Dominant-Negative Form of SIGIRR: SIGIRRΔE8 Promotes Tumor Growth Through Regulation of Metabolic Pathways.

Colorectal carcinoma is the leading cause of cancer-related death. Previously we have shown that tumor suppressor single immunoglobulin interleukin-1-related receptor (SIGIRR) is frequently inactivated in human colorectal cancer by the increased expression of a novel SIGIRR isoform (SIGIRRΔE8). SIGIRRΔE8 showed increased retention in the cytoplasm and loss of complex glycan modification compared to the full-length SIGIRR. Now we found that the arginine residues located in the C-terminus of SIGIRRΔE8 serve as an endoplasmic reticulum retention signal and are required for resident protein ribophorin 1 (RPN1) interaction. In addition, we found that SIGIRRΔE8 exerts a direct impact on cell metabolism through interaction with the adenosine triphosphate synthase in the colorectal cancer cells. SIGIRRΔE8 expression promoted the metabolic shift through upregulation of mammalian target of rapamycin signaling pathway and dysregulation of mitochondrial function to promote survival and proliferation of colon cancer cells in xenograft model.

IL-17A Recruits Rab35 to IL-17R to Mediate PKCα-Dependent Stress Fiber Formation and Airway Smooth Muscle Contractility.

IL-17A is a critical proinflammatory cytokine for the pathogenesis of asthma including neutrophilic pulmonary inflammation and airway hyperresponsiveness. In this study, by cell type-specific deletion of IL-17R and adaptor Act1, we demonstrated that IL-17R/Act1 exerts a direct impact on the contraction of airway smooth muscle cells (ASMCs). Mechanistically, IL-17A induced the recruitment of Rab35 (a small monomeric GTPase) and DennD1C (guanine nucleotide exchange factor [GEF]) to the IL-17R/Act1 complex in ASMCs, resulting in activation of Rab35. Rab35 knockdown showed that IL-17A-induced Rab35 activation was essential for protein kinase Cα (PKCα) activation and phosphorylation of fascin at Ser39 in ASMCs, allowing F-actin to interact with myosin to form stress fibers and enhance the contraction induced by methacholine. PKCα inhibitor or Rab35 knockdown indeed substantially reduced IL-17A-induced stress fiber formation in ASMCs and attenuated IL-17A-enhanced, methacholine-induced contraction of airway smooth muscle. Taken together, these data indicate that IL-17A promotes airway smooth muscle contraction via direct recruitment of Rab35 to IL-17R, followed by PKCα activation and stress fiber formation.

The dynamics of vaginal and rectal Lactobacillus spp. flora in subsequent trimesters of pregnancy in healthy Polish women, assessed using the Sanger sequencing method.

BACKGROUND: Lactobacilli play an important role in maintaining vaginal health and protection against bacterial infections in the genital tract. The aim of this study is to show the dynamics of changes of the vaginal and rectal Lactobacillus flora during pregnancy by using the Sanger sequencing method. METHOD: The study included 31 healthy pregnant women without clinical signs of genitourinary infections. The material was taken in the three trimesters of pregnancy by vaginal and rectal swabs and grown on the MRS agar quantitatively to estimate the number of Lactobacillus spp. [CFU/ml]. Afterwards, 3 to 8 morphologically different lactobacilli colonies were taken for identification. Bacterial species identification was performed by 16 s rDNA sequence fragment analyses using the Sanger method. RESULTS: Among the patients tested, the most common species colonizing the vagina in the first trimester were: L. crispatus 29%, L. gasseri 19.4% and L. rhamnosus 16.1%, in the second trimester: L. crispatus 51.6%, L. gasseri 25.8%, L. rhamnosus 19.4% and L. amylovorus 16.1%, and in the third trimester the most common Lactobacillus species were: L. crispatus 25.8%, L. gasseri 25.8% and L. johnsonii 19.4%. In rectal species, the number decreased in the second and third trimesters in comparison to the first trimester (p = 0.003). An analysis of rectal dynamics showed that in the first trimester, the most common species were: L. johnsonii 19.4%, and L. plantarum 9.7%, in the second trimester: L. crispatus 9.7% and L. mucosae 6.5%, and in the third trimester: L. casei 9.7% and L. rhamnosus 9.7%. Individual dynamics of the Lactobacillus species composition showed variability, characterized by continuous, intermittent, or periodic colonization. The patients examined were mostly colonized by three Lactobacillus species in vagina (32.3%), whereas for the rectum, one Lactobacillus species during the whole pregnancy duration was common (32.3%). CONCLUSION: This study showed that in the examined group of healthy, pregnant Polish women, the vaginal Lactobacillus flora, both qualitative and quantitative, was stable during the three subsequent trimesters. In contrast, the number of rectal Lactobacillus species dramatically decreased after the first trimester.

