Chiral separation of amino acids in biological fluids by micellar electrokinetic chromatography with laser-induced fluorescence detection

A method is presented for the chiral analysis of amino acids in biological fluids using micellar electrokinetic chromatography (MEKC) and laser-induced fluorescence (LIF). The amino acids are derivatized with the chiral reagent (+/−)-1-(9-anthryl)-2-propyl chloroformate (APOC) and separated using a mixed micellar separation system. No tedious pre-purification of samples is required. The excellent separation efficiency and good detection capabilities of the MEKC-LIF system are exemplified in the analysis of urine and cerebrospinal fluid. This is the first time MEKC has been reported for chiral analysis of amino acids in biological fluids. The amino acids -alanine, -glutamine, and -aspartic acid have been observed in cerebrospinal fluid, and -alanine and -glutamic acid in urine. To the best of our knowledge no measurements of either -alanine in cerebrospinal fluid or -glutamic acid in urine have been presented in the literature before.     

Nicotinamide Mononucleotide Adenylyltransferase 2 (Nmnat2) Regulates Axon Integrity in the Mouse Embryo

Using transposon-mediated gene-trap mutagenesis, we have generated a novel mouse mutant termed Blad (Bloated Bladder). Homozygous mutant mice die perinatally showing a greatly distended bladder, underdeveloped diaphragm and a reduction in total skeletal muscle mass. Wild type and heterozygote mice appear normal. Using PCR, we identified a transposon insertion site in the first intron of Nmnat2 (Nicotinamide mononucleotide adenyltransferase 2). Nmnat2 is expressed predominantly in the brain and nervous system and has been linked to the survival of axons. Expression of this gene is undetectable in Nmnat2^blad/blad mutants. Examination of the brains of E18.5 Nmnat2^blad/blad mutant embryos did not reveal any obvious morphological changes. In contrast, E18.5 Nmnat2^blad/blad homozygotes showed an approximate 60% reduction of spinal motoneurons in the lumbar region and a more than 80% reduction in the sensory neurons of the dorsal root ganglion (DRG). In addition, facial motoneuron numbers were severely reduced, and there was virtually a complete absence of axons in the hind limb. Our observations suggest that during embryogenesis, Nmnat2 plays an important role in axonal growth or maintenance. It appears that in the absence of Nmnat2, major target organs and tissues (e.g., muscle) are not functionally innervated resulting in perinatal lethality. In addition, neither Nmnat1 nor 3 can compensate for the loss of Nmnat2. Whilst there have been recent suggestions that Nmnat2 may be an endogenous modulator of axon integrity, this work represents the first in vivo study demonstrating that Nmnat2 is involved in axon development or survival in a mammal.     

Effects on water chemistry after drainage of a bog for forestry

Drainage for forestry has received increasing interest during recent decades. Generally, drainage concerns wet mineral soils while the utilization of peatlands is a matter of controversy. The peatlands mainly involved are fens, while forestry on bogs is an insignificant activity. Consequently, hydrology of bogs and effects of drainage on their hydrochemistry are little known.The investigation performed aimed at elucidating the parent conditions and the drainage impact on the hydrology and hydrochemistry of an ombrotrophic bog. Two bogs were first compared during a calibration period of two years and then, after drainage of one of them, during a period of three years. The second bog was kept virgin as a control.Considerable influences on runoff and stream water quality were found from the surrounding mineral soil uplands of the bog. Significant differences occurred between the chemical composition of the groundwater in the mineral soil and in the bog peat.Effects on runoff water from drainage of the bog deviate from drainage of minerotrophic peatlands with respect to decreased concentrations and losses of organic carbon and nitrogen. From two small bog catchments within the drained bog, there generally were greater losses of nutrients than from the catchment as a whole. Furthermore, the runoff from the drained bog decreased in comparison with the undrained condition. However, there were also similarities to drainage of other peatlands as regards increased pH, alkalinity and concentrations of sulphate. Also, concentrations of total-phosphorus increased in spite of a decreased phosphate (MRP) concentration.     

Cross-breeding of selected and mutated homokaryotic strains of Phanerochaete chrysosporium K-3: New cellulase deficient strains with increased ability to degrade lignin

Summary A strain of Phanerochaete chrysosporium, designated strain K-3, was isolated from a monosporous conidiospore culture of Sporotrichum pulverulentum. This strain produces fruit bodies with only four sterigmata. From basidiospores of this culture, the homokaryotic strain 31 with high lignin degrading capacity was selected and subjected to ultraviolet irradiation to obtain cellulase deficient (Cel^-) strains. By cross-breeding one of these Cel^- variants with selected Cel⁺ homokaryotic strains from K-3 with high lignin degrading capacity, new Cel^- mutants were isolated which exceeded K-3 in their capacity to degrade lignin.The Cel^- strains were totally incapable of degrading cellulose but were able to degrade xylan. Evolution of ¹⁴CO₂ from ¹⁴C-ring-labelled synthetic lignin a dehydrogenation polymerizate (DHP) was used to screen for strains with high lignin degrading capacity.Studies of weight loss on birch and spruce wood revealed that the weight losses caused by strain K-3 exceeded, in all cases, those caused by the Cel^- strains. However, higher lignin losses in birch wood were obtained with several of the Cel^- strains than with the K-3 strain. After 2 weeks, one strain caused a lignin loss in birch wood of 21% of the initial amount of lignin, while with another strain there was, after 3 weeks incubation, a 28.5% decrease in the lignin content.     

