Streptococcal tetracycline resistance mediated at the level of protein synthesis.

The mechanism of tetracycline resistance was examined in strains containing each of the three previously identified resistance determinants in Streptococcus spp. Uptake of tetracycline was measured in tetracycline-sensitive cells as well as in cells containing each of the three resistance determinants. In cells containing tetL, uptake was not observed. However, in sensitive cells and cells containing either tetM or tetN, tetracycline was accumulated approximately 25-fold against a concentration gradient. Furthermore, there was no evidence for modification of intracellular tetracycline recovered from sensitive, tetM, or tetN cells. Protein synthesis in extracts derived from organisms containing tetM or tetN was resistant to tetracycline. In contrast, extracts of sensitive and tetL cells were sensitive to tetracycline.     

Insulin-induced decrease in 5'-nucleotidase activity in skeletal muscle membranes

Insulin releases inositol phosphoglycans from myocytes in culture [(1986) Science 233, 967-972], which display insulinomimetic activity. Because 5'-nucleotidase is anchored to the membrane through inositol-containing phospholipid glycans, we investigated whether insulin could release the enzyme from the membrane. Membranes prepared from hindquarter muscles of rats perfused with insulin showed a 23% decrease in 5'-nucleotidase activity. Isolated membranes from muscle exposed to insulin in vitro also showed a small but reproducible decrease (9%) in 5'-nucleotidase activity relative to unexposed controls. Phospholipase C from Staphylococcus aureus released 60% of the membrane-bound 5'-nucleotidase. We propose that insulin may activate an endogenous phospholipase C that cleaves phospholipid-glycan-anchored proteins.     

Extracellularly truncated desmoglein 1 compromises desmosomes in MDCK cells

Bacteriorhodopsin and Halorhodopsin present in Halobacterium halobium strains have been investigated in relation to Na⁺/H⁺ exchange in isolated cell envelope vesicles. Upon illumination, these retinal proteins result in extrusion of sodium ions by either an electrogenic Na⁺/H⁺ antiporter and/or a direct sodium pump. Since a molecular characterization of these mechanism(s) of sodium extrusion has not yet been realized, it was of interest to measure directly the light- and sodium-dependent changes in ΔpH and membrane potential under nearly identical conditions in S₉ and R¹mR cell membrane vesicles to gain information on the relation of these retinal proteins to sodium extrusion. These activities were evaluated in terms of their dependence on light intensity, and on the inhibitory effect of chemical modifiers of carboxyl groups (carbodiimides); electroneutral exchanges (monensin and triphenyltin); digitoxin and some analogues; and phloretin. Under most of the conditions and treatments employed, light- and sodium-dependent ΔpH led to similar effects in both membrane vesicle types. Hence, it is concluded that the ΔpH and Δξ which arise from sodium transport occur by either a single mechanism or by one which shares common features.     

Purification and characterization of Tet(M), a protein that renders ribosomes resistant to tetracycline

The tet(M) tetracycline resistance gene has been found in a wide variety of clinically important bacteria. It has been shown previously (Burdett, V. (1986) J. Bacteriol. 165, 564-569) that the tet(M) gene product mediates resistance at the level of protein synthesis as judged by in vitro assay. Using this assay, large amounts of protein were purified from an Escherichia coli overproducer expressing the gene under control of a T7 promoter. The purified activity consists of a single polypeptide of molecular weight 68,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was confirmed to be the tet(M) gene product by amino-terminal sequence analysis. Purified Tet(M) has an associated ribosome-dependent GTPase with the specific activity being similar to that of the corresponding activity associated with elongation factor G. Since Tet(M) also displays substantial homology to elongation factor G throughout its length, Tet(M) may function as an analog of this elongation factor.     

Comparison of the leader sequences of four group A streptococcal M protein genes.

The 5' portions and flanking sequences of genes encoding types 1, 12, 24, and 6 M proteins were compared. Although the DNA sequences encoding the amino-termini of the mature M proteins had no obvious similarity, upstream sequences, and those encoding the signal peptides (leader sequences) of the four M protein genes had considerable similarity. In general, the 5' ends of all the leader sequences were more conserved than the 3' ends, although the M6 and M24 leader sequences had identical 3' ends. Sequence similarity among the deduced amino acid sequences of the four signal peptides was more extensive than the corresponding DNA sequences. We found that strict DNA similarity among all four sequences extended only to the ends of the hydrophilic amino-terminal regions of the signal peptides, but that amino acid sequence conservation continued to the ends of the respective hydrophobic cores. With the exception of the M6 and M24 sequences, the regions adjacent to the signal peptidase cleavage sites were highly variable.     

