Salmonella typhimurium LT2 was grown in batch culture (trypticase soy broth, with 0.3%(w/v) yeast extract, 1% (w/v) glucose and 0.5% (w/v/) NaCl, 20 °C) at a range of initial pH (4.4, 4.8, 5.0 and 7.0). The consumption of oxygen and glucose was found to be independent of initial pH, and stoichiometric with growth. Mean yield coefficients of 6.9×10−15 and 15.5×10−15 mol oxygen/cell were estimated. Calculation of the instantaneous state of carbon during the cultivation showed stoichiometric conversion of glucose into biomass, carbon dioxide and organic acids. The concentration of the undissociated form of the primary acidic product (acetic acid) was shown to be the factor limiting growth.

Succinyl-CoA:3-oxoacid CoA-transferase (SCOT) deficiency is an inborn error of ketone body utilization, characterized by intermittent ketoacidotic crises and persistent ketosis. The diagnosis was suspected in a patient who presented with hypoglycaemia, ketoacidosis and coma at 4 days of age. The hypoglycaemic tendency was only observed during the first month of life. A novel macromolecular labelling assay in cultured skin fibroblasts using D-3-hydroxy[3-14C]butyrate supported the diagnosis. Subsequently, 9% residual SCOT activity and undetectable cross-reactive protein were noted in fibroblasts and the patient was found to be homozygous for the G324E SCOT gene mutation. By 7 years of age, recurrent episodes of ketoacidosis superimposed on persistent hyperketonaemia had resulted in over 25 hospitalizations requiring intravenous fluid, glucose and sodium bicarbonate therapy. He has had normal growth but developmental delay and attention deficit–hyperactivity disorder. A continuous intravenous glucose infusion at 38 μmol (6.8 mg)/kg per min reduced plasma total ketone levels from greater than 1.5 mmol/L to less than 0.5 mmol/L after 48 h. This indicates that patients with SCOT deficiency do not always manifest ketosis with administration of a sufficient amount of carbohydrates, but that even under such conditions hyperketonaemia is difficult to eliminate completely. The presence of hypoglycaemia does not exclude the diagnosis of SCOT deficiency in infancy.

North American Indian childhood cirrhosis (NAIC, or CIRH1A) is an isolated nonsyndromic form of familial cholestasis reported in Ojibway-Cree children and young adults in northwestern Quebec. The pattern of transmission is consistent with an autosomal recessive mode of inheritance. To map the NAIC locus, we performed a genomewide scan on three DNA pools of samples from 13 patients, 16 unaffected siblings, and 22 parents from five families. Analysis of 333 highly polymorphic markers revealed 3 markers with apparent excess allele sharing among affected individuals. Additional mapping identified a chromosome 16q segment shared by all affected individuals. When the program FASTLINK/LINKAGE was used and a completely penetrant autosomal recessive mode of inheritance was assumed, a maximum LOD score of 4.44 was observed for a recombination fraction of 0, with marker D16S3067. A five-marker haplotype (D16S3067, D16S752, D16S2624, D16S3025, and D16S3106) spanning 4.9 cM was shared by all patients. These results provide significant evidence of linkage for a candidate gene on chromosome 16q22.

Stroke-like episodes, defined as periods of acute localized neurological dysfunction during which brain imagery suggests cerebral ischemia but vascular anatomy is normal, occurred in 3 patients with autosomal recessive Saguenay-Lac St-Jean (SLSJ) cytochrome oxidase (COX) deficiency. The patients developed focal neurological deterioration and frontal hypodensities on cerebral computerized tomography (CT). Arteriography, performed in 1 patient during an acute episode, showed normal vascular anatomy. Nevertheless, capillary shunting was evident both in regions that appeared abnormal on the initial cerebral CT study and in regions that appeared normal but subsequently developed Leigh disease. Stroke-like episodes did not exacerbate systemic acidosis, and acidotic decompensations occurred independently of stroke-like episodes. In conclusion, stroke-like episodes occur in autosomal recessively inherited congenital lactic acidoses as well as in those caused by mitochondrial DNA mutations. In some cases, acute localized neurovascular changes occur in regions that subsequently develop Leigh disease. Ann Neurol 1999;45:389–392

We report a 13-year-old girl with nephropathic cystinosis on chronic peritoneal dialysis who presented with two episodes of stroke. Laboratory evaluation showed severe hyperhomocysteinemia (108 mumol/l). Further testing revealed that she was homozygous for the thermolabile variant of the methylenetetrahydrofolate reductase (MTHFR) gene. Treatment with folic acid and vitamin B12 lowered plasma homocysteine to less than 20 mumol/l. No further episodes of stroke occurred over a follow-up of 12 months. Homocysteine levels should be measured in patients with chronic renal failure, since simple and safe treatment with folic acid and vitamin B12 is effective in lowering the plasma homocysteine level in patients with the thermolabile MTHFR allele.

