinia chrysanthemi involved in pectin degradation has been characterized. These four genes form independent transcription units and are regulated by the negative regulatory gene, kdgR. The functions of two of these genes are known: kduD codes for the 2-keto-3-deoxygluconate oxydoreductase and kdul for the 5-keto-4-deoxyuronate isomerase, two enzymes of the pectin degradation pathway. kdgC has 36% homology with pectate lyase genes of the periplasmic family but its product does not seem to have pectinolytic activity. The fourth gene, kdgF, could have a role in the pathogenicity of E. chrysanthemi. A comparison of the regulatory regions of all the genes controlled by kdgR allowed better definition of the KdgR-binding-site consensus.

Notes: To establish the molecular basis of the chromosomal virulence genes of Shigella flexneri 2a (YSH6000), a Notl restriction map of the chromosome was constructed by exploiting Notl-linking clones, partial Notl digestion and DNA probes from various genes of Escherichia coli K-12. The map revealed at least three local differences in the placements of genes between YSH6000 and E. coli K-12. Using the additional Notl sites introduced by Tn5 insertion, nine virulence loci Identified previously by random Tn5 insertions were physically mapped on the chromosome. To demonstrate the versatility of the Notl map in direct assignment of the virulence loci tagged by Tn5 to a known genetic region in E. coli K-12, the major class of avirulent mutants defective in the core structure of lipopolysaccharide (LPS) was examined for the sites of Tn5 insertions. The two Notl segments created by the Tn5 insertion in the Notl fragment were analysed by Southern blotting with two DNA probes for the 5′ and 3’flanking regions of the rfa region, and shown to hybridize separately with each of them, confirming the sites of Tn5 in the rfa locus. This approach will facilitate direct comparison of genetically mapped Tn5 insertion mutations of S. flexneri with genes physically determined in E. coli K-12.

Notes: The O antigen of Escherichia coli O111 is identical in structure to that of Salmonella enterics serovar adelaide. Another O-antigen structure, similar to that of E. coli O111 and S. enterica serovar adelaide is found in both E. coli O55 and S. enterica serovar greenside. Both O-antigen structures contain colitose, a 3,6 dideoxyhexose found only rarely in the Enterobacteriaceae. The O-antigen structure is determined by genes generally located in the rfb gene cluster. We cloned the rfb gene cluster from an E. coli O111 strain (M92), and the clone expressed O antigen in both E. coli K-12 and a K-12 strain deleted for rfb. Lipopoly-saccharide analysis showed that the O antigen produced by strains containing the cloned DNA is polymerized. The chain length of O antigen was affected by a region outside of rfb but linked to it and present on some of the plasmids containing rfb. The rfb region of M92 was analysed and compared, by DNA hybridization, with that of strains with related O antigens. The possible evolution of the rfb genes in these O antigen groups is discussed.

Notes: A 2 kb DNA fragment isolated from a cosmid library of Aquaspirillum magnetotacticum strain MS-1 complements the aromatic-metabolite requirements and iron-uptake deficiencies of Escherichia coli and Salmonella typhimurium strains that lack a functional aroD (biosynthetic dehydrodquinase) sequence. All recombinant cosmids selected for their aroD complementation property carry this sequence. No DNA sequence homology has, however, been detected by Southern hybridization between the cloned fragment and the aroD gene of E. coli or the qa2 (catabolic dehydroquinase) gene of Neurospora crassa.

Notes: Evidence for ptelotropic activation of virulence genes in Listeria monocytogenes is presented. A complementation study of a spontaneous prfA-deletion mutant and analysis of cassette and transposon insertion mutants showed that the gene prfA activates the transcription of four independent genes which code for a phosphatidyl-inositol-specific phospholipase C (gene plcA), listeriolysin O (gene hlyA), a metallo-protease (gene prtA) and a lecithinase (gene prtC). Transcription of prfA is not constitutive. During the growth phase, two peaks of prfA transcript accumulation were observed: the first was during exponential growth, and the second was at the beginning of the stationary phase. In addition, two prf4-specific transcripts of 2.2 kb and 1 kb are detected. Early in exponential growth, prfA is co-transcribed with plcA which lies upstream prfA, giving rise to the 2.2 kb plcA-prfA transcript. In late-exponential growth and at the beginning of the stationary phase, prfA transcripts of 1 kb are predominantly detected. Our results demonstrate that since prfA controls plcA transcription, it also regulates its own synthesis.

