GRACILE syndrome,a lethal metabolic disorder with iron overload,is caused by a point mutation in BCS1L

GRACILE (growth retardation, aminoaciduria, cholestasis, iron overload, lactacidosis, and early death) syndrome is a recessively inherited lethal disease characterized by fetal growth retardation, lactic acidosis, aminoaciduria, cholestasis, and abnormalities in iron metabolism. We previously localized the causative gene to a 1.5-cM region on chromosome 2q33-37. In the present study, we report the molecular defect causing this metabolic disorder, by identifying a homozygous missense mutation that results in an S78G amino acid change in the BCS1L gene in Finnish patients with GRACILE syndrome, as well as five different mutations in three British infants. BCS1L, a mitochondrial inner-membrane protein, is a chaperone necessary for the assembly of mitochondrial respiratory chain complex III. Pulse-chase experiments performed in COS-1 cells indicated that the S78G amino acid change results in instability of the polypeptide, and yeast complementation studies revealed a functional defect in the mutated BCS1L protein. Four different mutations in the BCS1L gene have been reported elsewhere, in Turkish patients with a distinctly different phenotype. Interestingly, the British and Turkish patients had complex III deficiency, whereas in the Finnish patients with GRACILE syndrome complex III activity was within the normal range, implying that BCS1L has another cellular function that is uncharacterized but essential and is putatively involved in iron metabolism.     

Excess copper predisposes photosystem II to photoinhibition in vivo by outcompeting iron and causing decrease in leaf chlorophyll

Photoinhibition of photosystem II was studied in vivo with bean (Phaseolus vulgaris) plants grown in the presence of 0.3 (control), 4, or 15 microM Cu(2+). Although photoinhibition, measured in the presence of lincomycin to block concurrent recovery, is faster in leaves of Cu(2+)-treated plants than in control leaves, thylakoids isolated from Cu-treated plants did not show high sensitivity to photoinhibition. Direct effects of excess Cu(2+) on chloroplast metabolism are actually unlikely, because the Cu concentration of chloroplasts of Cu-treated plants was lower than that of their leaves. Excess Cu in the growth medium did not cause severe oxidative stress, collapse of antioxidative defenses, or loss of photoprotection. Thus, these hypothetical effects can be eliminated as causes for Cu-enhanced photoinhibition in intact leaves. However, Cu treatment lowered the leaf chlorophyll (Chl) concentration and reduced the thylakoid membrane network. The loss of Chl and sensitivity to photoinhibition could be overcome by adding excess Fe together with excess Cu to the growth medium. The addition of Fe lowered the Cu(2+) concentration of the leaves, suggesting that Cu outcompetes Fe in Fe uptake. We suggest that the reduction of leaf Chl concentration, caused by the Cu-induced iron deficiency, causes the high photosensitivity of photosystem II in Cu(2+)-treated plants. A causal relationship between the susceptibility to photoinhibition and the leaf optical density was established in several plant species. Plant species adapted to high-light habitats apparently benefit from thick leaves because the rate of photoinhibition is directly proportional to light intensity, but photosynthesis becomes saturated by moderate light.     

Priming effect as determined by adding 14C-glucose to modified controlled composting test

The development of new biodegradable packaging materials, especially biodegradable plastics, has created a need for biodegradability testing. The European standard for controlled composting test was used in this study for assessing if the addition of a test material results in excess CO₂ production in compost. This effect, designated as the priming effect, would give an erroneous result for biodegradation, which is based on CO₂ formation from the test material. Glucose was selected as a test substrate because it is the degradation product of starch and cellulose, which are major compounds of many packaging materials. Both ¹⁴C-glucose and non-labelled glucose was applied to nine compost samples of variable stability and agefrom two weeks to 1.5 years. CO₂ and ¹⁴CO₂ evolution were measured during the incubation. Biodegradation of glucose in unstable composts (age leq6 months) was negative and ¹⁴CO₂ evolution was poor, although the respective composts without glucose produced relatively high amounts of CO₂. It was concluded that a negative priming effect was observed in unstable composts, in which glucose remained mostly non-degraded and apparently inhibited the mineralization of native organic matter in the compost. In stable composts (age 6 months), biodegradation of glucose was high and approximately equal to ¹⁴C-glucose mineralization, i.e., the composts showed no priming effect. Young composts were unsuitable for controlled composting test due to lack of stability. It is important to ensure that the compost inoculum used for the test is sufficiently stable.     

