Phosphoribosylpyrophosphate synthetase superactivity. A study of five patients with catalytic defects in the enzyme.

Superactive phosphoribosylpyrophosphate (PRPP) synthetases were characterized in fibroblasts and erythrocytes from 5 unrelated men with gout and/or hyperuricemia and uric acid overproduction. The kinetic basis of enzyme superactivity in all patients was increased maximal reaction velocity. Affinities of the enzymes for substrates and activators and responsiveness to inhibitors were normal, and levels of immunoreactive enzyme in patient and control fibroblast and erythrocyte extracts were comparable. Enzymes purified to homogeneity from 2 patients confirmed the presence of isolated catalytic defects. Altered physical properties of certain of the superactive enzymes suggested the presence of several distinctive structural defects among the aberrant forms. Fibroblasts from each affected patient showed increased PRPP concentration and generation, as well as accelerated rates of all PRPP-requiring purine nucleotide synthetic pathways. These findings support the concept that enzyme superactivity results in uric acid overproduction as a consequence of increased rates of PRPP and purine nucleotide synthesis. Cultured cells from female relatives of 2 patients showed evidence for the heterozygous carrier state, as measured both by enzyme activities and by rates of PRPP and purine synthesis. The clinical phenotype in 4 patients was limited to early adult-onset gout and its consequences, whereas the fifth patient expressed a familial constellation of hyperuricemia, sensorineural deafness, ataxia, and renal insufficiency. The severity of the derangements in PRPP synthetase and in PRPP and purine synthesis in cells from the 5 patients, however, was comparable. The neurologic accompaniments of enzyme superactivity found in 1 family described here, and in 2 others described previously, thus may not necessarily be consequences of primary defects in PRPP synthetase.

Improved enrichment for recovery of Shigella sonnei from foods.

Shigella species were recovered from foods by the procedure described in the Bacteriological Analytical Manual, 5th Ed. The method is effective if Shigella species are present at about 10(6) cells/g. A 25 g food portion was incubated in Gram-negative (GN) and selenite cystine broths for 16 h at 35 degrees C and streaked onto MacConkey, Levine's eosin methylene blue, desoxycholate citrate, and xylose lysine desoxycholate agars. S. sonnei cells were recovered quantitatively at 44.5 degrees C, and along with other Shigella species, were grown with Escherichia coli in a tryptone broth under anaerobic conditions. Shigella species were also grown in a mixed microflora from foods. S. sonnei cells were inoculated into an enrichment broth containing 20 g tryptone, 2 g K2HPO4, 2 g KH2PO4, 1 g glucose, 5 g NaCl, 1.5 mL Tween 80, and 0.5 mg novobiocin/L (pH 7.0) and incubated for 20 h at 44 degrees C. Enrichments were streaked onto MacConkey agar and the plates were incubated 20 h at 35 degrees C. Suspect Shigella colonies were screened in glucose, tryptone, and lysine broths and in triple sugar iron and motility agars. The sensitivity varied from 0.3 to 1000 bacteria/g. The method has been examined with artificially inoculated lettuce, celery, brussels sprouts, mushrooms, and hamburger. It is also applicable to S. flexneri if incubation is conducted at 42 degrees C.

Corticosteroid hormonal influence on cranial computerized tomography: observations in the Rhesus monkey.

Six adult male Rhesus monkeys (T), treated with 0.5 mg/kg dexamethasone for 106 days, and 6 weight-watched controls (U), were studied postmortem with pathologic examination and cranial computerized tomography (CT). Cortical atrophy, as defined by perisulcal atrophy and ventricular dilatation, was found in 50% of each group, but marked changes were noted only among T. Adrenal, brain, and body weights were all lower among T (p less than 0.05). No significant histopathologic abnormalities were found. Our data suggest that CT changes of cortical atrophy may be caused by corticosteroid hormones alone, but are inconsistent and probably dependent upon individual susceptibility to the effects of corticosteroids.

Detection of invasiveness of mammalian cells by Escherichia coli: collaborative study.

In a collaborative study of 2 standard procedures (Procedure I, presented in J. Assoc. Off. Anal. Chem. 60, 546-562 (1977) and Procedure II in the Bacteriological Analytical Manual, 1978), 7 laboratories evaluated the invasive potential of Escherichia coli. Monolayers of HeLa cultures were infected with E. coli (infectivity ratio 100 bacteria/mammalian cell) suspended in 2 menstrua: heat-inactivated fetal bovine serum (Procedure I) and 0.2% bovine albumin Fraction V dissolved in Earle's buffered salts (Procedure II). After uptake of the bacteria, intracellular growth, and differential staining, the percentages of HeLa cells containing a minimum of 5 bacteria were determined microscopically. All laboratories correctly identified invasiveness of E. coli by Procedure I; 5 of 6 laboratories recognized invasiveness by Procedure II. Although Procedure I was more reproducible than Procedure II, repeatability did not differ significantly. Procedure I was adopted official first action for presumptive recognition of invasiveness in E. coli.

