Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226

Background  

Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBPα) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBPα is a link between GSK3 and these gene promoters.     

The detection of Bifidobacterium adolescentis by colony hybridization as an indicator of human faecal pollution

AIMS: To develop an improved method for the detection of Bifidobacterium adolescentis as an indicator of human faecal pollution. METHODS AND RESULTS: Bifidobacterium medium (BFM) was identified as the optimal medium for the recovery of bifidobacteria from human effluent. Dilutions of faeces and effluent from both humans and animals were filtered, grown on BFM and human specific B. adolescentis identified via colony hybridization with a digoxigenin (DIG)-labelled oligonucleotide probe. CONCLUSIONS: The combination of BFM with colony probing allows the detection of B. adolescentis, a specific indicator of human faecal pollution. SIGNIFICANCE AND IMPACT OF THE STUDY: It is now technically feasible to use B. adolescentis as indicators of human faecal pollution, and studies to examine the survival and appropriateness of bifidobacteria in this role can be initiated.     

Tetranectin Is a Novel Marker for Myogenesis during Embryonic Development, Muscle Regeneration, and Muscle Cell Differentiationin Vitro

Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell differentiationin vitro.We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining is observed in normal adult muscle. However, during skeletal muscle regeneration induced by the intramuscular injection of the myotoxic anesthetic Marcaine, myoblasts, myotubes, and the stumps of damaged myofibers exhibit intense tetranectin immunostaining. Tetranectin is also present in regenerating muscle cells in dystrophicmdxmice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiationin vitro.Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis.     

Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment

When microbes evolve in a continuous, nutrient-limited environment, natural selection can be predicted to favor genetic changes that give cells greater access to limiting substrate. We analyzed a population of baker's yeast that underwent 450 generations of glucose-limited growth. Relative to the strain used as the inoculum, the predominant cell type at the end of this experiment sustains growth at significantly lower steady-state glucose concentrations and demonstrates markedly enhanced cell yield per mole glucose, significantly enhanced high-affinity glucose transport, and greater relative fitness in pairwise competition. These changes are correlated with increased levels of mRNA hybridizing to probe generated from the hexose transport locus HXT6. Further analysis of the evolved strain reveals the existence of multiple tandem duplications involving two highly similar, high- affinity hexose transport loci, HXT6 and HXT7. Selection appears to have favored changes that result in the formation of more than three chimeric genes derived from the upstream promoter of the HXT7 gene and the coding sequence of HXT6. We propose a genetic mechanism to account for these changes and speculate as to their adaptive significance in the context of gene duplication as a common response of microorganisms to nutrient limitation.      

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

The influence of hydration on the conformation of lysozyme studied by solid-state 13C-nmr spectroscopy

¹³C proton decoupled cross-polarization magic-angle spinning nmr spectra of lysozyme are reported as a function of hydration. Increases in hydration level enhance the resolution of the spectra, particularly in the aliphatic region, but has no significant effect on either the rotating frame proton spin–lattice relaxation time or the cross-relaxation time. The enhancement in spectral resolution with hydration is attributed to a decrease in the distribution of isotropic chemical shifts, which reflects a decrease in the distribution of conformational states sampled by the protein. Changes in the distribution of isotropic chemical shifts occur after the addition of water to the charged groups as coverage of the polar side chains and peptide groups takes place. The onset of this behavior occurs at a hydration level of about, 0.1–0.2 g water/g protein and is largely complete at about 0.3 g water/g protein, the same hydration range where changes in the heat capacity are observed. That hydrogen exchange of buried protons can occur at hydration levels significantly lower than those at which changes in the distribution of conformational states are first observed suggests that some motions that mediate exchange are already present in the dry protein. The preservation of efficient dipolar coupling indicates that the conformational rearrangements that do-occur on hydration are small and do not involve any significant overall expansion of free volume or weakening of interactions that would increase the reorientational freedom of protein groups. © 1993 John Wiley & Sons, Inc.     

Isolation and characterization of a mutant of Staphylococcus aureus deficient in autolytic activity.

A mutant of Staphylococcus aureus H (RUS3) uas isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The rate of autolysis of whole cells and isolated cell walls of RUS3 was less than 10% of the parent strain. In addition, the ability of the crude soluble enzyme isolated from RUS3 to degrade cell walls was negligible compared with the parent strain. The cell wall composition and the generation time of RUS3 were comparable to the parent strain. Unlike S. aureus H, RUS3 grew in clumps and did not undergo cell wall turnover. Both strains exhibited identical kinetics of killing by penicillin G. This may indicate that autolytic enzymes play a role in cell wall turnover and cell separation, but in S. aureus most of the autolytic activity is unrelated to the lethal effect of cell wall antibiotics.     

Adaptation of a stable L-form of Bacillus subtilis to minimal salts medium without osmotic stabilizers.

An L-form isolated from Bacillus subtilis 168 was adapted to growth in a 340 mOsm minimal salts medium without the addition of osmotically protective solutes. This L-form had no chemically detectable peptidoglycan residues on its surface, but 0.8% of the dry weight of washed membranes was hexosamine. The osmotic stability and susceptibility to bacitracin and vancomycin of the L-form adapted to growth in 340 mOsm osmotically unprotected medium was twice that of the L-form grown in 2,680 mOsm medium supplemented with 1.2 M NaCl.     

Examination of the “null” model of connor and simberloff for species co-occurrences on Islands

Summary Among birds of the Bismarck Archipelago, only certain combinations of the species in a guild coexist on islands, and some species that are very similar ecologically have mutually exclusive distributions. Diamond (1975) interpreted these patterns as biologically significant, involving effects such as competition. Connor and Simberloff (1979) claimed such patterns to be not recognizably different from random, because they were scarcely distinguishable from those generated by a null distribution supposedly not incorporating competition.On examining the analysis by Connor and Simberloff, we find that it actually yields the opposite conclusion: three faunas tested have grossly non-random structures, while their test is unworkable for the fourth fauna. The method of Connor and Simberloff for generating a null distribution has many fatal weaknesses: dilution of relevant data from guilds with irrelevant data from the whole species pool; hidden incorporation of effects of competition into the constraints; inability to recognize a checkerboard, the extreme example of a distribution produced by competition; reliance on inefficient Monte Carlo simulations; severe constraints that sometimes make generation of a null distribution impossible; failure to weight species combinations; and failure to identify the direction of non-randomness or the species combinations most responsible.Finally, we use other recent studies by Simberloff and coleagues to examine the value of constructing null hypotheses in community ecology.