Diagnosis of flavobacteriosis by direct amplification of rRNA genes

A broad-range bacterial PCR method with universal 16S rDNA targeting primers and bacterial cultivation was used to identify the putative pathogen in flavobacterial outbreaks. Restriction fragment length polymorphism (PCR-RFLP) analysis and sequencing of the partial 16S rDNA PCR products of 10 skin samples and 10 representative isolates derived from the same fish specimens revealed differences between direct molecular and cultivation-based analysis. Flavobacterium columnare-like sequences dominated in the direct molecular analysis in most cases, whereas most of the isolates belonged to a phylogenetically heterogeneous group of flavobacteria clustering with F. hibernum. F. columnare was isolated in only 1 outbreak. The possible explanations for the different results may be attributable to difficulties in the plate cultivation procedure of external flavobacterial samples. During plate cultivation, the dominating Flavobacterium species can be masked by saprophytic species of the same genus or other genera, or the growth of flavobacteria can be completely inhibited by antagonistic bacteria such as Pseudomonas. Direct analysis of the prevailing 16S rDNA sequences avoids the problems with cultivation and may thus be preferable for the diagnosis of flavobacterial diseases. When isolating flavobacteria from external samples, serial dilution of the sample before plating can improve the results.     

X-ray absorption spectroscopic investigation of the resting ferrous and cosubstrate-bound active sites of phenylalanine hydroxylase

Previous studies of ferrous wild-type phenylalanine hydroxylase, [Fe(2+)]PAH(T)[], have shown the active site to be a six-coordinate distorted octahedral site. After the substrate and cofactor bind to the enzyme ([Fe(2+)]PAH(R)[L-Phe,5-deaza-6-MPH(4)]), the active site converts to a five-coordinate square pyramidal structure in which the identity of the missing ligand had not been previously determined. X-ray absorption spectroscopy (XAS) at the Fe K-edge further supports this coordination number change with the binding of both cosubstrates to the enzyme, and determines this to be due to the loss of a water ligand.     

High-yield production of a bacterial xylanase in the filamentous fungus Trichoderma reesei requires a carrier polypeptide with an intact domain structure

A bacterial xylanase gene, Nonomuraea flexuosa xyn11A, was expressed in the filamentous fungus Trichoderma reesei from the strong cellobiohydrolase 1 promoter as fusions to a variety of carrier polypeptides. By using single-copy isogenic transformants, it was shown that production of this xylanase was clearly increased (up to 820 mg/liter) when it was produced as a fusion protein with a carrier polypeptide having an intact domain structure compared to the production (150 to 300 mg/liter) of fusions to the signal sequence alone or to carriers having incomplete domain structures. The carriers tested were the T. reesei mannanase I (Man5A, or MANI) core-hinge and a fragment thereof and the cellulose binding domain of T. reesei cellobiohydrolase II (Cel6A, or CBHII) with and without the hinge region(s) and a fragment thereof. The flexible hinge region was shown to have a positive effect on both the production of Xyn11A and the efficiency of cleavage of the fusion polypeptide. The recombinant Xyn11A produced had properties similar to those of the native xylanase. It constituted 6 to 10% of the total proteins secreted by the transformants. About three times more of the Man5A core-hinge carrier polypeptide than of the recombinant Xyn11A was observed. Even in the best Xyn11A producers, the levels of the fusion mRNAs were only approximately 10% of the level of cel7A (cbh1) mRNA in the untransformed host strain.     

Increased exopolysaccharide production in Lactococcus lactis due to increased levels of expression of the NIZO B40 eps gene cluster

Exopolysaccharides (EPS) play an important role in the rheology and texture of fermented food products. This is the first report demonstrating that homologous overexpression of a complete eps gene cluster in Lactococcus lactis leads to increased EPS production levels. A ninefold-elevated EPS plasmid copy number led to an almost threefold increase in the eps expression level, resulting in an almost fourfold increase in the NIZO B40 EPS production level. It was previously reported that increased EPS precursor levels did not influence NIZO B40 EPS production levels. However, the present results indicate that the maximal NIZO B40 EPS production level is limited by the activity level of the expression products of the eps gene cluster rather than by the level of EPS precursors.     

Mechanisms regulating adrenomedullin gene expression in the left ventricle: role of mechanical load

Xenin is a 25 amino acid peptide produced by specific endocrine cells of the duodenal mucosa. Xenin has multiple biological actions in the gastrointestinal tract. It modulates intestinal motility, affects exocrine pancreatic secretion, and gastric secretion of acid. In the present investigation, we studied plasma concentration of xenin in volunteers after modified sham feeding and after meals of different composition. Plasma xenin concentrations were determined by radioimmunoassay in unextracted plasmas and after acidic extraction using C-18 Sep-Pak chromatography and after neutral extraction using affinity filtration. Both extraction methods were followed by C 18 r.p. HPLC chromatography. Xenin plasma concentrations in unextracted and in extracted plasma rose significantly after modified sham feeding when the food was brought to the volunteers from another room immediately before sham feeding started. When the volunteers had the opportunity to observe the preparation of the meal, xenin plasma concentrations during fasting were high and no further rise was observed after sham feeding. Isocaloric feeding resulted in elevated xenin concentrations in unextracted plasma and after high-pressure liquid chromatography. The methods of extraction, acidic or neutral, did not affect the results. CONCLUSION: Cephalic factors, investigated by modified sham feeding, stimulate release of xenin into the circulation. Xenin may participate in the central nervous regulation of gastrointestinal function.