[Age related molecular-genetic preconditions for dental caries in pregnant women]. / Molekuliarno-geneticheskie predposylki kariesa zubov u beremennykh raznogo vozrasta.

The aim of the study was to evaluate the allelic polymorphisms kallikrein-4 (KLK-4) gene at the mutant points: G2664153A and G2142A in pregnant women under and over 30 of age. In pregnant women with KLK-4 gene polymorphisms A/A and G/A genotypes the rate of tooth decay growth increases in spite of applying the ternary calcium-phosphate-fluoride-containing gel. This genotype is also associates with unfavorable alteration of such oral fluid indicators as pH, concentrations of inorganic phosphorus, the active concentrations of calcium and potassium, as well as the ratio of total calcium and phosphorus concentrations, the active concentrations of electrolytes, and demineralizing activity of oral fluid.

Omicseq: a web-based search engine for exploring omics datasets.

The development and application of high-throughput genomics technologies has resulted in massive quantities of diverse omics data that continue to accumulate rapidly. These rich datasets offer unprecedented and exciting opportunities to address long standing questions in biomedical research. However, our ability to explore and query the content of diverse omics data is very limited. Existing dataset search tools rely almost exclusively on the metadata. A text-based query for gene name(s) does not work well on datasets wherein the vast majority of their content is numeric. To overcome this barrier, we have developed Omicseq, a novel web-based platform that facilitates the easy interrogation of omics datasets holistically to improve 'findability' of relevant data. The core component of Omicseq is trackRank, a novel algorithm for ranking omics datasets that fully uses the numerical content of the dataset to determine relevance to the query entity. The Omicseq system is supported by a scalable and elastic, NoSQL database that hosts a large collection of processed omics datasets. In the front end, a simple, web-based interface allows users to enter queries and instantly receive search results as a list of ranked datasets deemed to be the most relevant. Omicseq is freely available at http://www.omicseq.org.

Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells.

Osteosarcoma (OS) is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a) inflammation and immunity; (b) formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium), quantity of gap junctions and skeletogenesis; (c) bone mineral density; and (d) cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 (FGF1 and FGF12), bone morphogenetic factor-1 (BMP1), SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 (SMARCA4), Matrix extracellular phosphoglycoprotein (MEPE), Integrin, ß4 (ITGBP4), Matrix Metalloproteinase -1, -28 (MMP1 and MMP28), and signal transducer and activator of transcription-4 (STAT4) in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1, MMP28 and kallikrein related peptidase-7 (KLK7), but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology.

Red Maca (Lepidium meyenii) did not affect cell viability despite increased androgen receptor and prostate-specific antigen gene expression in the human prostate cancer cell line LNCaP.

We examined whether aqueous extract of Lepidium meyenii (red Maca) could inhibit growth, potentiate apoptotic activity of two anticancer drugs Taxol and 2-methoxyestradiol (2ME) or change mRNA expression for the androgen target genes, androgen receptor (Ar) and prostate-specific antigen (Psa) in the human prostate cancer cell line LNCaP. Red Maca aqueous extract at 0, 10, 20, 40 or 80 µg/ml was added to LNCaP cells, and viability was evaluated by the MTS assay at 24 or 48 hr after treatment. Furthermore, LNCaP cells were treated with 80 µg/ml of red Maca plus Taxol or 2ME 5 µM and viability was assessed 48 hr later. Finally, LNCaP cells were treated with red Maca 0, 20, 40 or 80 µg/ml, and 12 hr later, mRNA level for Ar or Psa was assessed by real-time PCR. Treatment with red Maca did not affect viability of LNCaP cells. Apoptotic activity induced by Taxol and 2ME in LNCaP cells was not altered with red Maca treatment. Relative expression of the mRNA for Ar and Psa increased with red Maca 20 and 40 µg/ml, but not at 80 µg/ml. We conclude that red Maca aqueous extract does not have toxic effects, but stimulates androgen signalling in LNCaP cells.

LPS/TLR4 Signaling Enhances TGF-ß Response Through Downregulating BAMBI During Prostatic Hyperplasia.