Selection and analysis of a DNA aptamer binding α-amanitin from Amanita phalloides.

Mushroom foraging is very popular in some regions of the world. Sometimes toxic and edible mushrooms are mistaken by mushroom collectors, leading to serious human poisoning. The group of mushrooms highly dangerous for human health includes Amanita phalloides. This mushroom produces a toxic octapeptide called α-amanitin which is an inhibitor of nuclear RNA polymerase II. The inhibition of this polymerase results in the abortion of mRNA synthesis. The ingestion of A. phalloides causes liver failure due to the fact that most of the toxin is uptaken by hepatocytes. The hospitalization of poisoned patients involves the removal of the toxin from the digestive tract, its dilution in the circulatory system and the administration of therapeutic adjuvants. Since there is no effective antidote against amanitin poisoning, in this study we developed a DNA aptamer exhibiting specific binding to α-amanitin. This aptamer was selected using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method. Next, its ability of toxin removal from aqueous solution was confirmed by pull-down assay. The aptamer region sufficient for α-amanitin binding was determined. Finally, the dissociation constant of the α-amanitin/DNA aptamer complex was calculated.

Adherence of group B streptococci to human rectal and vaginal epithelial cell lines in relation to capsular polysaccharides as well as alpha-like protein genes - pilot study.

Streptococcus agalactiae (Group B Streptococci, GBS) constitutes a risk factor for infections of the newborns born by colonized mothers. The adherence of GBS to epithelial cells has been proved to be an important factor in the colonization of mucus membranes of both human rectum and vagina. The objective of the study was to assess the adhesion of the selected GBS strains to the human colon adenocarcinoma cell line (HT-29) and human epidermoid vulvo-vaginal cells (A-431) in relation to the capsular polysaccharides and alpha-like protein genes. GBS strains from the human sources belonging to Ia, Ib, II, III and V serotypes possessing different surface alpha-like protein genes such as the alp 2, alp 3, bca, epsilon and rib in the conventional adherence assay were examined. The adherence of GBS strains to the HT-29 cell line was considerably higher than to the A-431 cell line. For GBS serotype Ia and III, a significant difference between the adhesion to the HT-29 and A-431 cell lines was presented. The adhesion of GBS strains to the HT-29 cell line depended on alpha-like protein genes. The most adhesive ones were the GBS strains containing the rib and alp 2 genes. The adherence of GBS strains to the A-431 cell line depended on both their serotype and alpha-like protein genes. Serotype III adhered to the A-431 cells most tightly, particularly the strains containing the rib and alp 2 genes. GBS strains containing the rib gene adhered to the HT-29 and A-431 cell lines more firmly than GBS strains containing other alpha-like protein genes.

Antibacterial activity of selected standard strains of lactic acid bacteria producing bacteriocins--pilot study.

INTRODUCTION: In this paper, an attempt was made to evaluate the antibacterial potential of standard strains of lactic acid bacteria (LAB) producing bacteriocins of various classes, thus demonstrating various mechanisms of cell membrane damages against the Streptococcus agalactiae strains (Group B Streptococcus, GBS), depending on surface polysaccharides and surface alpha-like protein genes. MATERIALS/METHODS: Antimicrobial property of the strains of L. plantarum C 11, L. sakei DSMZ 6333, and L. lactis ATCC 11454 producing bacteriocins: JK and EF plantaricins, sakacin and nisin, respectively, against the GBS strains was evaluated. The chosen to the study GBS strains were represented by serotypes Ia, Ib, II, III, V and they had bca, epsilon, rib, alp2 or alp3 alpha-like protein genes. The experiment was conducted by means of suspension culture and the bacteria count was determined using the serial dilution method. RESULTS: A great ability of L. plantarum C 11 strain was proven to inhibit the GBS growth. The strain of L. sakei DSMZ 6333 did not demonstrate any ability to inhibit the growth of GBS, whereas L. lactis ATCC 11454 inhibited the growth of S. agalactiae indicator strains to a minor extent. Statistically significant differences were demonstrated between the GBS strains representing various serotypes against the antimicrobial activity of model LAB strains. The least sensitive to the activity of bacteriocins were the strains representing serotypes Ib and III, whereas the strains representing serotype II were the most sensitive. The sensitivity of the GBS strains to the antimicrobial activity of LAB was not dependent on alpha-like protein genes. DISCUSSION: Among the LAB standard strains producing bacteriocins, the strongest antimicrobial property was observed in the strain of L. plantarum C 11. Because of the generally known and verified strong antagonistic property of the strains of L. plantarum species against indicator bacteria, it is necessary to further pursue the research presented in this paper.