Notes: It is now well documented that some enteric bacteria which cause diarrhoeal and/or dysenteric disease produce, at high levels, one or more of a family of protein toxins referred to as Shiga toxin and Shiga-like toxins (SLTs; alternatively called verocytotoxins or VTs). Within the past few years, there have been considerable advancements made in our understanding of the biochemistry and molecular biology of Shiga toxin and SLTs. However, the precise role of the toxins in mediating colonic disease, as well as their contribution to the development of extra-intestinal sequelae (e.g. the haemolytic uraemic syndrome and neurological disorders), remain less clear. In this MicroReview, we will briefly summarize recent progress in Shiga toxin- and SLT-related research and present evidence supporting the concept that these toxins contribute to pathogenesis by directly damaging vascular endothelial cells, thereby disrupting the homeostatic properties of these cells. We will also discuss data which suggest that toxin-mediated damage in the kidney may not be limited to glomerular endothelial cells but may include tubular epithelial cells. Thus, the role of the toxins in renal disease may not be limited to the glomeruli, as was initially hypothesized when the association of infection with toxin-producing strains and the development of acute renal failure was established.

Notes: Retrotransposons are a widely distributed group of eukaryotic mobile genetic elements that transpose through an RNA intermediate. The element Ty (Transposon yeast), found in the yeast Saccharomyces cerevisiae, is a model system for the study of retrotransposons because of the experimental tools that exist to manipulate and detect transposition. Ty transposition can be elevated to levels exceeding one transposition event per cell when an element is expressed from an inducible yeast promoter. In addition, individual genomic Ty elements can be tagged with a retrotransposition indicator gene that allows transposition events occurring at at a rate of 10−5 to 10−7 per element per cell division to be detected phenotypically. These systems are being used to elucidate the mechanism of Ty transposition and clarify how Ty transposition is controlled.

Notes: Oligonucleotide primers derived from the ipaH7.8 sequence have been used to determine the boundaries of DNA sequence homology among five lpaH genes on the invasion plasmid (pWR100) of Shigella flexneri 5, strain M9OT-W. The primary structure of lpaH4.5 has been established from DNA sequence analysis. The first 197 amino acids in lpaH7.8 were replaced in lpaH4.5 by a unique set of 251 amino acids, generating two related proteins with variable and conserved sequences. The amino-terminal region of lpaH4.5 displayed an internal repeat structure, also seen in lpaH7.8, characteristic of members of the leucine-rich glycoprotein (LRG) family. The DNA sequences of ipaH2.5 and ipaH1.4 indicate that these genes are truncated versions of lpaH7.8. Western blot analysis of a λgt11 ipaH recombinant (W7) subclone demonstrated that the antigenicity of lpaH7.8 resides outside the leucine-rich repetitive region.

