Lean and obese dietary phenotypes: differences in energy and substrate metabolism and appetite.

This study aimed to characterise lean and obese phenotypes according to diet and body composition, and to compare fasting and postprandial appetite and metabolic profiles following a high-fat test meal. A total of ten lean (BMI40 and 30 kg/m2) high-fat consumers (OHF; >40 % energy from fat) were recruited. Before and following the test meal (4727 kJ (1130 kcal), 77 % fat, 20 % carbohydrate (CHO) and 3 % protein), fasting plasma glucose, insulin, leptin, ghrelin, peptide YY (PYY), RER, RMR and subjective appetite ratings (AR) were measured for 6 h. Thereafter, subjects consumed a self-selected portion of a standardised post-test meal (40 % fat, 45 % CHO and 15 % protein) and reported AR. Fasting (P=0·01) and postprandial (P<0·001) fat oxidation was significantly higher in LHF than in LLF but was not different between LHF and OHF. Although similar between the lean groups, fasting and postprandial energy expenditures were significantly higher in OHF compared with LHF (P<0·01). Despite similar AR across groups, LLF consumed a relatively greater quantity of the post-test meal than did LHF (7·87 (sd 2·96) v. 7·23 (sd 2·67) g/kg, P=0·013). The lean groups showed appropriate changes in plasma ghrelin and PYY following the test meal, whereas the OHF group showed a blunted response. In conclusion, the LHF phenotype had a greater capacity for fat oxidation, which may be protective against weight gain. OHF individuals had a blunted appetite hormone response to the high-fat test meal, which may subsequently increase energy intake, driving further weight gain.

Development and application of an assay for uranyl complexation by fungal metabolites, including siderophores.

An assay to detect UO(2)(2+) complexation was developed based on the chrome azurol S (CAS) assay for siderophores (B. Schwyn and J. B. Neilands, Anal. Biochem. 160:47-56, 1987) and was used to investigate the ability of fungal metabolites to complex actinides. In this assay the discoloration of two dyed agars (one containing a CAS-Fe(3+) dye and the other containing a CAS-UO(2)(2+) dye) caused by ligands was quantified. The assay was tested by using the siderophore desferrioxamine B (DFO), and the results showed that there was a regular, reproducible relationship between discoloration and the amount of siderophore added. The ratio of the discoloration on the CAS-UO(2)(2+) agar to the discoloration on the CAS-Fe(3+) agar was independent of the amount of siderophore added. A total of 113 fungi and yeasts were isolated from three soil samples taken from the Peak District National Park. The fungi were screened for the production of UO(2)(2+) chelators by using the CAS-based assay and were also tested specifically for hydroxamate siderophore production by using the hydroxamate siderophore auxotroph Aureobacterium flavescens JG-9. This organism is highly sensitive to the presence of hydroxamate siderophores. However, the CAS-based assay was found to be less sensitive than the A. flavescens JG-9 assay. No significant difference between the results for each site for the two tests was found. Three isolates were selected for further study and were identified as two Pencillium species and a Mucor species. Our results show that the new assay can be effectively used to screen fungi for the production of UO(2)(2+) chelating ligands. We suggest that hydroxamate siderophores can be produced by mucoraceous fungi.

Glucoamylase::green fluorescent protein fusions to monitor protein secretion in Aspergillus niger.

A glucoamylase::green fluorescent protein fusion (GLA::sGFP) was constructed which allows the green fluorescent protein to be used as an in vivo reporter of protein secretion in Aspergillus niger. Two secretory fusions were designed for secretion of GLA::sGFP which employed slightly different lengths of the glucoamylase protein (GLA499 and GLA514). Expression of GLA::sGFP revealed that fluorescence was localized in the hyphal cell walls and septa, and that fluorescence was most intense at hyphal apices. Extracellular GLA::sGFP was detectable by Western blotting only in the supernatant of young cultures grown in soya milk medium. In older cultures, acidification of the medium and induction of proteases were probably responsible for the loss of extracellular and cell wall fluorescence and the inability to detect GLA::sGFP by Western analysis. A strain containing the GLA::sGFP construct was subjected to UV mutagenesis and survivors screened for mutations in the general secretory pathway. Three mutants were isolated that were unable to form a halo on either starch or gelatin medium. All three mutants grew poorly compared to the parental strain. Fluorescence microscopy revealed that for two of the mutants, GLA::sGFP accumulated intracellularly with no evidence of wall fluorescence, whereas for the third mutant, wall fluorescence was observed with no evidence of intracellular accumulation. These results indicate that the GLA::sGFP fusion constructs can be used as convenient fluorescent markers to study the dynamics of protein secretion in vivo and as a tool in the isolation of mutants in the general secretory pathway.

