Overexpression of Toll-like receptor 4 amplifies the host response to lipopolysaccharide and provides a survival advantage in transgenic mice

Toll-like receptors are transmembrane proteins that are involved in the innate immune recognition of microbial constituents. Among them, Toll-like receptor 4 (Tlr4) is a crucial signal transducer for LPS, the major component of Gram-negative bacteria outer cell membrane. The contribution of Tlr4 to the host response to LPS and to infection with virulent Salmonella typhimurium was studied in four transgenic (Tg) strains including three overexpressing Tlr4. There was a good correlation between the level of Tlr4 mRNA expression and the sensitivity to LPS both in vitro and in vivo: Tg mice possessing the highest number of Tlr4 copies respond the most to LPS. Overexpression of Tlr4 by itself appears to have a survival advantage in Tg mice early during infection: animals possessing more than two copies of the gene survived longer and in a greater percentage to Salmonella infection. The beneficial effect of Tlr4 overexpression is greatly enhanced when the mice present a wild-type allele at natural resistance-associated macrophage protein 1, another critical innate immune gene involved in resistance to infection with SALMONELLA: Tlr4 and natural resistance-associated macrophage protein 1 exhibit functional epistatic interaction to improve the capacity of the host to control bacterial replication. However, this early improvement in disease resistance is not conducted later during infection, because mice overexpressing Tlr4 developed an excessive inflammatory response detrimental to the host.     

Protein kinase D: a family affair

The protein kinase D family of enzymes consists of three isoforms: PKD1/PKCmu PKD2 and PKD3/PKCnu. They all share a similar architecture with regulatory sub-domains that play specific roles in the activation, translocation and function of the enzymes. The PKD enzymes have recently been implicated in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, metastasis, immune responses, apoptosis and cell proliferation.     

Composition,acquisition, and distribution of the Vi exopolysaccharide-encoding Salmonella enterica pathogenicity island SPI-7

Vi capsular polysaccharide production is encoded by the viaB locus, which has a limited distribution in Salmonella enterica serovars. In S. enterica serovar Typhi, viaB is encoded on a 134-kb pathogenicity island known as SPI-7 that is located between partially duplicated tRNA(pheU) sites. Functional and bioinformatic analysis suggests that SPI-7 has a mosaic structure and may have evolved as a consequence of several independent insertion events. Analysis of viaB-associated DNA in Vi-positive S. enterica serovar Paratyphi C and S. enterica serovar Dublin isolates revealed the presence of similar SPI-7 islands. In S. enterica serovars Paratyphi C and Dublin, the SopE bacteriophage and a 15-kb fragment adjacent to the intact tRNA(pheU) site were absent. In S. enterica serovar Paratyphi C only, a region encoding a type IV pilus involved in the adherence of S. enterica serovar Typhi to host cells was missing. The remainder of the SPI-7 islands investigated exhibited over 99% DNA sequence identity in the three serovars. Of 30 other Salmonella serovars examined, 24 contained no insertions at the equivalent tRNA(pheU) site, 2 had a 3.7-kb insertion, and 4 showed sequence variation at the tRNA(pheU)-phoN junction, which was not analyzed further. Sequence analysis of the SPI-7 region from S. enterica serovar Typhi strain CT18 revealed significant synteny with clusters of genes from a variety of saprophytic bacteria and phytobacteria, including Pseudomonas aeruginosa and Xanthomonas axonopodis pv. citri. This analysis suggested that SPI-7 may be a mobile element, such as a conjugative transposon or an integrated plasmid remnant.     

Inflammatory microcrystals alter the functional phenotype of human osteoblast-like cells in vitro: synergism with IL-1 to overexpress cyclooxygenase-2

Chronic crystal-associated arthropathies such as gout and pseudogout can lead to local bone destruction. Because osteoblasts, which orchestrate bone remodeling via soluble factors and cell-to-cell interactions, have been described in contact with microcrystals, particularly in uratic foci of gout, we hypothesized that microcrystals of monosodium urate monohydrate (MSUM) and of calcium pyrophosphate dihydrate (CPPD) could alter osteoblastic functions. MSUM and CPPD adhered to human osteoblastic cells (hOB) in vitro and were partly phagocytized as shown by scanning electron microscopy. MSUM and CPPD dose-dependently stimulated the production of PGE(2) in hOB as assessed by enzyme immunoassay, a response that was synergistically enhanced in the presence of IL-1. The mechanism of this synergism was, at least in part, at the level of the expression of cyclooxygenase-2 as evaluated by immunoblot analysis. MSUM and CPPD also stimulated the expression of IL-6 and IL-8 and reduced the 1,25-dihydroxyvitamin D(3)-induced activity of alkaline phosphatase and osteocalcin in hOB (with no synergism with IL-1). MSUM- or CPPD-stimulated expression of IL-6 in hOB pretreated with the selective cyclooxygenase-2 inhibitor NS-398 was increased, unlike that induced by IL-1 alone which was partially reduced. MSUM-, CPPD- or IL-1-induced expression of IL-8 was unchanged by pretreating hOB with NS-398. These results suggest that inflammatory microcrystals alter the normal phenotype of hOB, redirecting them toward reduced bone formation and amplified osteoblast-mediated bone resorption, abnormalities that could play a role in the bone destruction associated with chronic crystal-induced arthritis.     