Notes: The meta operon of the Pseudomonas putida TOL plasmid (pWW0) encodes all enzymes of a meta-cleavage pathway for the metabolism of benzoic acids to Krebs-cycle intermediates. We have determined and analysed the nucleic acid sequence of a 3442 bp region of the meta operon containing the xylGFJ genes whose products are involved in the post meta-ring fission transformation of catechols. Homology analysis of the xylGFJ gene products revealed evidence of biochemical relatedness, suggested enzymatic mechanisms, and permitted us to propose evolutionary events which may have generated the current variety of aromatic degradative pathways.The xylG gene, which specifies 2-hydroxymuconic semialdehyde dehydrogenase (HMSD), was found to encode a protein of 51.7 kDa. The predicted protein sequence exhibits significant homology to eukaryotic aldehyde dehydrogenases (ADHs) and to the products of two other Pseudomonas catabolic genes, i.e. xylC and alkH. Expansion of the ADH superfamily to include these prokaryotic enzymes permitted a broader analysis of functionally critical ADH residues and phylogenetic relationships among superfamily members. The importance of three regions of these enzymes previously thought to be critical to ADH activity was reinforced by this analysis. However glutamine-487, also thought to be critical, is less well conserved. The revised ADH phylogeny proposed here suggests early catabolic ADH divergence with subsequent interkingdom gene exchange.The xylF gene, which specifies 2-hydroxymuconic semialdehyde hydrolase (HMSH), was delineated by N-terminal sequence analysis of the purified gene product and is shown to encode a protein of 30.6 kDa. Homology analysis revealed sequence similarity to a chromosomally encoded serine hydrolase, especially in the region of the previously identified active-site serine residue, suggesting that HMSH may also possess a serine hydrolytic enzymatic mechanism.Likewise, the xylJ gene, which specifies 2-hydroxypent-2,4-dienoate hydratase (HPH), was delineated by N-terminal sequence analysis of purified HPH, and was found to encode a 23.9 kDa protein.Sequence comparisons revealed that both HMSH and HPH have analogues in the tod gene cluster, which specifies a toluene/benzene degradative pathway. Although the newly identified todF and todJ genes had been at least partially sequenced (Zylstra and Gibson, 1989), the open reading frames had not been positively identified. The presence of todJ provides strong evidence that the reactions following ring fission in the tod pathway are identical to those of the TOL pathway.The meta operon is one of the longest prokaryotic operons known. The structure of the xylGFJ genes displays short intergenic regions and overlapping translational signals. This genetic organization suggests a mechanism of post-transcriptional regulation whereby translational competency of downstream genes might be enhanced and lengthy transcripts stabilized against ribonucleolytic attack. Such an operon structure may have evolved to facilitate adequate expression of promoter distal genes of long operons.

Notes: Vibrio cholerae is known to secrete DNase(s) into the extracellular environment. These proteins have been thought to be responsible for the difficulties in transforming this organism. In this work we demonstrate that the dns and xds genes differ and that their products are solely responsible for the extracellular DNase activity. By site-directed mutagenesis, strains have been constructed which are mutant in one or both genes. These strains have been assessed for their ability to be transformed with plasmid DNA and for their virulence in the infant mouse cholera model. DNase-deficient mutants can be readily transformed and the product of dns appears to be the more significant barrier. No effect on virulence was observed with the mutants.

Notes: As an additional system for analysing mutations that appear to be specifically induced or directed, we have used a plasmid that contains the mnt repressor gene inserted as an operon fusion with the tet gene of the plasmid pBR322. Thus, the mnt gene product acts as a negative transcriptional regulator of tet gene expression. Mutations inactivating the Mnt repressor are recessive while those destroying operator recognition (Oc) are dominant in conferring tetracycline resistance on the host. When resistance mutations were isolated on plates with high levels of tetracycline they were preferentially mnt- and the plasmids were monomers. Pre-exposure to low concentrations increased the frequency of resistant mutants by 100- to 1000-fold, and the mutations were now mostly Oc, located on one unit of a plasmid multimer. Recessive repressor mutations on one unit would not have been selected. We suggest that the high frequency of mutation in tandem multimeric plasmids may be caused by the formation of single-stranded and hence highly mutable regions by homologous pairing out of register. The role of tetracycline in promoting mutations is discussed.

Notes: At the onset of starvation Escherichia coli undergoes a temporally ordered program of starvation gene expression involving 40–80 genes which some four hours later yields cells possessing an enhanced general resistance. Two classes of genes are induced upon carbon starvation: the cst genes, requiring cyclic AMP, and the pex genes, not requiring this nucleotide for induction. The cst genes are not involved in the development of the resistant state and are concerned with escape from starvation, while the pex gene induction appears to be associated with resistance. Many of the latter are induced in response to a variety of starvation conditions. They include heat shock and oxidation resistance genes, and some utilize minor, stationary-phase-specific sigma factors for induction during starvation. The protective role of stress proteins may be due to their ability to rescue misfolded macromolecules. The starvation promoters can be potentially useful for selective expression of desired genes in metabolically sluggish populations, e.g. in high-density industrial fermentations and in situ bioremediation.

