The response regulator CpxR directly regulates expression of several Legionella pneumophila icm/dot components as well as new translocated substrates

Legionella pneumophila has been shown to utilize the icm/dot type IV secretion system for pathogenesis. This system was shown to be composed of icm/dot complex components and accessory proteins, as well as a large number of translocated substrates. Bioinformatic analysis of the regulatory regions of all the genes revealed that several icm/dot genes, as well as two genes encoding icm/dot translocated substrates, contain the conserved CpxR regulatory element, a regulator that has been shown previously to control the expression of the icmR gene. An experimental analysis, which included a comparison of gene expression in a L. pneumophila wild-type strain and gene expression in a cpxR deletion mutant, construction of mutants with mutations in the CpxR conserved regulatory elements, controlled expression studies, and mobility shift assays, demonstrated the direct relationship between the CpxR regulator and the expression of the genes. Furthermore, genomic analysis identified nine additional genes that contain a putative CpxR regulatory element; five of these genes (two legA genes and three ceg genes) were suggested previously to be putative icm/dot translocated substrates. The three ceg genes identified, which were shown previously to contain a putative PmrA regulatory element, were found here to be regulated by both CpxR and PmrA. The other six genes (two legA genes and four new genes products were found to be regulated by CpxR. Moreover, using the CyaA translocation assay, these nine gene products were found to be translocated into host cells in an Icm/Dot-dependent manner. Our results establish that the CpxR regulator is a fundamental regulator of the icm/dot type IV secretion system in L. pneumophila.     

A New Avian Fibroblast Growth Factor Receptor in Myogenic and Chondrogenic Cell Differentiation

We studied the expression of FREK (fibroblast growth factor receptor-like embryonic kinase), a new receptor recently cloned from quail embryo, during the differentiation of skeletal muscle satellite cells and epiphyseal growth-plate chondrocytes. Although FREK mRNA was expressed in both cell types, satellite cells expressed higher levels of this mRNA than chondrocytes. FREK gene expression was found to be modulated by b-FGF in a biphasic manner: low concentrations increased expression, whereas high concentrations attenuated it. In both cell cultures, the levels of FREK mRNA declined during terminal differentiation. Moreover, retinoic acid (RA), which induces skeletal muscle satellite cells to differentiate, also caused a reduction in FREK gene expression in these cells. Induction of chondrocyte differentiation with ascorbic acid was monitored by a decrease in collagen type II gene expression and an increase in alkaline phosphatase activity. Satellite cell differentiation was marked by morphological changes as well as by increased sarcomeric myogenin content and creatine kinase activity and changes in the expression of the regulatory muscle-specific genes, MyoD and myogenin. DNA synthesis in both cell types was stimulated by b-FGF. However, in satellite cells, the response was bell-shaped, peaking at 1 ng/ml b-FGF, whereas in chondrocytes, higher levels of b-FGF were needed. b-FGF-dependent DNA synthesis in satellite cells was decreased by RA at concentrations over 10^-7M . The observed correlation between the level of FREK gene expression and various stages of differentiation, its modulation by b-FGF and RA, as well as the correlation between FREK gene expression and the physiological response to b-FGF, suggest that this specific FGF receptor plays an important role in muscle and cartilage cell differentiation.     

Oxidation of 2-mercaptoacetyl-phenylalanyl-leucine,a specific inhibitor ofPseudomonas aeruginosa elastase,in the presence of the bacteria and its prevention

The compound 2-mercaptoacetyl-l-phenylalanyl-l-leucine (HSAc-Phe-Leu) is a specific and potent inhibitor ofPseudomonas aeruginosa elastase with demonstrated potential as a drug for the treatment ofPseudomonas eye infections. The thiol moiety of this compound is lost in the presence of the bacterial at a rate greater than that seen in the absence of the organisms. Tritium-labeled inhibitor was prepared and used to examine whether this accelerated loss is due to the uptake of the inhibitor by the bacteria or due to its modification by some bacterial products. Our results excluded by possibility of uptake and indicated that the loss of the thiol resulted from its oxidation to the inactive disulfide form. The oxidation reaction is probably catalyzed by a low molecular weight extracellular bacterial product, and is effectively prevented in the presence of sodium bisulfite. It is suggested that HSAc-Phe-Leu preparations used for further investigation of the inhibitor's therapeutic potential should include antoxidants such as sodium bisulfite to provide maximal inhibitory capacity.     

