Inhibition of surgical trauma-enhanced peritoneal dissemination of tumor cells by human catalase derivatives in mice

Surgical trauma, which is inevitably associated with the surgical removal of cancer, has been reported to accelerate tumor metastasis. The close association of reactive oxygen species with the trauma and tumor metastasis supports the possibility of using antioxidants for the inhibition of metastasis. To inhibit surgical trauma-enhanced peritoneal dissemination, human catalase (hCAT) derivatives, i.e., hCAT-nona-arginine peptide (hCAT-R9) and hCAT-albumin-binding peptide (hCAT-ABP), were designed to increase the retention time of the antioxidant enzyme in the abdominal cavity after intraperitoneal administration. Both ¹²⁵I-labeled derivatives showed significantly prolonged retention in the cavity compared to ¹²⁵I-hCAT. Cauterization of the cecum of mice with a hot iron, an experimental model of surgical trauma, induced abdominal adhesions. In addition, cauterization followed by colon26 tumor cell inoculation increased lipid peroxidation in the cecum and mRNA expression of molecules associated with tissue repair/adhesion and inflammation in the peritoneum. hCAT derivatives significantly suppressed the increased mRNA expression. The cauterization also increased the number of tumor cells in the abdominal organs, and the number was significantly reduced by hCAT-R9 or hCAT-ABP. These results indicate that hCAT-R9 and hCAT-ABP, both of which have a long retention time in the peritoneal cavity, can be effective at inhibiting surgery-induced peritoneal metastasis.     

Increase in CpG DNA-induced inflammatory responses by DNA oxidation in macrophages and mice

Unmethylated CpG dinucleotide (CpG motif) is involved in the exacerbation of DNA-associated autoimmune diseases. We investigated the effect of DNA containing 8-hydroxydeoxyguanosine (oxo-dG), a representative DNA biomarker for oxidative stress in the diseases, on CpG motif-dependent inflammatory responses. ODN1668 and ODN1720 were selected as CpG-DNA and non-CpG DNA, respectively. Deoxyguanosine in the CpG motif (G9) or outside the motif (G15) of ODN1668 was substituted with oxo-dG to obtain oxo(G9)-1668 and oxo(G15)-1668, respectively. Oxo(G15)-1668 induced a significantly higher amount of tumor necrosis factor (TNF)-α from RAW264.7 macrophage-like cells than ODN1668, whereas oxo(G9)-1668, oxo(G8)-1720, or oxo(G15)-1720 hardly did. CpG DNA-induced TNF-α production was significantly increased by addition of oxo(G8)-1720 or oxo(G15)-1720, but not of ODN1720. This oxo-dG-containing DNA-induced increase in TNF-α production was also observed in primary cultured macrophages isolated from wild-type mice, but not observed in those from Toll-like receptor (TLR)-9 knockout mice. In addition, TNF-α production by ligands for TLR3, TLR4, or TLR7 was not affected by oxo-dG-containing DNA. Then, the footpad swelling induced by subcutaneous injection of ODN1668 into mice was increased by coinjection with oxo(G8)-1720, but not with ODN1720. These results indicate that oxo-dG-containing DNA increases the CpG motif-dependent inflammatory responses, which would exacerbate DNA-related autoimmune diseases.     

<Emphasis Type="Italic">Rubus rosaefolius</Emphasis> Extract as a Natural Preservative Candidate in Topical Formulations

Even though the synthetic preservatives may offer a high antimicrobial efficacy, they are commonly related to adverse reactions and regarded as having potentially harmful effects caused by chronic consumption. The development of natural preservatives provides a way of reducing the amount of synthetic preservatives normally used in pharmaceutical and cosmetic preparations. In addition, these agents have less toxic effects and represent a possible natural and safer alternative of the preservatives. The purpose of this research was to evaluate the Rubus rosaefolius Smith extract efficiency as a natural preservative in base formulations. Of the extract, 0.2% (w/w) was assayed for its effectiveness of antimicrobial protection in two different base formulations (emulsion and gel). The microbial challenge test was performed following the standard procedures proposed by The United States Pharmacopoeia 33nd, European Pharmacopoeia 6th, Japanese Pharmacopoeia 15th, and the Cosmetics, Toiletries, and Fragrance Association using standardized microorganisms. The results demonstrated that R. rosaefolius extract at the studied concentration reduced the bacterial inocula, satisfying the criterion in all formulations, even though it was not able to present an effective preservative behavior against fungi. Thus, the investigation of new natural substances with preservative properties that could be applied in pharmaceutical and cosmetic products is relevant due to the possibility of substituting or decreasing the concentration of synthetic preservatives, providing a way for the development of safer formulas for the use of consumers.     