Notes: During carbon-starvation-induced entry into stationary phase, Escherichia coli cells exhibit a variety of physiological and morphological changes that ensure survival during periods of prolonged starvation. Induction of 30–50 proteins of mostly unknown function has been shown under these conditions. In an attempt to identify C-starvation-regulated genes we isolated and characterized chromosomal C-starvation- induced csi::lacZ fusions using the λplac Mu system. One operon fusion (csi 2::lacZ) has been studied in detail. csi 2::lacZ was induced during transition from exponential to stationary phase and was negatively regulated by cAMP. It was mapped at 59 min on the E. coli chromosome and conferred a pleiotropic phenotype. As demonstrated by two-dimensional gel electrophoresis, cells carrying csi 2::lacZ did not synthesize at least 16 proteins present In an isogenic csi 2+ strain. Cells containing csi 2::lacZ or csi 2::Tn10 did not produce glycogen, did not develop thermo-tolerance and H2O2 resistance, and did not induce a stationary-phase-specific acidic phosphatase (AppA) as well as another csi fusion (csi5::lacZ). Moreover, they died off much more rapidly than wild-type cells during prolonged starvation. We conclude that csi 2::lacZ defines a regulatory gene of central importance for stationary phase E. coli cells. These results and the cloning of the wild-type gene corresponding to csi 2 demonstrated that the csi 2 locus is allelic with the previously identified regulatory genes katF and appR. The katF sequence indicated that its gene product is a novel sigma factor supposed to regulate expression of catalase HPII and exonuclease III (Mulvey and Loewen, 1989). We suggest that this novel sigma subunit of RNA polymerase defined by csi 2/katF/appR is a central early regulator of a large starvation/stationary phase regulon in E. coli and propose ‘rpoS’ (‘σs’) as appropriate designations.

Notes: Rhizobium leguminosarum biovar phaseol CFN23 loses its ability to nodulate beans at a high frequency because of a deletion of part of its symbiotic (pSym) plasmid (Soberón-Chávez et al., 1986). We report here that at least 80kb of pSym are deleted upon loss of the symbiotic phenotype; the deletion removes the nitrogenase structural nifHDK and the common nod ABC genes. The size of the deleted pSym is not reduced, since it is accompanied by an amplification of other pSym plasmid sequences. This genetic rearrangement is similar to the deletion and amplification of yeast mitochondrial DNA leading to‘petite’mutations.

Notes: The 58/59 min region of the Escherichia coli chromosome contains two divergently oriented gene clusters coding for proteins with a function in hydrogenase formation. One cluster (the hyc operon), transcribed counterclockwise with respect to the E. coli chromosome, codes for gene products with a structural role in hydrogenase 3 formation (Böhm et al., 1990). The nucleotide sequence of the divergently transcribed operon (hyp) has been determined. It contains five genes, all of which are expressed in vivo in a T7 promoter/polymerase system, and the sizes of the synthesized products correspond with those predicted from the amino acid sequence. Complementation analysis of previously characterized mutants showed that the hypB, hypC and hypD genes have a function in the formation of all three hydrogenase isoenzymes, lesions in hypB being complemented by high nickel ion concentration in the medium. Prevention of hypBCDE gene expression led to an altered electrophoretic pattern of hydrogense 1 and 2 constituent subunits, indicating increased chemical or proteolytic susceptibility. Under fermentative growth conditions, operon expression was governed by an NtrA-dependent promoter lying upstream of hypA working together with an fnr gene product-dependent promoter which was localized within the hyp A gene. The latter (operon-internal) promoter is responsible for hypBCDE transcription under non-fermentative conditions when the -24/-12 NtrA-dependent promoter upstream of hyp A is silent.

Notes: In order to investigate the basis of functional diversity among the pyridine nucleotide-oxidoreductases the gor gene from Pseudomonas aeruginosa PAO, which encodes glutathione reductase, was analysed. The P. aeruginosa gor gene was identified by hybridization with a short DNA sequence from the gene encoding mercuric reductase in transposon Tn501. The gene was cloned, sequenced and overexpressed in Escherichia coli. Expression of the gene enabled rescue of an E. coli gor- mutant, confirming the identity of the cloned gene. The predicted sequence of the gene product showed homology with other members of the pyridine nucleotide-disulphide oxidoreductase family, and allowed determination of positions that may be involved in substrate specificity. These predictions provided information on the relationship of sequence to function, independently of structural data used in previous studies.