Biophysics of pole formation of gram-positive rods

During pole formation in Bacillus subtilis the inner and outer surfaces of the nascent pole are enlarged by almost exactly the same extent. This means that the stress is almost uniformly distributed throughout the polar wall. This differs from the situation in the cylindrical side wall, where most of the stress is exerted in the outer portions of the intact wall. Because the stress is shared more uniformly, the maximum strain in any part of the polar wall is reduced, compared with the maximum strain within the side wall. The lowered stress may account, in part, for the resistance of the polar wall to hydrolysis by autolytic enzymes under certain conditions. The shape of the newly completed pole is significantly different from the spherical shape that the hydrostatic pressure would tend to produce. It does, however, achieve the shape that maximizes the polar volume under the restrictions arising due to expansion along the circumference not being possible near the junction of cylindrical and polar wall.     

Effects of cyclophosphamide on the secondary palate development in golden Syrian hamster: teratology, morphology, and morphometry

Cyclophosphamide (CP), when injected in hamster mother between days 9 and 11 of pregnancy, was teratogenic in fetuses. On the basis of a morphological study it was deduced that CP delayed the reorientation of hamster palatal shelves by 16-20 h. In a subsequent experiment, in both control and CP-treated palatal shelves, the numbers of epithelial and mesenchymal cells were counted and cross-sectional area was measured. DNA synthesis, measured by 3H-thymidine incorporation, was used as an index of growth by cell proliferation. The results showed that during the vertical development of palatal shelves, the mesenchymal cells reached their peak number during the initial 24 hours, i.e., at the end of the second peak in DNA synthesis, and remained unchanged thereafter throughout reorientation. The shelf area also showed rapid increase during the initial 24 h followed by a spurt 2 h prior to reorientation. Cyclophosphamide prolonged the acquisition of these features by affecting the mesenchymal cells and consequently delayed the reorientation of the vertical shelves until such time that the number of healthy mesenchymal cells and shelf area were restored to the control values. The data lend further support to the hypothesis that the acquisition of a specific number of cells and shelf volume, during vertical palatal development, may be essential for palatal shelf reorientation.     

Genotypes and haplotypes in the insulin-like growth factors, their receptors and binding proteins in relation to plasma metabolic levels and mammographic density

Background

Chronic inflammatory diseases including inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis), psoriasis and rheumatoid arthritis (RA) afflict millions of people worldwide, but their pathogenesis is still not well understood. It is also not well known if distinct changes in gene expression characterize these diseases and if these patterns can discriminate between diseased and control patients and/or stratify the disease. The main focus of our work was the identification of novel markers that overlap among the 3 diseases or discriminate them from each other.

Methods

Diseased (n = 13, n = 15 and n = 12 in IBD, psoriasis and RA respectively) and healthy patients (n = 18) were recruited based on strict inclusion and exclusion criteria; peripheral blood samples were collected by clinicians (30 ml) in Venous Blood Vacuum Collection Tubes containing EDTA and peripheral blood mononuclear cells were separated by Ficoll gradient centrifugation. RNA was extracted using Trizol reagent. Gene expression data was obtained using TaqMan Low Density Array (TLDA) containing 96 genes that were selected by an algorithm and the statistical analyses were performed in Prism by using non-parametric Mann-Whitney U test (P-values < 0.05).

Results

Here we show that using a panel of 96 disease associated genes and measuring mRNA expression levels in peripheral blood derived mononuclear cells; we could identify disease-specific gene panels that separate each disease from healthy controls. In addition, a panel of five genes such as ADM, AQP9, CXCL2, IL10 and NAMPT discriminates between all samples from patients with chronic inflammation and healthy controls. We also found genes that stratify the diseases and separate different subtypes or different states of prognosis in each condition.

Conclusions

These findings and the identification of five universal markers of chronic inflammation suggest that these diseases have a common background in pathomechanism, but still can be separated by peripheral blood gene expression. Importantly, the identified genes can be associated with overlapping biological processes including changed inflammatory response. Gene panels based on such markers can play a major role in the development of personalized medicine, in monitoring disease progression and can lead to the identification of new potential drug targets in chronic inflammation.