Extracellular proteases produced by the Quorn® myco-protein fungus Fusarium graminearum in batch and chemostat culture.

Fusarium graminearum was grown in batch and continuous (chemostat) culture on a glucose-mineral salts medium in the presence and absence of casein. In the absence of casein no protease activity was detected in the culture filtrate from either batch or chemostat culture. For batch cultures grown on medium containing casein, most of the proteolytic activity detected in the supernatant during exponential growth had an optimum at ca pH 5.0. However, as the cultures passed from late exponential into stationary phase, the pH profile of the protease activity broadened until most of it was in the alkaline pH region. For glucose-limited chemostat cultures grown on media containing casein, protease activity had a narrow pH optimum with maximum activity at pH 5.0. For all concentrations of casein examined, protease activity was greater in chemostat culture than in batch culture. Extracellular proteases from batch and chemostat cultures were purified by bacitracin-Sepharose affinity chromatography. At least seven proteins were purified from batch cultures but chemostat cultures contained only a single aspartic protease with a molecular mass of 40 kDa.

How do highly branched (colonial) mutants of Fusarium graminearum A3/5 arise during Quorn myco-protein fermentations?

Chlorate-resistant, highly branched (colonial) mutants and auxotrophic mutants were used to study the nuclear distribution, morphology and growth of heterokaryons of the Quorn myco-protein fungus, Fusarium graminearum A3/5. The results showed that for several complementary homokaryons, even a strong selective pressure was insufficient to maintain heterokaryons in a 'balanced' condition (i.e. exhibiting a wild-type or near wild-type phenotype). Furthermore, the margins of heterokaryotic colonies generally contained nuclei from only one of the parental homokaryons, indicating imperfect nuclear mixing within the mycelium. These observations suggest that recessive, colonial mutants may appear during Quorn myco-protein production following shear-induced separation of hyphal fragments which contain a sufficiently high ratio of colonial:wild-type nuclei for the colonial phenotype to be expressed.

Choline- and acetylcholine-induced changes in the morphology of Fusarium graminearum: evidence for the involvement of the choline transport system and acetylcholinesterase.

The response of Fusarium graminearum to choline, acetylcholine and a number of related analogues was investigated and their ability to induce a morphological response quantified. A number of mutants resistant to the alkylating agent nitrogen mustard (nim strains) were generated and found to have lost the ability to transport choline. These mutants were found to be insensitive to choline and acetylcholine but not to betaine, ethanolamine and other analogues. In addition, the non-competitive inhibitor hemicholinium-3 was also found to reduce the morphological effect of choline, proving that transport of choline into the hypha is essential for the morphological response. Acetylcholinesterase inhibitors blocked the morphological response to acetylcholine but had no effect on the response to choline, suggesting the presence of a membrane- or wall-bound acetylcholinesterase that hydrolyses acetylcholine to choline which subsequently induces the morphological response. Studies on the in vivo chitin synthase activity revealed that addition of choline caused a transient 75% increase in chitin synthase activity within 30 s, the rate rapidly returning to that observed before the addition of choline. No such effect was observed with the nim mutants.

Transannular GORE-TEX patch with pericardial unicusp for total correction of tetralogy of fallot.

When corrective surgery for tetralogy of Fallot was accomplished through the use of a transannular GORE-TEX patch with a pericardial unicusp, the right-ventricular end-diastolic volumes of all patients studied within a year of the surgery were within the normal ranges because of decreased pulmonary valve regurgitation. The right-ventricular ejection fraction was also only slightly depressed, indicating preservation of right-ventricular function. All patients were noted to maintain normal stroke volumes and normal systolic indices. In contrast, the patients who had transannular patches placed without unicusps showed significantly elevated right-ventricular end-diastolic volumes and lower right-ventricular ejection fractions. These resulted from markedly dilated right-ventricular outflow regions in conjunction with enlarged right-ventricular chambers, which manifested as large dyskinetic areas in the anterior right-ventricular walls.

Heat capacity of Matsushita carbon resistance thermometers below 1 K.

Matsushita resistors have a much smaller heat capacity than Speer resistors, and thus are preferable in some thermometry applications at temperatures below 1 K.