Notes: A 1.3kb cDNA (cDNA52) was derived from Trypanosoma cruzi trypomastigote mRNA. Using single stranded probes in Northern blots, we identified the putative coding strand of cDNA52. In addition, a minor band was detected in RNA from epimastigotes that was absent in RNA from trypomastigotes. Nucleotide sequence analysis revealed that cDNA52 was highly homologous to T. cruzi kinetoplast DNA minicircle sequences. All four conserved regions of T. cruzi minicircles were identified in cDNA52. Using several criteria, we demonstrated that the hybridization signals were not caused by contaminating minicircle DNA in the RNA preparations. The data provide direct evidence for the unprecedented finding that the entire length of a kDNA minicircle is transcribed in T. cruzi.

Notes: Transcription of the Vibrio cholerae hlyA gene, which encodes a cytotoxic haemolysin, has been investigated. The hlyA transcript initiates 430 nucleotides (nt) upstream of the translational start site, hlyA–cat transcriptional fusion constructs were active in V. cholerae but not in Escherichia coli. An hlyA–cat fusion was used to select, from a V. cholerae O17 plasmid library, a clone that could activate the hlyA promoter in E coli. This regulatory locus has been designated hlyU. hlyU appears to be distinct from the previously described hlyR locus.

Notes: The ctaBCDEF genes coding for cytochrome c oxidase were found to reside adjacent to a regulatory gene ctaA at 127° on the Bacillus subtilis chromosome. The structural genes for subunits I and II, ctaD and ctaC, were deleted by gene-replacement using a phleomycin-resistance marker. The mutant was unable to oxidize N,N,N′,N′-tetramethyl-p-phenylene-diamine and oxidized cytochrome c at a significantly lower rate. Absorption spectra of the mutant and wild-type membranes confirmed the presence of two haem A-containing enzymes in B. subtilis. Another mutant, with a spontaneous deletion upstream from CtaC, was found to express neither of these enzymes. Radioactive haem-labelling was used to identify sub-unit ii, which contains a haem C, and cytochrome c-550 among the membrane-bound c-type cytochromes of B. subtilis.

Notes: Synthesis of the capsular polysaccharide colanic acid in Escherichia coli K12 Is regulated by a complex network of regulatory proteins. This regulation is expressed at the level of transcription of the cps (capsular polysaccharide synthesis) genes. Two positive regulators, RcsA and RcsB, are necessary for maximal capsule expression. The availability of RcsA is normally limited because the RcsA protein is rapidly degraded by the Lon ATP-dependent protease. Therefore Lon acts, indirectly, as a negative regulator of capsule synthesis. The sequence predicted for RcsB suggests that it is the effector component of a two-component system; a protein with homology to sensors, RcsC, also plays a role in capsule regulation. We propose a model for capsule synthesis in which RcsA interacts with RcsB to stimulate transcription of the cps genes. The mechanism of regulation of colanic acid synthesis in E. coli may apply to other capsules in a variety of Gram-negative bacteria.

Notes: Filamentous phages present a genetically well-defined system for studying the ordered membrane assembly of five different phage-encoded proteins around the circular single-stranded DNA phage genome. Assembly occurs at high efficiency in vivo, catalysed by two phage-encoded membrane proteins and al least one host protein, thioredoxin. This review presents a description of the virion and its cytoplasmic precursor and summarizes the results of genetic and biochemical experiments that are beginning to elucidate the rote of the three morphogenetic proteins. The recent discovery of bacterial transport proteins with homology to a phage morphogenetic protein located in the outer membrane suggests the existence of a common mechanism for moving complex macromolecules across bacterial membranes.

Notes: The chromosomal gef gene of Escherichia coli is a member of the gef gene family which encodes strongly toxic proteins of about 50 amino acids. We demonstrate here that the Gef protein is detectable by anti-peptide antibodies. Furthermore, we show that Gef is anchored in the cytoplasmic membrane by the N-terminal part of the protein, and that the C-terminal part is localized in the periplasm in a dimeric form with at least one disulphide bond. By mutagenesis of gef it is shown that the periplasmic portion of Gef encodes the toxic domain and that the dimerization of Gef is not essential for the toxic effect.

