Linker insertion analysis of the FimH adhesin of type 1 fimbriae in an Escherichia coli fimH-null background

Abstract The gene encoding the Escherichia coli FimH adhesin of type 1 fimbriae has been subjected to linker insertion mutagenesis. Amino acid changes were introduced at a number of positions spanning the entire sequence in order to probe the structure-function relationship of the FimH protein. The effect of these mutations on the ability of bacteria to express a D-mannose binding phenotype was assessed in a fimH null mutant (MS4) constructed by allelic exchange in the E. coli K-12 strain PC31. Mutations mapping at amino acid residues 36, 58 and 279 of the mature FimH protein were shown to completely abolish binding to D-mannose receptors. Differences in the level of fimbriation were also observed as a result of some of the mutations in the fimH gene. These mutants may prove useful in dissecting receptor-ligand interactions by defining regions of the FimH protein that are important in erythrocyte binding.     

Genomic organization of a mouse MHC class II region including theH2-M andLmp2 loci

The region encompassing theMa, Mb1, Mb2, andLmp2 genes of the mouse class II major histocompatibility complex (MHC) was sequenced. Since this region contains clusters of genes required for efficient class I and class II antigen presentation, it was interesting to search for putative additional genes in the 21 kilobase gap between theMb1 andLmp2 genes. Computer predictions of coding regions and CpG islands, exon trapping experiments, and cross-species comparison with the corresponding human sequence indicate that no additional functional gene is present in that stretch. However, computer analysis revealed the possible existence of an alternative 3 exon forMb1. Except for the fact that the mouse MHC contains twoMb genes, the genomic organization of theH2-M loci was found to be almost identical to the organization of the humanHLA-DM genes. The promoter regions of theMa andMb genes also resemble classical class II promoters, containing typical S, X, and Y boxes. Like the human genes, the threeH2-M genes displayed very limited polymorphism when we compared the cDNA sequences from six haplotypes. Finally, comparison ofDMB withMb1 andMb2, both at the genomic level and in their coding regions, suggests that theMb gene was recently duplicated, probably only in certain rodents.     

Power and work produced in different leg muscle groups when rising from a chair

The aim of this study was to determine the power output and work done by different muscle groups at the hip and knee joints during a rising movement, to be able to tell the degree of activation of the muscle groups and the relationship between concentric and eccentric work. Nine healthy male subjects rose from a chair with the seat at knee level. The moments of force about the hip and knee joints were calculated semidynamically. The power output (P) and work in the different muscle groups surrounding the joints was calculated as moment of force times joint angular velocity. Work was calculated as: work = f Pdt. The mean peak concentric power output was for the hip extensors 49.9 W, hip flexors 7.9 W and knee extensor 89.5 W. This power output corresponded to a net concentric work of 20.7 J, 1.0 J and 55.6 J, respectively. There was no concentric power output from the knee flexor muscles. Energy absorption through eccentric muscle action was produced by the hip extensors and hip flexors with a mean peak power output of 4.8 W and 7.4 W, respectively. It was concluded that during rising, the hip and knee muscles mainly worked concentrically and that the greatest power output and work were produced during concentric contraction of the knee and hip extensor muscles. There was however also a demand for eccentric work by the hip extensors as well as both concentric and eccentric work by the hip flexors. The knee flexor muscles were unloaded.     

Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes studies using the selective inhibitor wortmannin

Incubation of rat adipocytes with wortmannin, a potent and selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, completely blocked the antilipolytic action of insulin (IC₅₀≈ 100 nM), the insulin-induced activation and phosphorylation of cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) as well as the activation of the insulin-stimulated cGI-PDE kinase (IC₅₀≈ 10–30 nM). No direct effects of the inhibitor on the insulin-stimulated cGI-PDE kinase, the cGI-PDE and the hormone-sensitive lipase were observed. These data suggest that activation of PI 3-kinase upstream of the insulin-stimulated cGI-PDE kinase in the antilipolytic insulin signalchain has an essential role for insulin-induced cGI-PDE activation/ phosphorylation and anti-lipolysis.     

Comparative sensitivity analysis of stable stage structures and reproductive values

New formulas for deriving the sensitivities of stable stage structures and reproductive values to changes in vital rates are presented. They enable comparison of the sensities to changes of different elements in the projection matrix; in other words, comparison of partial derivatives of the eigenvectors. These kinds of sensitivities can be used in applied problems such as an analysis of the effect of harvesting on the population structure. However, in this paper, we examine the application of the sensitivities in a more general ecological context. We investigate why the stable stage structure of the mustard aphid,Lipaphis erysimi, changes very little in the temperature interval 10–30°C. The sensitivities of the stable stage structure at 15°C and 25°C were derived. The character of the sensitivites were the same in both temperatures although the stage structure was more sensitive to changes at 15°C than at 25°C. The sensitivity analysis also revealed that the temperature variation results in changes in fecundity and developmental rate that have a counteractive effect on the population structure.     

Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes

Glycinamide ribonucleotide (GAR) synthetase, GAR transformylase and aminoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 10-step de novo purine biosynthetic pathway. From a cDNA library of Arabidopsis thaliana, cDNAs encoding the above three enzymes were cloned by functional complementation of corresponding Escherichia coli mutants. Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transformylase or AIR synthetase. Comparisons of the three Arabidopsis purine biosynthetic enzymes with corresponding enzymes/polypeptide-fragments from procaryotic and eucaryotic sources indicate a high degree of conserved homology at the amino acid level, in particular with procaryotic enzymes. Assays from extracts of E. coli expressing the complementing clones verified the specific enzymatic activity of Arabidopsis GAR synthetase and GAR transformylase. Sequence analysis, as well as Northern blot analysis indicate that Arabidopsis has single and monofunctional enzymes. In this respect the organization of these three plant purine biosynthesis genes is fundamentally different from the multifunctional purine biosynthesis enzymes characteristic of other eucaryotes and instead resembles the one gene, one enzyme relationship found in procaryotes.     

