Fatal inanition in reindeer (<Emphasis Type="Italic">Rangifer tarandus tarandus</Emphasis>): Pathological findings in completely emaciated carcasses

Background  

In a project to determine the causes of winter mortality in reindeer in Finnmark County, northern Norway, the most frequent diagnosis turned out to be complete emaciation, despite several of the reindeer having been given silage for up to 4 weeks before they died. The present paper describes autopsy results and other findings in these animals.     

A database of mutants and effects of site-directed mutagenesis experiments on G protein-coupled receptors

A database system and computer programs for storage and retrieval of information about guanine nucleotide-binding protein (G protein) -coupled receptor mutants and associated biological effects have been developed. Mutation data on the receptors were collected from the literature and a database of mutants and effects of mutations was developed. The G protein-coupled receptor, family A, point mutation database (GRAP) provides detailed information on ligand-binding and signal transduction properties of more than 2130 receptor mutants. The amino acid sequences of receptors for which mutation experiments have been reported were aligned, and from this alignment mutation data may be retrieved. Alternatively, a search form allowing detailed specification of which mutants to retrieve may be used, for example, to search for specific amino acid substitutions, substitutions in specific protein domains or reported biological effects. Furthermore, ligand and bibliographic oriented queries may be performed. GRAP is available on the Internet (URL: http://www-grap.fagmed.uit.no/GRAP/homepage.html ) using the World-Wide Web system. © 1996 Wiley-Liss, Inc.     

Mannuronan C-5-epimerases and their application for in vitro and in vivo design of new alginates useful in biotechnology

The industrially important polysaccharide alginate is a linear copolymer of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). It is produced commercially by extraction from brown seaweeds, although some of the bacteria belonging to the genera Azotobacter and Pseudomonas also synthesize alginates. Alginates are synthesized as mannuronan, and varying amounts of the M residues in the polymer are then epimerized to G residues by mannuronan C-5-epimerases. The gel-forming, water-binding, and immunogenic properties of the polymer are dependent on the relative amount and sequence distribution of M and G residues. A family of seven calcium-dependent, secreted epimerases (AlgE1-7) from Azotobacter vinelandii have now been characterized, and in this paper the properties of all these enzymes are described. AlgE4 introduces alternating M and G residues into its substrate, while the remaining six enzymes introduce a mixture of continuous stretches of G residues and alternating sequences. Two of the enzymes, AlgE1 and AlgE3, are composed of two catalytically active domains, each introducing different G residue sequence patterns in alginate. These results indicate that the enzymes can be used for production of alginates with specialized properties.     

The recombinant Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 epimerizes alginate by a nonrandom attack mechanism

The Ca2+-dependent mannuronan C-5-epimerase AlgE4 is a representative of a family of Azotobacter vinelandii enzymes catalyzing the polymer level epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. The reaction product of recombinantly produced AlgE4 is predominantly characterized by an alternating sequence distribution of the M and G residues (MG blocks). AlgE4 was purified after intracellular overexpression in Escherichia coli, and the activity was shown to be optimal at pH values between 6.5 and 7.0, in the presence of 1-3 mM Ca2+, and at temperatures near 37 degrees C. Sr2+ was found to substitute reasonably well for Ca2+ in activation, whereas Zn2+ strongly inhibited the activity. During epimerization of alginate, the fraction of GMG blocks increased linearly as a function of the total fraction of G residues and comparably much faster than that of MMG blocks. These experimental data could not be accounted for by a random attack mechanism, suggesting that the enzyme either slides along the alginate chain during catalysis or recognizes a pre-existing G residue as a preferred substrate in its consecutive attacks.     

Effects of estrogen treatment on glutamate uptake in cultured human astrocytes derived from cortex of Alzheimer's disease patients

Estrogen is thought to play a protective role against neurodegeneration through a variety of mechanisms including the activation of growth factors, the control of synaptic plasticity, and the reduction of response to various insults, such as iron and glutamate. Increasing evidence indicates an increased level of extracellular glutamate and a down-regulation of glutamate transporters in Alzheimer's disease (AD). In this study, we show that glutamate uptake in astrocytes derived from Alzheimer's patients is significantly lower than that from non-demented controls. Estrogen treatment increases glutamate uptake in a dose-dependent pattern. Two glutamate transporters, GLT-1 and GLAST, are expressed in the astrocytes. Up-regulation of the glutamate transporters is induced by estrogen treatment in AD astrocytes only. Our data suggest that the action of estrogen on glutamate uptake by astrocytes might contribute to its potential neuroprotective role in AD.     