Drosophila JAB1/CSN5 acts in photoreceptor cells to induce glial cells.

Different classes of photoreceptor neurons (R cells) in the Drosophila compound eye form connections in different optic ganglia. The R1-R6 subclass connects to the first optic ganglion, the lamina, and relies upon glial cells as intermediate targets. Conversely, R cells promote glial cell development including migration of glial cells into the target region. Here, we show that the JAB1/CSN5 subunit of the COP9 signalosome complex is expressed in R cells, accumulates in the developing optic lobe neuropil, and through the analysis of a unique set of missense mutations, is required in R cells to induce lamina glial cell migration. In these CSN5 alleles, R1-R6 targeting is disrupted. Genetic analysis of protein null alleles further revealed that the COP9 signalosome is required at an earlier stage of development for R cell differentiation.     

Zinc-desferrioxamine attenuates retinal degeneration in the rd10 mouse model of retinitis pigmentosa

Iron-associated oxidative injury plays a role in retinal degeneration such as age-related macular degeneration and retinitis pigmentosa. The metallo-complex zinc-desferrioxamine (Zn/DFO) may ameliorate such injury by chelation of labile iron in combination with release of zinc. We explored whether Zn/DFO can affect the course of retinal degeneration in the rd10 mouse model of retinitis pigmentosa. Zn/DFO-treated animals showed significantly higher electroretinographic responses at 3 and 4.5 weeks of age compared with saline-injected controls. Corresponding retinal (photoreceptor) structural rescue was observed by quantitative histological and immunohistochemical techniques. When administered alone, the components of the complex, Zn and DFO, showed a lesser, partial effect. TBARS, a marker of lipid peroxidation, and levels of oxidative DNA damage as quantified by 8-OHdG immunostaining were significantly lower in Zn/DFO-treated retinas compared with saline-injected controls. Reduced levels of retinal ferritin as well as reduced iron content within ferritin molecules were measured in Zn/DFO-treated retinas. The data, taken together, suggest that the protective effects of the Zn/DFO complex are mediated through modulation of iron bioavailability, leading to attenuation of oxidative injury. Reducing iron-associated oxidative stress using complexes such as Zn/DFO may serve as a “common pathway” therapeutic approach to attenuate injury in retinal degeneration.     

The influence of a male twin on birthweight of its female co-twin - a population-based study.

Fetal growth is influenced by various determinants, with males being heavier than females. It was recently suggested that female birthweight tends to be increased by the presence of a male co-twin. We evaluated this hypothesis in the Israeli population. We reviewed a retrospective population database of the 1993-1998 Israeli Birth Registry. We compared male birthweight in the male-male and male-female combination sets and female birthweight in the female-female and male-female combination sets. We compared the mean birthweight of males and females in three groups of total twin birthweight. The sample of 12,686 sets included 50.6% males. The male-male combination was found in 31.3% sets, female-female in 38.7% and unlike-sex combination in 30.0%. A total twin birthweight of less than 3000g was found in 7.2% sets, of 3001-5000g in 50.7% sets, and of over 5000g in 42.1% sets. The mean male birthweight was 2398 +/- 602 g and the mean female birthweight was 2316 +/- 566g, p <.0001 (95% CI of 67, 96). The mean birthweight of males in the male-male combination was significantly lower than in the unlike-sex combination, p <.0001, in total birthweights over 3000g. No significant difference was found between the mean birthweight of females in the female-female and in the unlike-sex combinations in all total birthweight strata. Our findings do not substantiate a male-related inter-twin influence on female birthweight. Population differences might underlie the opposing observations in the literature.