Inhibition by 2,4-D of somatic embryogenesis in carrot as explored by its reversal by difluoromethylornithine

The development of somatic embryos is, in many plants, inhibited by 2,4-dichlorophenoxyacetic acid (2,4-D) and other auxins. The finding that difluoromethylornithine (DFMO) can counteract this inhibition has been used to test some of the hypotheses for the mechanism of inhibition.
Inhibition of somatic embryogenesis in carrot ( Daucus carota L.) by exogenous ethylene (from ethephon), antioxidants (ascorbic acid and glutathione), ethanol/acetaldehyde and abscisic acid was not counteracted by DFMO, indicating that the inhibitory effect of 2,4-D is not manifest through the formation of these compounds. Embryogenesis was abolished by micromolar concentrations of the polar auxin transport inhibitors 2, 3, 5-triiodobenzoic acid (TIBA), N-1-naphthylphthalamic acid (NPA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA). This inhibition was counteracted to a considerable extent by DFMO. Inhibition by relatively high concentrations of the antiauxin 2-( p -chlorophenoxy)-isobutyric acid (CPIB), which does not affect polar auxin transport, was in contrast not counteracted by DFMO. These findings indicate that exogenous auxins may inhibit embryogenesis by interfering with the ability of postglobular embryos to set up internal auxin gradients necessary for polarized growth.     

Whole inactivated SIV vaccine grown on human cells fails to protect against homologous SIV grown on simian cells

Several groups have reported protection against experimental SIV infection in macaques immunized with a whole inactivated virus vaccine. The aim of the current study was to investigate whether five macaques vaccinated with whole inactivated SIV and previously shown to be protected against challenge with two divergent strains of SIV grown on human cells could resist challenge with a subsequent homologous SIV grown on macaque cells. We show here that this same vaccine did not protect when the challenge virus was grown on primary cells of monkey origin.     

Evidence for the Colonization of Lactic Acid Bacteria in the Gastrointestinal Tract of Suckling Mink

Lactic acid bacteria are considered indigenous members of the gastrointestinal microflora in a number of animal species (Savage 1977a). Some intestinal strains of lactobacilli and streptococci are aWe to adhere to stratified squamous epithelium of some animals (Tannock et al. 1987), in the non-secreting part of the stomach of piglets (Barrow et al. 1980, Fuller et al. 1978) and rodents (Tannock et al. 1982), and in the crop of poultry (Fuller 1978). The presence of lactic acid bacteria in the digestive tract is believed to be of beneficial value to the host animal (Fuller 1989). The production of organic acids in the stomach or the crop helps maintaining a low pH which may be important for inhibiting the colonization of potentially pathogenic bacteria, particularly in the newborn animal (Barrow et al 1980, Fuller 1977, Fuller 1978). The adhesion of lactobacilli to squamous epithelium is host specific: strains capable of adhering to the epithelium of piglets are usually not able to adhere in rodents or poultry and vice versa (Fuller 1978, Lin & Savage 1984, Tannock et al 1982). Adhesion of lactic acid bacterial strains to other epithelia than stratified squamous epithelium has been reported. Thus, the attachment of lactobacilli to cells from the secreting epithelium of the murine stomach (Kotarski & Savage 1979), to intestinal cells of humans (Goldin & Gorbach 1987), and to columnar epithelial cells of piglets and calves (Mäyrä-Mäkinen et al 1983) has been demonstrated using in vitro methods. In another study the in vivo attachment of Enterococcus faecium to duodenal epithelium of gnotobi-otic chickens was demonstrated (Fuller et al 1981). Recent research indicated that in adult mink lactic acid bacteria are not indigenous members of the intestinal flora, and they do not attach to epithelium in any part of the gastrointestinal tract (Federsen & Jørgensen 1992). The present paper presents evidence that Gram positive cocci may colonize the gut of suckling mink kits and attach to the gut mucosa.     

Growth Hormone Secretion after Testosterone Administration to Men with Visceral Obesity

Visceral obese men were characterized by a decreased total GH secretion and diminished peak amplitude, size, and number. T-substitution was followed by elevation of IGF-I levels. The IGF-I increase correlated with the elevation of T-concentration, and was most pronounced in men with the lowest concentrations of free T from the outset. There were no detectable changes in total quantity, amplitude, size or number of peaks of GH secretion. Glucose, cholesterol and triglycerides as well as diastolic blood pressure decreased. There were no changes in thyroid or hematology variables. Visceral obesity in men has been reported to be characterized by low testosterone (T) and insulin-like growth factor I (IGF-I) concentrations, the latter suggesting a relative growth hormone (GH) deficiency. Since T and GH-secretions are interrelated, men with visceral obesity were substituted with T for 14 days, and diurnal secretion pattern of GH as well as IGF-I concentrations, and metabolic variables were followed. T-substitution of visceral obese men is followed by an elevation of IGF-I concentrations. It is suggested that this might be due either to minor, non-detectable increases in GH secretion, or to direct effects of T on IGF-I concentrations. The regulatory mechanisms by which T-administration are leading to metabolic and anthropometric improvements, might be direct effects of T, with or without mediation via GH secretion.