Human alpha-fetoprotein binds to primary macrophages

We have previously reported that alpha-fetoprotein (AFP) inhibits infection of human monocyte-derived macrophages (MDM) by R5-HIV-1 strains and that a peptide mimicking the clade B HIV-1 gp120 consensus V3 domain (V3Cs) binds to CCR5. We demonstrate here that AFP binds high- and low-affinity binding sites of MDM, characterized, respectively, by 5.15 and 100nM K(d) values. Heat denaturation or neuraminidase treatment of AFP inhibits this binding, suggesting the involvement of protein-protein and lectin-carbohydrate interactions. Moreover, AFP displaces V3Cs binding to MDM. In addition, MIP-1beta, the most specific CCR5 ligand, displaces AFP binding to MDM (IC(50)=4.3nM). Finally, we demonstrate that AFP binds to a ligand of HIV-gp120 V3Cs domain, CCR5, expressed by MDM and by HeLa cells expressing CCR5. Such binding is not observed in the presence of HeLa cells lacking CCR5. The present results provide strong evidence that AFP directly binds to CCR5 expressed by human primary macrophages and by transfected CCR5+ HeLa cells.     

Pitx3 is required for motor activity and for survival of a subset of midbrain dopaminergic neurons

Mesencephalic dopaminergic (MesDA) neurons play crucial roles in motor and behavioral processes; their loss in Parkinson's disease (PD) results in striatal dopamine (DA) deficiency and hypokinetic movement disorder. The Pitx3 homeobox gene is expressed in the MesDA system. We now show that only a subset of MesDA neurons express Pitx3 and that in Pitx3-deficient aphakia mice, this subset is progressively lost by apoptosis during fetal (substantia nigra, SN) and postnatal (ventral tegmental area) development, resulting in very low striatal DA and akinesia. Similar to human PD, dorsal SN neurons (which are Pitx3 negative) are spared in mutant mice. Thus, Pitx3 defines a pathway for survival of neurons that are implicated in PD and that are required for spontaneous locomotor activity.     

The comparative amino acid sequences, substrate specificities and gene or cDNA nucleotide sequences of some prokaryote and eukaryote amidinotransferases: implications for evolution

The amino acid sequences of the amidinotransferases and the nucleotide sequences of their genes or cDNA from four Streptomyces species (seven genes) and from the kidneys of rat, pig, human and human pancreas were compared. The overall amino acid and nucleotide sequences of the prokaryotes and eukaryotes were very similar and further, three regions were identified that were highly identical. Evidence is presented that there is virtually zero chance that the overall and high identity regions of the amino acid sequence similarities and the overall nucleotide sequence similarities between Streptomyces and mammals represent random match. Both rat and lamprey amidinotransferases were able to use inosamine phosphate, the amidine group acceptor of Streptomyces. We have concluded that the structure and function of the amidinotransferases and their genes has been highly conserved through evolution from prokaryotes to eukaryotes. The evolution has occurred with: (1) a high degree of retention of nucleotide and amino acid sequences; (2) a high degree of retention of the primitive Streptomyces guanine+cytosine (G+C) third codon position composition in certain high identity regions of the eukaryote cDNA; (3) a decrease in the specificities for the amidine group acceptors; and (4) most of the mutations silent in the regions suggested to code for active sites in the enzymes.     

High Expression of KCa3.1 in Patients with Clear Cell Renal Carcinoma Predicts High Metastatic Risk and Poor Survival

Background

Ca²⁺-activated K⁺ channels have been implicated in cancer cell growth, metastasis, and tumor angiogenesis. Here we hypothesized that high mRNA and protein expression of the intermediate-conductance Ca²⁺-activated K⁺ channel, KCa3.1, is a molecular marker of clear cell Renal Cell Carcinoma (ccRCC) and metastatic potential and survival.

Methodology/Principal Findings

We analyzed channel expression by qRT-PCR, immunohistochemistry, and patch-clamp in ccRCC and benign oncocytoma specimens, in primary ccRCC and oncocytoma cell lines, as well as in two ccRCC cell lines (Caki-1 and Caki-2). CcRCC specimens contained 12-fold higher mRNA levels of KCa3.1 than oncocytoma specimens. The large-conductance channel, KCa1.1, was 3-fold more highly expressed in ccRCC than in oncocytoma. KCa3.1 mRNA expression in ccRCC was 2-fold higher than in the healthy cortex of the same kidney. Disease specific survival trended towards reduction in the subgroup of high-KCa3.1-expressing tumors (p<0.08 vs. low-KCa3.1-expressing tumors). Progression-free survival (time to metastasis/recurrence) was reduced significantly in the subgroup of high-KCa3.1-expressing tumors (p<0.02, vs. low-KCa3.1-expressing tumors). Immunohistochemistry revealed high protein expression of KCa3.1 in tumor vessels of ccRCC and oncocytoma and in a subset of ccRCC cells. Oncocytoma cells were devoid of KCa3.1 protein. In a primary ccRCC cell line and Caki-1/2-ccRCC cells, we found KCa3.1-protein as well as TRAM-34-sensitive KCa3.1-currents in a subset of cells. Furthermore, Caki-1/2-ccRCC cells displayed functional Paxilline-sensitive KCa1.1 currents. Neither KCa3.1 nor KCa1.1 were found in a primary oncocytoma cell line. Yet KCa-blockers, like TRAM-34 (KCa3.1) and Paxilline (KCa1.1), had no appreciable effects on Caki-1 proliferation in-vitro.

Conclusions/Significance

Our study demonstrated expression of KCa3.1 in ccRCC but not in benign oncocytoma. Moreover, high KCa3.1-mRNA expression levels were indicative of low disease specific survival of ccRCC patients, short progression-free survival, and a high metastatic potential. Therefore, KCa3.1 is of prognostic value in ccRCC.