P38 mitogen-activated protein kinase inhibitor, FR167653, inhibits parathyroid hormone related protein-induced osteoclastogenesis and bone resorption

p38 mitogen-activated protein kinase (MAPK) acts downstream in the signaling pathway that includes receptor activator of NF-κB (RANK), a powerful inducer of osteoclast formation and activation. We investigated the role of p38 MAPK in parathyroid hormone related protein (PTHrP)-induced osteoclastogenesis in vitro and PTHrP-induced bone resorption in vivo. The ability of FR167653 to inhibit osteoclast formation was evaluated by counting the number of tartrate-resistant acid phosphatase positive multinucleated cells (TRAP-positive MNCs) in in vitro osteoclastgenesis assays. Its mechanisms were evaluated by detecting the expression level of c-Fos and nuclear factor of activated T cells c1 (NFATc1) in bone marrow macrophages (BMMs) stimulated with sRANKL and M-CSF, and by detecting the expression level of osteoprotegerin (OPG) and RANKL in bone marrow stromal cells stimulated with PTHrP in the presence of FR167653. The function of FR167653 on bone resorption was assessed by measuring the bone resorption area radiographically and by counting osteoclast number per unit bone tissue area in calvaria in a mouse model of bone resorption by injecting PTHrP subcutaneously onto calvaria. Whole blood ionized calcium levels were also recorded. FR167653 inhibited PTHrP-induced osteoclast formation and PTHrP-induced c-Fos and NFATc1 expression in bone marrow macrophages, but not the expression levels of RANKL and OPG in primary bone marrow stromal cells treated by PTHrP. Furthermore, bone resorption area and osteoclast number in vivo were significantly decreased by the treatment of FR167653. Systemic hypercalcemia was also partially inhibited. Inhibition of p38 MAPK by FR167653 blocks PTHrP-induced osteoclastogenesis in vitro and PTHrP-induced bone resorption in vivo, suggesting that the p38 MAPK signaling pathway plays a fundamental role in PTHrP-induced osteoclastic bone resorption.     

Morphological discrimination of two genetic groups of a Japanese salamander, Hynobius naevius (Amphibia, Caudata)

Hynobius naevius, distributed on western Honshu, Shikoku, and Kyushu Islands of Japan, includes two genetically distinct groups (Groups A and B) that have never been delimited morphologically. Using specimens from the entire species range, we investigated the possibility of distinguishing these groups morphologically. Multivariate analyses of morphometric characters resulted in recognition of two groups that corresponded well to the two genetic groups. One (Group A) was characterized by larger body, compressed tail, shallower vomerine tooth series, bluish- or reddish-purple ground color, and pale-white lateral markings. In contrast, another (Group B) was characterized by smaller body, cylindrical tail, longer vomerine tooth series, reddish-brown ground color, and white lateral markings. Group A was composed of populations from the Chugoku District of Honshu and northern Kyushu, and could not be divided into subgroups, while Group B encompassed populations from the Chubu and Kinki Districts of Honshu, Shikoku and Kyushu, and was subdivided into three local subgroups that are geographically separated by marine straits. Morphometric differentiation in Group A is presumed to have been less affected by genetic factors than by other factors, such as ecological relationships with other, coexisting species. Differentiation in Group B is assumed to have been enhanced not only by genetic but also by climatological factors.     

Indoor air microbiological evaluation of offices, hospitals, industries, and shopping centers

In this study it was compared the MAS-100 and the Andersen air samplers' performances and a similar trend in both instruments was observed. It was also evaluated the microbial contamination levels in 3060 samples of offices, hospitals, industries, and shopping centers, in the period of 1998 to 2002, in Rio de Janeiro city. Considering each environment, 94.3 to 99.4% of the samples were the allowed limit in Brazil (750 CFU/m3). The industries' results showed more important similarity among fungi and total heterotrophs distributions, with the majority of the results between zero and 100 CFU/m3. The offices' results showed dispersion around 300 CFU/m3. The hospitals' results presented the same trend, with an average of 200 CFU/m3. Shopping centers' environments showed an average of 300 CFU/m3 for fungi, but presented a larger dispersion pattern for the total heterotrophs, with the highest average (1000 CFU/m3). It was also investigated the correlation of the sampling period with the number of airborne microorganisms and with the environmental parameters (temperature and air humidity) through the principal components analysis. All indoor air samples distributions were very similar. The temperature and air humidity had no significant influence on the samples dispersion patterns.     