Methylpurine DNA glycosylase of the hyperthermophilic archaeon Archaeoglobus fulgidus

Base excision repair of DNA alkylation damage is initiated by a methylpurine DNA glycosylase (MPG) function. Such enzymes have previously been characterized from bacteria and eukarya, but not from archaea. We identified activity for the release of methylated bases from DNA in cell-free extracts of Archaeoglobus fulgidus, an archaeon growing optimally at 83 degrees C. An open reading frame homologous to the alkA gene of Escherichia coli was overexpressed and identified as a gene encoding an MPG enzyme (M(r) = 34 251), hereafter designated afalkA. The purified AfalkA protein differs from E. coli AlkA by excising alkylated bases only, from DNA, in the following order of efficiency: 3-methyladenine (m(3)A) > 3-methylguanine approximately 7-methyladenine > 7-methylguanine. Although the rate of enzymatic release of m(3)A is highest in the temperature range of 65-75 degrees C, it is only reduced by 50% at 45 degrees C, a temperature that does not support growth of A. fulgidus. At temperatures above 75 degrees C, nonenzymatic release of methylpurines predominates. The results suggest that the biological function of AfalkA is to excise m(3)A from DNA at suboptimal and maybe even mesophilic temperatures. This hypothesis is further supported by the observation that the afalkA gene function suppresses the alkylation sensitivity of the E. coli tag alkA double mutant. The amino acid sequence similarity and evolutionary relationship of AfalkA with other MPG enzymes from the three domains of life are described and discussed.     

New broad-host-range promoter probe vectors based on the plasmid RK2 replicon

Broad-host-range plasmid RK2-based promoter probe vectors with a known nucleotide sequence were constructed. In the absence of an upstream promoter, the expression of two tested reporter genes (luc and lacZ) in Escherichia coli was virtually zero, while insertion of the Ptrc promoter resulted in strong inducer-dependent expression. The lacZ-based vectors were mobilized into Pseudomonas fluorescens ST, Pseudomonas putida KT2442, Sphingomonas spp. and Burkholderia spp. LB400, and expression analyses indicated that the properties observed in E. coli are maintained across the species barriers. In addition, the previously established knowledge of RK2 molecular biology allows easy manipulations of features such as plasmid copy number, further extending the application potential of the vectors.     

Mannuronan C-5 epimerases and cellular differentiation of Azotobacter vinelandii

Differentiation in Azotobacter vinelandii involves the encystment of the vegetative cell under adverse environmental circumstances and the germination of the resting cell into the vegetative state when growth conditions are satisfactory again. Morphologically, the encystment process involves the development of a protective coat around the resting cell. This coat partly consists of multiple layers of alginate, which is a co-polymer of β- d -mannuronic acid (M) and α- l -guluronic acid (G). Alginate contributes to coat rigidity by virtue of a high content of GG blocks. Such block structures are generated through a family of mannuronan C-5 epimerases that convert M to G after polymerization. Results from immunodetection and light microscopy, using stains that distinguish between different cyst components and types, indicate a correlation between cyst coat organization and the amount and appearance of mannuronan C-5 epimerases in the extracellular medium and attached to the cells. Specific roles of individual members of the epimerase family are indicated. Calcium and magnesium ions appear to have different roles in the structural organization of the cyst coat. Also reported is a new gene sharing strong sequence homology with parts of the epimerase-encoded R-modules. This gene is located within the epimerase gene cluster of Azotobacter vinelandii .     

Mutational spectrum in the cardioauditory syndrome of Jervell and Lange-Nielsen

Jervell and Lange-Nielsen syndrome (JLNS) is an autosomal recessive syndrome characterised by profound congenital sensorineural deafness and prolongation of the QT interval on the electrocardiogram, representing abnormal ventricular repolarisation. In a study of ten British and Norwegian families with JLNS, we have identified all of the mutations in the KCNQ1 gene, including two that are novel. Of the nine mutations identified in this group of 10 families, five are nonsense or frameshift mutations. Truncation of the protein proximal to the recently identified C-terminal assembly domain is expected to preclude assembly of KCNQ1 monomers into tetramers and explains the recessive inheritance of JLNS. However, study of a frameshift mutation, with a dominant effect phenotypically, suggests the presence of another assembly domain nearer to the N-terminus.     

Parameters affecting gene expression from the Pm promoter in gram-negative bacteria

The Pm promoter inserted chromosomally or in broad-host-range replicons based on plasmid RSF1010 or RK2 are useful systems for both high- and low-level expression of cloned genes in several gram-negative bacterial species. The positive Pm regulator XylS is activated by certain substituted benzoic acid derivatives, and here we show that these effectors induce expression of Pm at similar relative ranking levels in both Escherichia coli and Pseudomonas aeruginosa However, the kinetics of expression was not the same in the two organisms. Different carbon sources and dissolved oxygen levels displayed limited effects on expression, but surprisingly the pH of the growth medium was found to be of major importance. By combining the effects of genetic and environmental parameters, expression from Pm could be varied over a ten-thousand- to a hundred-thousand-fold continuous range, and as an example of its applications we showed that Pm can be used to control the xanthan biosynthesis in Xanthomonas campestris.