Notes: We have cloned a gene encoding a polygalacturonase (PG) in the filamentous fungus Aspergillus niger RH5344. The structural gene comprises 1141 bp codIng for 362 amino acids and the open reading frame is disrupted by one intron of 52 bp. Eukaryotic consensus sequences for transcription regulation are found only in deviated forms. The biological functionality of the isolated PG gene was established by retransformation in A. niger and Aspergillus awamori. In addition, we have found that the PG protein of A. niger shares significant similarities with PG proteins from tomato and Erwinia carotovora. Comparison of the three enzymes revealed a highly conserved region in their C-terminal region probably comprising the elements of substrate binding and the catalytic centre.

Notes: The plasmid pBRD026, which directs expression of the B subunrt of the Escherichia coli heat-labile toxin (LTB), was modified so that DNA encoding epitopes could be inserted at the 3 end of the gene. An oligonucleotide linker containing restriction sites for BglH and Spel was inserted at the Spel site at the 3′ end of the LTB gene to form plasmid pFV1. This linker also encodes the amino acid sequence Gly–Pro–Gly–Pro which we propose acts as a ‘hinge’ between the LTB and the foreign epitope. Oligonucleotides specifying an epitope from the Bordetelia pertussis P.69 outer membrane protein were cloned into pFV1 to form pFV169. The resultant fusion protein (LTB69) was partially purified from the periplasm of E. coli strains in a soluble pentameric form which could bind GM1 gangliosides. Mice immunized intranasally with purified LTB69 produced antibodies against both LTB and the P.69 protein. In addition, ELISPOT assays demonstrated the presence of LTB-specific and P.69-specific antibody-secreting cells in the lungs of immunized mice.

Notes: Class 5 outer membrane proteins of Neisseria meningitidis show both phase- and antigenic variation of expression. The proteins are encoded by a family of opa genes that share a conserved framework interspersed with three variable regions, designated the semivariable (SV) region and hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of all of the opa genes of meningococcal strain FAM18, to assess the structural and antigenic variability in the family of proteins made by one strain. Pulsed field electrophoresis and Southern blotting showed that there are four opa genes in the FAM18 chromosome, and that they are not tightly clustered. DNA sequence analysis of the four cloned genes showed a modest degree of diversity in the SV region and more extensive differences in the HV1 and HV2 regions. There were four versions of HV1 and three versions of HV2 among the four genes. Each of the FAM18 opa loci contained a gene with a unique combination of SV, HV1, and HV2 sequences. We used λgt11 cloning and synthetic peptides to demonstrate that HV2 sequences completely encode the epitopes for two monoclonal antibodies specific for different class 5 proteins of FAM18.

Notes: The population of capsulate Haemophilus influenzae is divided into two phylogenetic divisions. Here we show that in division I strains the capsulation (cap) gene cluster lies between direct repeats of a novel insertion sequence (IS)-like element, IS 1016. cap has apparently been mobilized in the chromosome as a compound transposon by IS 1016, and the repeats have provided a molecular substrate for reversible cap gene amplification, with augmentation of capsule production, through unequal homologous recombination. Such amplification has occurred in serotype b strains, but in these a large direct repeat of cap genes has become fixed in the population. We have found a 1.2kb deletion at one end of this duplicated cap b locus, removing most of one copy of the polysaccharide export gene bexA. We have shown that this makes capsulation dependent on preservation of the direct repeat structure in order to avoid recombination-mediated loss of the other copy of bexA. Type b strains with this cap configuration are disseminated worldwide and currently cause nearly all invasive Haemophilus infections, leading us to speculate that the 1.2 kb deletion occurred in an ancestral type b strain and conferred significant biological advantage.