Genetic connectivity of the scleractinian coral Goniastrea aspera around the Okinawa Islands

Genetic variation and gene flow in the scleractinian coral Goniastrea aspera (Verrill), found around the north–south Okinawa Islands, were studied using allozyme and starch gel electrophoresis. The relative contribution of sexual and asexual reproduction to recruitment was determined. Analysis of multilocus genotypes of samples, collected at least 3 m apart, identified a high number of unique genotypes (N_G) relative to the number of individuals sampled (N) (mean N_G: N=0.97±0.03 [SD]), and also highly observed genotypic diversity (G_O) relative to expected genotypic diversity under Hardy–Weinberg equilibrium (G_E) (mean G_O: G_E=0.95±0.06 [SD]). These results suggest that the collected G. aspera propagated predominantly by sexual reproduction in Okinawan populations. The UPGMA grouping of five populations in the Okinawa, based on Neis unbiased genetic distance, showed two clusters that were south and north Okinawa populations. AMOVA analyses that incorporated the data of populations from the Kerama and the Ishigaki Islands detected significant F_SC values among the populations within groups in analyses distinguishing north–south Okinawa, Okinawa–Kerama, and Okinawa–Kerama–Ishigaki. Significant F_CT values were neither detected between north–south Okinawa nor between the Okinawa–Kerama groups. The local-recruits hypothesis, which assumes that a substantial proportion of recruits is produced locally, appears to be applicable to coral populations in the Ryukyu Archipelago. On the other hand, detection of nonsignificant F_CT values indicated the presence of genetic connectivity between north–south Okinawa and Kerama–Okinawa groups.     

A novel mechanism of autolysis in Helicobacter pylori: possible involvement of peptidergic substances

BACKGROUND: Helicobacter pylori survival in a hostile acidic environment is known to be caused by its production of urease, which is not released by known secretion pathways. It has been proposed that H. pylori cells undergo spontaneous autolysis during cultivation and that urease becomes surface-associated only concomitant with bacterial autolysis. The aim of this study was to elucidate mechanisms by which H. pylori cells undergo autolysis during cultivation. MATERIALS AND METHODS: Autolysis of H. pylori KZ109 cells was estimated by measuring the turbidity of the culture, by detection of cytoplasmic protein release into the culture supernatant and by scanning electron microscopic observation of H. pylori cells during cultivation. An autolysis-inducing factor (AIF) was partially purified from the culture supernatant by a partition method using ethyl acetate. RESULTS: Bacterial turbidity of KZ109 cells was drastically decreased after late-log phase accompanying release of urease and HspB into the extracellular space. Concomitantly, cell lytic activity was detected in the culture supernatant. Scanning electron microscopic observation suggested that partially purified AIF induced cell lysis. It was also shown that the AIF is different from other autolytic enzymes or substances so far reported. CONCLUSIONS: This study demonstrated the presence of the peptidergic autolytic substances in the culture supernatant of H. pylori KZ109 cells. The results of this study should be useful for further studies aimed at elucidation of the strategy of survival of H. pylori in the gastric environment and elucidation of the mechanisms of pathogenesis induced by H. pylori.     

Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells

Preparation of specific lineages at high purities from embryonic stem (ES) cells requires both selective culture conditions and markers to guide and monitor the differentiation. In this study, we distinguished definitive and visceral endoderm by using a mouse ES cell line that bears the gfp and human IL2R alpha (also known as CD25) marker genes in the goosecoid (Gsc) and Sox17 loci, respectively. This cell line allowed us to monitor the generation of Gsc+ Sox17+ definitive endoderm and Gsc- Sox17+ visceral endoderm and to define culture conditions that differentially induce definitive and visceral endoderm. By comparing the gene expression profiles of definitive and visceral endoderm, we identified seven surface molecules that are expressed differentially in the two populations. One of the seven markers, Cxcr4, to which a monoclonal antibody is available allowed us to monitor and purify the Gsc+ population from genetically unmanipulated ES cells under the condition that selects definitive endoderm.