Sequence structure and expression of a cloned beta-glucosidase gene from an extreme thermophile

Summary The gene for a -glucosidase from the extremely thermophilic bacterium Caldocellum saccharolyticum has been isolated from a genomic library and sequenced. An open reading frame identified by computer analysis of the sequence could encode a protein of M_r 54400, which is close to the size of the polypeptide experimentally determined using maxicells. Analysis of the amino-terminal residues of the protein produced in Escherichia coli suggests that it is processed by a methionine aminopeptidase. A sequence within C. saccharolyticum DNA upstream of the -glucosidase gene was found to act as a promoter for expression of the thermophile gene in E. coli. The protein has been overproduced in E. coli and Bacillus subtilis where it retains its enzymatic activity and heat stability. There appears to be a single copy of the gene in Caldocellum DNA.     

Cloning,sequencing and overexpression in Escherichia coli of a xylanase gene,xynA from the thermophilic bacterium Rt8B.4 genus Caldicellulosiruptor

 A genomic library of the extremely thermophilic eubacterial strain Rt8B.4 was constructed in λZapII and screened for the expression of xylanase activity. One recombinant bacteriophage showed xylanase, xylosidase and arabinosidase activity. Sequence analysis and homology comparisons showed that this plasmid derivative, pNZ2011, was composed of 6.7 kb thermophilic DNA and contained what appeared to be an operon-like structure involving genes associated with xylose metabolism. The xylanase gene, xynA was shown to code for a multi-domain protein. Xylanase activity was shown to be associated with the carboxy-terminal domain (domain 2) by deletion analysis and also by selective polymerase chain reaction (PCR) amplification and expression of the individual domains. Denaturing polyacrylamide gel analysis of the protein encoded by the PCR product showed three main overexpressed proteins to be present in cell extracts, presumably caused by proteolytic degradation in the Escherichia coli host. The xylanase activity from domain 2 is associated with a 36-kDa protein, which is stable at 70^°C for at least 12 h at pH 7. The small size of this active enzymatic domain and its temperature stability suggest that it may be of value in the enzyme-enhanced bleaching of kraft pulp. Received: 18 April 1995/Received revision: 4 August 1995/Accepted: 22 August 1995     

celA,another gene coding for a multidomain cellulase from the extreme thermophile Caldocellum saccharolyticum

Caldocellum saccharolyticum is an extremely thermophilic anaerobic bacterium capable of growth on cellulose and hemicellulose as sole carbon sources. Cellulase together on its genome. The gene for one of the cellulases (celA) was isolated on a genomic library clone, sequenced and found to comprise a large open-reading frame of 5253 base pairs that could be translated into a peptide of 1751 amino acids. To date, it is the largest cellulase gene sequenced. The translated product is a multidomain structure composed of two catalytic domains and two cellulose-binding domains linked by proline-threonine-rich regions (PT linkers). The N-terminal domain of celA encodes for an endoglucanase activity on carboxymethylcellulose, consistent with its high homology to the sequences of several other endo-1,4--d-glucanases. The carboxylterminal domain shows sequence homology with a cellulase from Clostridium thermocellum (CelS), which is known to act synergistically with a second component to hydrolyze crystalline cellulose. In the absence of a Caldocellum homologue for this second protein, we can detect no activity from this domain.     

The H3 chaperone function of NASP is conserved in Arabidopsis

Histones are abundant cellular proteins but, if not incorporated into chromatin, they are usually bound by histone chaperones. Here, we identify Arabidopsis NASP as a chaperone for histones H3.1 and H3.3. NASP interacts in vitro with monomeric H3.1 and H3.3 as well as with histone H3.1–H4 and H3.3–H4 dimers. However, NASP does not bind to monomeric H4. NASP shifts the equilibrium between histone dimers and tetramers towards tetramers but does not interact with tetramers in vitro. Arabidopsis NASP promotes [H3–H4]₂ tetrasome formation, possibly by providing preassembled histone tetramers. However, NASP does not promote disassembly of in vitro preassembled tetrasomes. In contrast to its mammalian homolog, Arabidopsis NASP is a predominantly nuclear protein. In vivo, NASP binds mainly monomeric H3.1 and H3.3. Pulldown experiments indicated that NASP may also interact with the histone chaperone MSI1 and a HSC70 heat shock protein.