Improved growth medium for Campylobacter species.

Campylobacter species were grown in a base containing proteose peptone no. 3, yeast extract, K2HPO4, (NH4)2SO4, NA2SO3, soluble starch, and agar. Concentrations and sources of organic nitrogen and growth factors were critical, and the optimal pH range was 7.0 to 7.5. Cultures tolerated 0.7% NaCl in addition to the salt present in the organic constituents and were sensitive to surface-active agents at concentrations recommended for enrichment of other gram-negative bacteria. Cultures were maintained on the proposed medium for 1 year with transfer every 2 weeks.

Pituitary and thyroid function in male cynomolgus monkeys.

In four male cynomolgus monkeys, serum thyroxine was 6.2 +/- 0.9 micrograms/dl, and triiodothyronine was 207 +/- 12 ng/dl (mean +/- SE). Kinetic studies using 131I-thyroxine and 125I-triiodothyronine showed that the disappearance of both hormones was non-linear and best fit a biexponential equation. The metabolic clearance rates and production rates for thyroxine were 21.5 +/- 0.6 ml/kg/day and 1.34 +/- 0.23 micrograms/kg/day, respectively, and T1/2(beta) = 29.6 +/- 2.0 hours. For triiodothyronine, the metabolic clearance rate was 156.6 +/- 12.0 ml/kg/day, the production rate was 0.33 +/- 0.04 micrograms/kg/day, and T1/2 (beta) was 13.3 +/- 1.3 hours. Basel serum thyrotropin levels in five euthyroid animals were 1.4 +/- 0.6 microU/ml and increased after thyrotropin-releasing hormone to 6.7 +/- 2.2 microU/ml. Serum prolactin was 5.8 +/- 0.7 ng/ml, and it increased to 26.6 +/- 4.5 ng/ml after thyrotropin-releasing hormone. Four animals received chronic dexamethasone therapy (1 mg twice daily for 5.5 months). While baseline and thyrotropin-releasing hormone stimulated thyrotropin values were lower (0.8 +/- 0.2 microU/ml and 3.2 +/- 0.5 microU/ml, respectively), these reductions were not significant.

Hyperventilating the hypoventilator.

A 65-year-old man had chronic hypoventilation and was demonstrated to have primary neuromuscular disease with major involvement of the thoracic bellows. By use of accessory muscles, he was able to voluntarily hyperventilate and reduce his PCO2 to normal. Hyperventilation gases must be interpreted with care in neuromuscular disease; the ability to reduce PCO2 to normal range does not exclude neuromuscular disease as a cause of chronic respiratory failure.

Alkali method for rapid recovery of Yersinia enterocolitica and Yersinia pseudotuberculosis from foods.

A new culture method employing a potassium hydroxide treatment was compared with the conventional cold enrichment method for efficacy in recovering Yersinia sp. from naturally and artificially contaminated food. The new method increased the yield of Yersinia sp. fourfold and the sensitivity 100-fold, shortened the incubation period, and appreciably decreased the growth of non-Yersinia bacteria from a variety of meats, shellfish, and vegetables.

Methodology for recognition of invasive potential of Escherichia coli.

Surveillance for dysentery-related invasive potential in bacteria using the Sereny keratoconjunctivitis test is restricted by expense, time factor, and necessity for confirmation. Primary screening of isolates in a standardized mammalian cell culture system is recommended. Bacteria are grown 20 hr in veal infusion, washed, and resuspended in 20% heat-inactivated fetal bovine serum (FBS) supplemented with 0.12% brain heart infusion and 0.1% bile salts. The HeLa culture is grown 20 hr as a monolayer in chamber slides with 90% minimal essential medium (MEM)-10% FBS. The host culture is infected at a ratio of 10 bacteria/mammalian cell for 3 hr at 35 degrees C. The infection medium is replaced with MEM-FBS supplemented with 300 microng lysozyme and 5 microng gentamycin/ml. The infected monolayer is incubated 5 hr at 35 degrees C to permit intracellular multiplication. Specimens are washed, fixed with methanol, and stained successively with May-Grunwald and Giemsa dyes. Bacteria occur within the cytoplasm if invasion has occurred. The criterion for a positive test is that 1% of the host cells possesses at least 5 bacteria in 2 of 3 trials. Invasiveness is correlated with and possibly preconditioned by cytotoxic principle(s). Infectivity rates vary from 0 to 30%. The cytopathic effect is noted in 5-50% of HeLa cells. Positive results must be confirmed by the Sereny test.

