Liver-specific Commd1 knockout mice are susceptible to hepatic copper accumulation

Canine copper toxicosis is an autosomal recessive disorder characterized by hepatic copper accumulation resulting in liver fibrosis and eventually cirrhosis. We have identified COMMD1 as the gene underlying copper toxicosis in Bedlington terriers. Although recent studies suggest that COMMD1 regulates hepatic copper export via an interaction with the Wilson disease protein ATP7B, its importance in hepatic copper homeostasis is ill-defined. In this study, we aimed to assess the effect of Commd1 deficiency on hepatic copper metabolism in mice. Liver-specific Commd1 knockout mice (Commd1(Δhep)) were generated and fed either a standard or a copper-enriched diet. Copper homeostasis and liver function were determined in Commd1(Δhep) mice by biochemical and histological analyses, and compared to wild-type littermates. Commd1(Δhep) mice were viable and did not develop an overt phenotype. At six weeks, the liver copper contents was increased up to a 3-fold upon Commd1 deficiency, but declined with age to concentrations similar to those seen in controls. Interestingly, Commd1(Δhep) mice fed a copper-enriched diet progressively accumulated copper in the liver up to a 20-fold increase compared to controls. These copper levels did not result in significant induction of the copper-responsive genes metallothionein I and II, neither was there evidence of biochemical liver injury nor overt liver pathology. The biosynthesis of ceruloplasmin was clearly augmented with age in Commd1(Δhep) mice. Although COMMD1 expression is associated with changes in ATP7B protein stability, no clear correlation between Atp7b levels and copper accumulation in Commd1(Δhep) mice could be detected. Despite the absence of hepatocellular toxicity in Commd1(Δhep) mice, the changes in liver copper displayed several parallels with copper toxicosis in Bedlington terriers. Thus, these results provide the first genetic evidence for COMMD1 to play an essential role in hepatic copper homeostasis and present a valuable mouse model for further understanding of the molecular mechanisms underlying hepatic copper homeostasis.     

Die Entwicklung isolierter Keimfragmente frühester Stadien von Triton taeniatus

Ohne Zusammenfassung     

Die Entwicklung isolierter dorsaler und lateraler Gastrulahälften von Triton taeniatus und alpestris,ihre Regulation und Postgeneration

Ohne Zusammenfassung     

Directed evolution of an industrial biocatalyst: 2-deoxy-D-ribose 5-phosphate aldolase

Aldolases are emerging as powerful and cost efficient tools for the industrial synthesis of chiral molecules. They catalyze enantioselective carbon-carbon bond formations, generating up to two chiral centers under mild reaction conditions. Despite their versatility, narrow substrate ranges and enzyme inactivation under synthesis conditions represented major obstacles for large-scale applications of aldolases. In this study we applied directed evolution to optimize Escherichia coli 2-deoxy-D-ribose 5-phosphate aldolase (DERA) as biocatalyst for the industrial synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside. This versatile chiral precursor for vastatin drugs like Lipitor (atorvastatin) is synthesized by DERA in a tandem-aldol reaction from chloroacetaldehyde and two acetaldehyde equivalents. However, E. coli DERA shows low affinity to chloroacetaldehyde and is rapidly inactivated at aldehyde concentrations useful for biocatalysis. Using high-throughput screenings for chloroacetaldehyde resistance and for higher productivity, several improved variants have been identified. By combination of the most beneficial mutations we obtained a tenfold improved variant compared to wild-type DERA with regard to (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside synthesis, under industrially relevant conditions.     

Learning and anticipatory behaviour in a “sit-and-wait” predator: The Atlantic halibut

We studied the learning capacities and anticipatory behaviour in a “sit-and-wait” predatory fish, the Atlantic halibut, Hippoglossus hippoglossus. In Experiment 1 two groups of halibut received series of light flashes (conditioned stimulus, CS) that started before delivery of food (unconditioned stimulus, US) and persisted until after food delivery, i.e. delay conditioning. Control groups received unpaired CS and US presentations. The anticipatory behaviour of delay conditioned halibut consisted mainly of take-offs towards the surface shortly after onset of the CS. In Experiment 2 six groups of halibut were trained in three trace conditioning procedures: Two groups with 20 s, two groups with 60 s and two groups with 120 s trace interval. Learning was evident in the 20 and 60 s trace groups and in one of the 120 s trace groups. In contrast to delay conditioning the anticipatory behaviour of trace conditioned halibut was characterized by subtle movements near the tank floor with orientation towards the CS. The cautious responses of halibut after trace conditioning differed markedly from what is observed in other fish species and are suggested to reflect a “sit-and-wait” foraging strategy that requires the predator to remain undetected until the prey is within lunging range.     

BAC to the future! or oligonucleotides: a perspective for micro array comparative genomic hybridization (array CGH)

The array CGH technique (Array Comparative Genome Hybridization) has been developed to detect chromosomal copy number changes on a genome-wide and/or high-resolution scale. It is used in human genetics and oncology, with great promise for clinical application. Until recently primarily PCR amplified bacterial artificial chromosomes (BACs) or cDNAs have been spotted as elements on the array. The large-scale DNA isolations or PCR amplifications of the large-insert clones necessary for manufacturing the arrays are elaborate and time-consuming. Lack of a high-resolution highly sensitive (commercial) alternative has undoubtedly hindered the implementation of array CGH in research and diagnostics. Recently, synthetic oligonucleotides as arrayed elements have been introduced as an alternative substrate for array CGH, both by academic institutions as well as by commercial providers. Oligonucleotide libraries or ready-made arrays can be bought off-the-shelf saving considerable time and efforts. For RNA expression profiling, we have seen a gradual transition from in-house printed cDNA-based expression arrays to oligonucleotide arrays and we expect a similar transition for array CGH. This review compares the different platforms and will attempt to shine a light on the ‘BAC to the future’ of the array CGH technique.     

Production of cyanophycin in <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> through the expression of a cyanophycin synthetase encoding gene

Cyanophycin or cyanophycin granule peptide is a protein that results from non-ribosomal protein synthesis in microorganisms such as cyanobacteria. The amino acids in cyanophycin can be used as a feedstock in the production of a wide range of chemicals such as acrylonitrile, polyacrylic acid, 1,4-butanediamine, and urea. In this study, an auxotrophic mutant (Rhizopus oryzae M16) of the filamentous fungus R. oryzae 99-880 was selected to express cyanophycin synthetase encoding genes. These genes originated from Synechocystis sp. strain PCC6803, Anabaena sp. strain PCC7120, and a codon optimized version of latter gene. The genes were under control of the pyruvate decarboxylase promoter and terminator elements of R. oryzae. Transformants were generated by the biolistic transformation method. In only two transformants both expressing the cyanophycin synthetase encoding gene from Synechocystis sp. strain PCC6803 was a specific enzyme activity detected of 1.5 mU/mg protein. In one of these transformants was both water-soluble and insoluble cyanophycin detected. The water-soluble fraction formed the major fraction and accounted for 0.5% of the dry weight. The water-insoluble CGP was produced in trace amounts. The amino acid composition of the water-soluble form was determined and constitutes of equimolar amounts of arginine and aspartic acid.