Notes: Genetic studies have identified a number of genes whose products appear to be required for the transport of the group A colicins and the single-stranded DNA of certain filamentous bacteriophages into Escherichia coli. Mutations in these genes allow normal binding of the colicins to their outer-membrane receptors and of the bacteriophage to the tip of specific conjugative pili, but do not allow translocation of the macromolecules to their target. These mutations have been designed‘tolerant’(tol) mutations and the protein products specified by these genes appear to comprise part of a transport system known as the Tol import system. Some of these genes have been isolated, sequenced and their protein products localized to the membranes or periplasm of E. coli. Information is also available regarding the domains of the colicins or phage proteins which interact with the Tol proteins. A preliminary model of the location and possible interactions of the Tol proteins is presented.

Notes: Previous work in our laboratory suggested that DNA topology could be implicated in the regulation of the division gene ftsZ. To settle this question, we have selected and characterized mutants in the gyrB gene able to phenotypically suppress the defects of the ftsZ84 mutation. No strict correlation was found between the degree of plasmid DNA relaxation and the level of suppression of the thermosensitivity of the ftsZ84 strain. Interestingly, the class of mutants that shows maximal suppression is substantially unaffected in DNA topology. In addition, the amount of ffsZ-specific mRNA in this class of mutants is comparable to that present in the ftsZ84 strain. These results hint that the ability of these gyrB mutants to correct the effects of the ftsZ84 mutation is largely unrelated to the function of the GyrB (as a part of DNA gyrase) in the control of DNA superhelicity and suggest hitherto unsuspected interaction between the ftsZ and gyrB gene products.

Notes: A previous study in our laboratory identified a surface-exposed peptidoglycan-associated protein of Neisseria gonorrhoeae which had an apparent molecular mass of 44000 daltons (44kDa) (Hill and Judd, 1989). This paper reports results which confirm that the 44 kDa protein is surface-exposed, and that the protein is expressed in, and is structurally invariant among, 14 strains of N. gonorrhoeae. The fact that the 44kDa outer-membrane protein is found in a conserved form in all gonococci examined strongly suggests that ft is crucial to the bacterium's survival. Moreover, it appears that this protein is a penicillin-binding protein (PBP3) (Shafer and Judd, 1991). This invariant, surface-exposed, peptidoglycan-associated outer-membrane protein deserves further investigation to elucidate its role in the immunobiology of N. gonorrhoeae, and its possible use as an immunoprophylactic reagent.

Notes: A 34 kb fragment of the Nocardia lactamdurans DNA carrying the cluster of early cephamycin biosynthetic genes was cloned in λ EMBL3 by hybridization with probes internal to the pcbAB and pcbC genes of Penicillium chrysogenum and Streptomyces griseus. The pcbAB and pcbC genes were found to be closely linked together in the genome of N. lactamdurans. The pcbAB gene of N. lactamdurans showed the same orientation as the pcbC gene, in contrast to the divergent expression of the genes in the pcbAB-pcbC cluster of P. chrysogenum and Acremonium chrysogenum. The pcbAB gene encodes a large (3649 amino acids) multidomain δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase with a deduced Mr, of 404134. This enzyme contains three repeated domains and a consensus thioesterase active-site sequence. The pcbC gene encodes a protein of 328 amino acids with a deduced Mr of 37469, which is similar to other isopenicillin N synthases except that it lacks one of two cysteine residues conserved in all other isopenicillin N synthases. The different organization of the pcbAB-pcbC gene cluster in N. lactamadurans and Streptomyces clavuligerus relative to P. chrysogenum and A. chrysogenum is intriguing in relation to the hypothesis of horizontal transference of these genes from actinomycetes to filamentous fungi by a single transfer event.

Notes: Selenocysteyl-tRNAs that decode UGA were identified previously in animal and bacterial cells and the genes for these tRNAs have been shown to be widespread in animals and eubacteria. In the present study, we identify a selenocysteyl-tRNA that codes for UGA in Thalassiosira pseudonana, which is a diatom, and in Tetrahymena borealis, which is a ciliate. The fact that these very diverse unicellular organisms also contain a selenocysteyl-tRNA suggests that selenocysteine-containing proteins and the use of UGA as a codon for selenocysteine are widespread, if not ubiquitous, in nature.