Enterotoxigenic Escherichia coli isolated from food.

Approximately 8% of 240 isolates of Escherichia coli obtained from food of animal origin in the United States were found to be enterotoxigenic, as determined in adrenal cells, rabbit ileal loops, and assays in infant mice. These organisms were of diverse serotypes that are not included among the so-called enteropathogenic serotypes and would not have been identified by usual laboratory methods. These enterotoxigenic E. coli are of potential importance to public health.

Pathogenicity of Escherichia coli recovered from food.

In Western nations, pathogenic biotypes are sporadically encountered in foodborne gastroentetis. Cholera, dysentery, and chronic ulcerative colitis syndromes are recognized. E. coli has been associated on the average with 2% of annual food outbreaks and 5% of total cases. In developing nations, the incidence may be greater. In contrast with Salmonella and Shigella, significance can be assessed only by fulfillment of Koch's postulates. Preliminary studies using vascular permeability reaction for the detection of heart-labile toxin and the Sereny keratoconjunctivities test for demonstration of invasiveness indicate limited incidence of these pathogenicity markers in cultures from foods. Two per cent of isolates from cheeses involved in recent outbreaks were toxigenic; 14% were invasive. Corresponding values for food isolates not associated with illness were 10 and 0, respectively. To facilitate examination of multiple isolates, 10 may be pooled for the detection of heat-labile toxin, and 5 for invasiveness. Present model pathogenicity systems require standardization, estimation of specificity and sensitivity limits, examination by collaborative study, and ascertainment of human equivalence. Supplemental tests include capacity for colonization of intestinal epithelium and intracellular growth. Stereotypes based upon serology, host range, and recognized toxic factors may require modification.

Microbiological methods. Methodology for enteropathogenic Escherichia coli.

Pathogenic biotypes of Escherichia coli grow poorly at temperatures greatly different from that of the host. Percentages quantitatively recovered at 42.0, 44.0, 44.5, and 45.5 degrees C in lauryl tryptose broth were 100, 76, 76, and 42, respectively. Corresponding values for 175 strains of varied origin were 98, 89, 82, and 65%. Maximal growth temperature is dependent upon medium. Lauryl tryptose and elevated coliform broths were equivalent in the recovery of small inocula (100 cells/ml) at 41.5-44.5 degrees. MacConkey, enteric enrichment, and Gram-negative broths were inhibitory at corresponding values. Growth at elevated temperature in nutrient broth is enhanced by carbohydrate. Standard lactose enrichment media fail to recover slow lactose fermenters. An acidified glutamic acid medium was unsuitable for recovery of E. coli. The data suggest modification of standard temperatures for the recovery of pathogenic biotypes. Previously recommended analytical methods have been simplified and supplemented. The enhancement of motility in indole-nitrite broth at 35 degrees C is recommended. A 4-tube semiquantitative test is offered for tentative identification of somatic and capsular antigens. Inclusion of Alkalescens-Dispar strains is warranted by their pathogenic behavior. Examination in Shigella and Alkalescens-Dispar sera is required to cover the dysentery-like biotypes. Pathogenic potential cannot be inferred from serotype.

Vibrio parahaemolyticus gastroenteritis in Maryland: laboratory aspects.

Vibrio parahaemolyticus was the etiological agent in three food-related epidemics of gastroenteritis in Maryland, during August 1971. These outbreaks involved crab food products. Fifteen isolates of V. parahaemolyticus were made which included 11 from patients and 4 from foods. Serotype 04:K11 was the cause of the outbreaks. It was recovered from patients in each outbreak and gave a positive Kanagawa reaction, an indication of enteropathogenicity. Other patient isolates included types 03:K30, 03:K33, and an untypable isolate, all of which were Kanagawa negative. Food isolates included serotypes 03:K30, 02:K28, and two untypable isolates, all of which were Kanagawa negative. The outbreaks reported in this paper constitute the first confirmed foodborne epidemics due to V. parahaemolyticus in the United States. Methods for the isolation and identification of V. parahaemolyticus are presented, including a procedure for the simple conversion of conventional laboratory media into suitable culture media for this halophilic organism.