Hormone-sensitive lipase (Lipe) catalyzes both the release of fatty acids from storage triglycerides in adipocytes and the liberation of cholesterol from cholesterol esters in steroidogenic tissues. Lipe activity is regulated in a tissue-, development- and hormone-specific fashion, the latter in large part by serine phosphorylation. We cloned and sequenced the Lipe gene from the 129Sv strain mouse, including 2.7 kb of the 5′ nontranslated region. The primary transcript of the 129Sv Lipe locus spans 9.6 kb and contains 9 exons. We studied the curious hypervariable region immediately 5′ to the regulatory serine residues by aligning the peptide and nucleic acid sequences of mouse, human, and rat Lipe. We propose that much of the variability is attributable to differences in the copy number of a 12-nucleotide repeat that shifts the intron 7 acceptor splice site. Introns 1 and 7 contain B1 elements, which in intron 7 are immediately adjacent to a tetranucleotide repeat. The mouse Lipe promoter region contains numerous potential binding motifs for factors implicated in adipose tissue expression and hormone responsiveness including adipocyte determination- and differentiation-dependent factor 1 (ADD1/SREBP1).

A method is presented for the growth of bacteria on the surfaces of gels as a model of the changes that occur on food surfaces, and is demonstrated forSalmonella typhimurium. The method is based on a gel cassette, which consists of a frame holding a layer of gel, in this case gelatin, formed between two PVC windows. After formation of a sterile gel, one of the PVC windows is removed to expose the gel surface for inoculation. Using this technique, the effect of sucrose and sodium chloride on the growth rate ofS. typhimuriumgrowing as surface colonies was compared with that of immersed colonies and planktonic cells in broth and was found to follow the order: broth>immersed colonies>surface colonies. The maximum numbers of cells decreased with increasing sucrose concentration and this decrease was more marked for surface colonies than for immersed colonies or planktonic cells. These results indicate that predictions of the growth rate of bacteria on food surfaces may be erroneous where these predictions are based on data collected in broth.

The growth of colonies of Salmonella typhimurium derived from single immobilised cells was studied while subjected to constant and sinusoidally-varying temperatures. The bacteria grew in microbiological culture media adjusted to different pH and sodium chloride (NaCl) concentration and solidified with gelatin that was contained within a cassette formed between sheets of PVC film that allowed gaseous exchange. At pH 7.0 and 0.5% (w/v) NaCl and either 12 degrees C or 20 degrees C, S. typhimurium grew at a rate similar to that in liquid medium. The decrease in growth rate at 20 degrees C at a lower pH or higher NaCl concentration was greater in the case of immobilised cells than for cells in liquid medium. The change in the numbers of viable bacteria was measured with time under sinusoidally-varying temperatures between 4 and 22 degrees C and between 12 and 22 degrees C of period in the range 12 to 480 min. The experimental growth curves were compared with predictions based on isothermal growth in liquid medium. The discrepancies between experiment and prediction were greater for gels stressed by NaCl or pH than for gels at pH 7.0 and containing 0.5% (w/v) NaCl, consistent with the isothermal observations.

Water activity is a method of preservation that can affect microbial growth in foods and that may fluctuate during their processing, distribution and storage. Sucrose has been used to change the water activity of microbiological culture media. Suspensions of Salmonella typhimurium LT2 in the exponential phase of growth have been subjected to step changes in sucrose concentration at 20 degrees C. The changes in the numbers of viable bacteria were measured with time and the experimental growth curves compared with predictions based on growth data obtained at constant sucrose concentrations. Steps down in sucrose concentration showed some apparent loss of viability after the step followed by growth at a rate close to the expected value. Steps up in sucrose concentration resulted in a greater apparent loss of viability after the step and either growth or the inducement of lag, depending on the final concentration of sucrose. A series of small steps up in sucrose concentration to 45% (w/v) was able to sustain growth where it was not possible by inoculation directly into this concentration. Improved recovery of bacteria subject to osmotic stress was possible with a medium containing sodium chloride.