Notes: OmpC, a major outer-membrane protein, is highly expressed when Salmonella typhi is grown in nutrient broth (NB) of either low (NB + 0% sucrose) or high (NB + 20% sucrose) osmolarity. This contrasts with the expression of Escherichia coli OmpC, which is inhibited in low osmolarity and enhanced in high osmolarity, as has been described previously (van Alphen and Lugtenberg, 1977; Verhoef et al., 1979; Kawaji et al., 1979). Nevertheless, expression of S. typhi OmpC is dependent on the E. coli OmpR transcriptional activator. These findings suggest differences between the mechanisms of osmoregulation of gene expression in both bacteria, although common effectors appear to be shared.

Notes: Glucose is taken up in Bacillus subtilis via the phosphoenolpyruvate:glucose phosphotransferase system (glucose PTS). Two genes, orfG and ptsX, have been implied in the glucose-specific part of this PTS, encoding an Enzyme IIGlc and an Enzyme IIIGlc, respectively. We now show that the glucose permease consists of a single, membrane-bound, polypeptide with an apparent molecular weight of 80000, encoded by a single gene which will be designated ptsG. The glucose permease contains domains that are 40-50% identical to the IIGlc and IIIGlc proteins of Escherichia coli. The B. subtilis IIIGlc domain can replace IIIGlc in E. coli crr mutants in supporting growth on glucose and transport of methyl α-glucoside.Mutations in the IIGlc and IIIGlc domains of the B. subtilis ptsG gene impaired growth on glucose and in some cases on sucrose. ptsG mutants lost all methyl α-glucoside transport but retained part of the glucose-transport capacity. Residual growth on glucose and transport of glucose in these ptsG mutants suggested that yet another uptake system for glucose existed, which is either another PT system or regulated by the PTS.The glucose PTS did not seem to be involved in the regulation of the uptake or metabolism of non-PTS compounds like glycerol. In contrast to ptsl mutants in members of the Enterobacteriaceae, the defective growth of B. subtilis ptsl mutants on glycerol was not restored by an insertion in the ptsG gene which eliminated IIGlc. Growth of B. subtilis ptsG mutants, lacking IIGlc, was not impaired on glycerol. From this we concluded that neither non-phosphorylated nor phosphorylated IIGlc was acting as an inhibitor or an activator, respectively, of glycerol uptake and metabolism.

Notes: In the life cycle of a bacterium there are several key processes: cellular growth, chromosome replication and decatenation, nucleoid partition, septum formation, and cell division. These processes have to be carefully controlled and co-ordinated both with respect to each other and to the growth of the cell, and could be viewed as parts of a single cycle in which each step is dependent upon the previous one. Alternatively, they could be independently controlled and carefully tuned to each other without actually constituting a true cycle. In this review, using Escherichia coli as model system, we discuss these two ways of describing the bacterial life cycle. The evidence supporting independent control of the processes is presented, and some of the key questions in the elucidation of the regulation of the bacterial life cycle are discussed.

Notes: Calcium and calcium-binding proteins including those resembling calmodulin are implicated in numerous diverse processes in bacteria. These processes include chemotaxis, sporulation, virulence, the transport of sugars and proteins, phosphorylation, heat shock, the initiation of DNAS replication, septation, nucleoid structure, nuclease activity and recombination, the stability of the envelope, and phospholipids synthesis and configuration. That such varied processes should have a common factor, calcium, suggests major underlying principles of calcium metabolism metabolism which have yet to be discovered.

Notes: We describe a family of highly conserved, Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences, 14 of which have been identified in Escherichia coli and Salmonella typhimurium and a further three in other enterobacterial species (Yersinia pseudotubercuiosis, Kiebsiella pneumoniae and Vibrio cholerae). ERIC sequences are 126 bp long and appear to be restricted to transcribed regions of the genome, either in intergenic regions of polycistronic operons or in untranslated regions upstream or downstream of open reading frames. ERIC sequences are highly conserved at the nucleotide sequence level but their chromosomal locations differ between species. Several features of ERIC sequences resemble those of REP sequences (Stern et al., 1984) although the nucleotide sequence is entirely different. The question of whether ERICs have a specific function, or represent a form of 'selfish’DNA, is discussed.