Ketone bodies are produced in the liver, mainly from the oxidation of fatty acids, and are exported to peripheral tissues for use as an energy source. They are particularly important for the brain, which has no other substantial non-glucose-derived energy source. The 2 main ketone bodies are 3-hydroxybutyrate (3HB) and acetoacetate (AcAc). Biochemically, abnormalities of ketone body metabolism can present in 3 fashions: ketosis, hypoketotic hypoglycemia, and abnormalities of the 3HB/AcAc ratio. Normally, the presence of ketosis implies 2 things: that lipid energy metabolism has been activated and that the entire pathway of lipid degradation is intact. In rare patients, ketosis reflects an inability to utilize ketone bodies. Ketosis is normal during fasting, after prolonged exercise, and when a high-fat diet is consumed. During the neonatal period, infancy and pregnancy, times at which lipid energy metabolism is particularly active, ketosis develops readily. Pathologic causes of ketosis include diabetes, ketotic hypoglycemia of childhood, corticosteroid or growth hormone deficiency, intoxication with alcohol or salicylates, and several inborn errors of metabolism. The absence of ketosis in a patient with hypoglycemia is abnormal and suggests the diagnosis of either hyperinsulinism or an inborn error of fat energy metabolism. An abnormal elevation of the 3HB/AcAc ratio usually implies a non-oxidized state of the hepatocyte mitochondrial matrix resulting from hypoxia-ischemia or other causes. We summarize the differential diagnosis of abnormalities of ketone body metabolism, as well as pertinent recent advances in research.

The effect of fluctuating temperatures on microbial growth is important in the passage of foods from production to consumption. Suspensions of Salmonella typhimurium have been subjected to sinusoidally time-varying temperatures of periods from 60 to 240 min between 4 degrees and 22 degrees C, that is within and below the growth temperature range. The suspensions were prepared with two concentrations of sodium chloride and adjusted to two different values of pH. The change in the numbers of viable bacteria was measured with time and the experimental growth curves and average generation times compared with predictions based on isothermal growth data. Generally, the experimental average generation times exceeded the predictions by not more than 10%. In enumerating viable bacteria in the suspensions containing 3.5% (w/v) sodium chloride it was necessary to use sodium chloride in the diluent and recovery medium in order to recover the bacteria quantitatively.

3-hydroxy-3-methylglutaryl CoA lyase (HL) is a mitochondrial matrix enzyme which catalyzes the last step of leucine catabolism and of ketogenesis. Autosomal recessive HL deficiency in humans results in episodes of hypoglycemia and coma. We are interested in the pathophysiology of HL deficiency as a model for both amino acid and fatty acid inborn errors. We have cloned the human and mouse HL genes. In order to analyze the 5{prime} nontranslated region of mouse HL gene, we cloned and sequenced a 1.8 kb fragment containing the 5{prime} extremity including exon 1 and about 1.6 kb of 5{prime} nontranslated sequence. The region surrounding exon 1 is CpG-rich (66.4%). Using the criteria of West, the Observed/Expected ratio for CpG dinucleotides is 0.7 ({ge}0.6 is consistent with a CpG island). We are carrying out primer extension and RNase protection experiments to determine the transcription initiation site. We constructed a gene targeting vector by introducing the neomycin resistance gene into exon 2 of a 7.5 kb genomic subclone of the mouse HL gene. Targeting was performed by electroporating 10 mg linearized vector into 10{sup 7} ES cells and selecting for 12 days with G418. 5/228 colonies (2.2%) had homologous recombination as shown by PCR screeningmore » and Southern analysis. We are microinjecting the 5 targeted clones into blastocysts to create an HL-deficient mouse. To date we have obtained two chimeras with contributions of 95% and 55% from 129, by coat color estimates. Three of 27 (11%) of the HL-deficient patients studied were suggested by genomic Southern analysis to be homozygous for large intragenic deletions. We confirmed this and defined the boundaries using exonic PCR.« less

SCOT (EC 2.8.3.5) mediates the activation and utilization of ketone bodies in extrahepatic tissues, especially brain, heart and kidney. Children with hereditary SCOT deficiency have episodes of severe ketoacidosis. Using a partial human heart SCOT cDNA, hSCOT-G, we detected a single {approximately}3 Kb mRNA in human heart and leukocytes, but not in liver. The length of the mouse SCOT mRNA detected with hSCOT-G in muscle, heart, kidney and brain is {approximately}3 Kb. We mapped the human SCOT gene to chromosome 5p13 by in situ hybridization. To date we have isolated human heart cDNAs spanning 2.9 Kb and including a 1248 bp open reading frame. The 3{prime} nontranslated region of the human SCOT mRNA extends at least 1712 bp, in contrast to the 209 bp sequence reported for pig SCOT cDNA. In one heart cDNA clone we detected a 58 bp insertion 258 bp downstream from the stop codon. We performed RT-PCR using a 5{prime} degenerate-sequence primer designed from the pig SCOT leader peptide sequence and 3{prime} human-specific primers. We obtained a fragment of the expected 320 bp length which strongly hybridizes to an internal oligonucleotide and which we are now characterizing. Human genomic Southern blot analysis with a partial humanmore » cDNA as probe suggests that the length of the human SCOT gene is about 40 K. Using hSCOT-G as a probe, we screened a human leukocyte genomic library in EMBL-3 phage and isolated two genomic clones. One of them contains a processed pseudogene. The other contains at least two exons of the human SCOT gene spanning cDNA residues 431 to 734. These findings will be useful for mutation analysis in SCOT-deficient patients.« less

The effect of fluctuating temperatures on microbial growth is important in the passage of foods through the food chain. Suspensions of Salmonella typhimurium were subjected to sinusoidally time-varying temperatures of periods from 40 to 480 min within their growth temperature range. The change in the numbers of viable bacteria was measured with time and the experimental growth curves and average generation times compared with predictions based on isothermal growth data. The experimental average generation times exceeded the predictions by less than 30%, although the discrepancy increased with cycle frequency. Instantaneous growth rates obtained for the 480 min cycles were in agreement with those predicted from isothermal behaviour.

Journal of Inherited Metabolic DiseaseVolume 17, Issue 3 p. 291-294 Short Communication Fatal cardiomyopathy associated with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency K. M. Gibson, K. M. Gibson Kimberly H. Courtwright and Joseph W. Summers Metabolic Disease Center, Baylor University Medical Center and Baylor Research Institute, Dallas, TX, USASearch for more papers by this authorS. B. Cassidy, S. B. Cassidy Department of Pediatrics, College of Medicine, University of Arizona Health Sciences Center, Tucson, AZ, USASearch for more papers by this authorL. H. Seaver, L. H. Seaver Department of Pediatrics, College of Medicine, University of Arizona Health Sciences Center, Tucson, AZ, USASearch for more papers by this authorR. J. A. Wanders, R. J. A. Wanders Department of Pediatrics, Academic Medical Center, University Hospital Amsterdam, The NetherlandsSearch for more papers by this authorN. G. Kennaway, N. G. Kennaway Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, OR, USASearch for more papers by this authorG. A. Mitchell, G. A. Mitchell Department of Pediatrics and Biochemistry and Medical Genetics Service, Hopital Sainte-Justine, Montreal, CanadaSearch for more papers by this authorR. P. Spark, R. P. Spark Departments of Pathology, Tucson Medical Center and College of Medicine, University of Arizona Health Sciences Center, Tucson, AZ, USASearch for more papers by this author K. M. Gibson, K. M. Gibson Kimberly H. Courtwright and Joseph W. Summers Metabolic Disease Center, Baylor University Medical Center and Baylor Research Institute, Dallas, TX, USASearch for more papers by this authorS. B. Cassidy, S. B. Cassidy Department of Pediatrics, College of Medicine, University of Arizona Health Sciences Center, Tucson, AZ, USASearch for more papers by this authorL. H. Seaver, L. H. Seaver Department of Pediatrics, College of Medicine, University of Arizona Health Sciences Center, Tucson, AZ, USASearch for more papers by this authorR. J. A. Wanders, R. J. A. Wanders Department of Pediatrics, Academic Medical Center, University Hospital Amsterdam, The NetherlandsSearch for more papers by this authorN. G. Kennaway, N. G. Kennaway Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, OR, USASearch for more papers by this authorG. A. Mitchell, G. A. Mitchell Department of Pediatrics and Biochemistry and Medical Genetics Service, Hopital Sainte-Justine, Montreal, CanadaSearch for more papers by this authorR. P. Spark, R. P. Spark Departments of Pathology, Tucson Medical Center and College of Medicine, University of Arizona Health Sciences Center, Tucson, AZ, USASearch for more papers by this author First published: 01 May 1994 https://doi.org/10.1007/BF00711810Citations: 31 Baylor Research Institute, 3812 Elm Street, Dallas, TX, 75226, USA AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume17, Issue3May 1994Pages 291-294 RelatedInformation