Compelling evidence suggests that benign prostatic hyperplasia (BPH) development involves accumulation of mesenchymal-like cells derived from the prostatic epithelium by epithelial-mesenchymal transition (EMT). Transforming growth factor (TGF)-ß induces EMT phenotypes with low E-cadherin and high vimentin expression in prostatic epithelial cells. Here we report that LPS/TLR4 signalling induces down-regulation of the bone morphogenic protein and activin membrane-bound inhibitor (BAMBI), which enhances TGF-ß signalling in the EMT process during prostatic hyperplasia. Additionally, we found that the mean TLR4 staining score was significantly higher in BPH tissues with inflammation compared with BPH tissues without inflammation (5.13 ± 1.21 and 2.96 ± 0.73, respectively; P < 0.001). Moreover, patients with inflammatory infiltrate were more likely to have a higher age (P = 0.020), BMI (P = 0.026), prostate volume (P = 0.024), total IPSS score (P = 0.009) and IPSS-S (P < 0.001). Pearson's correlation coefficient and multiple regression analyses demonstrated that TLR4 mRNA expression level was significantly positively associated with age, BMI, serum PSA levels, urgency and nocturia subscores of IPSS in the inflammatory group. These findings provide new insights into the TLR4-amplified EMT process and the association between TLR4 levels and storage LUTS, suggesting chronic inflammation as vital to the pathogenesis of BPH.

Maternal plasma folate impacts differential DNA methylation in an epigenome-wide meta-analysis of newborns.

Folate is vital for fetal development. Periconceptional folic acid supplementation and food fortification are recommended to prevent neural tube defects. Mechanisms whereby periconceptional folate influences normal development and disease are poorly understood: epigenetics may be involved. We examine the association between maternal plasma folate during pregnancy and epigenome-wide DNA methylation using Illumina's HumanMethyl450 Beadchip in 1,988 newborns from two European cohorts. Here we report the combined covariate-adjusted results using meta-analysis and employ pathway and gene expression analyses. Four-hundred forty-three CpGs (320 genes) are significantly associated with maternal plasma folate levels during pregnancy (false discovery rate 5%); 48 are significant after Bonferroni correction. Most genes are not known for folate biology, including APC2, GRM8, SLC16A12, OPCML, PRPH, LHX1, KLK4 and PRSS21. Some relate to birth defects other than neural tube defects, neurological functions or varied aspects of embryonic development. These findings may inform how maternal folate impacts the developing epigenome and health outcomes in offspring.

Anti-Androgenic Activity of Icarisid II from Epimedium Herb in Prostate Cancer LNCaP Cells.

Anti-androgens are regarded as potential therapeutic agents for the treatment of prostate cancer. We determined that an epimedium herb (EH) extract exhibited anti-androgenic activity in a luciferase assay using androgen receptor-positive prostate cancer LNCaP cells. Nine EH-derived flavonoids were examined. The results identified icarisid II as a very potent anti-androgenic EH-derived flavonoid. A quantitative RT-PCR analysis confirmed that the flavonol suppressed the expression of the androgen-responsive KLK3 gene.

Anti-androgenic activity of hydroxyxanthones in prostate cancer LNCaP cells.

Anti-androgens are used to treat prostate cancer. Here, we report that hydroxyxanthones from a plant extract act as anti-androgens in androgen receptor (AR)-positive prostate cancer LNCaP cells. Anti-androgenic activity of the ethanol extract from Garcinia subelliptica was observed in a luciferase assay using LNCaP/MMTV cells with a stably integrated mouse mammary tumor virus (MMTV) promoter. HPLC-based activity profiling followed by a chemical library-based assay strategy enabled the rapid identification of several active principles bearing a xanthone core substituted with hydroxyl and isoprenyl groups. Among the active compounds, 2-(1,1-dimethyl-allyl)-1,4,5,6-tetrahydroxyxanthone (subelliptenone F) was identified as a potent inhibitor of AR transcriptional activity. The structure-activity relationship of some substituents on the xanthone core was also determined using the chemical library-based bioassay. A quantitative RT-PCR analysis revealed that treatment with the compound resulted in a significant reduction in AR-induced gene (KLK3) expression. Hydroxyxanthone may be a possible candidate for the development of a new anti-androgenic molecule.

Effects of a phytoestrogen-containing soy extract on the growth-inhibitory activity of ICI 182 780 in an experimental model of estrogen-dependent breast cancer.

The study reported here was designed to determine whether a phytoestrogen-containing soy extract (SSE) could negate/overwhelm the inhibitory effects of ICI 182 780 on the growth of estrogen-sustained human breast cancer xenografts (MCF-7), in ovariectomized athymic mice. As expected, estradiol-supplemented tumors did not grow over the study period in ICI 182 780-treated females; concomitant administration of 50 mg/kg per day SSE slightly potentiated the inhibitory activity of the drug, while at 100 mg/kg per day, SSE partially negated ICI 182 780 activity. In keeping with these in vivo outcomes, we observed that the level of cyclin D1 (and progesterone receptor) in MCF-7 xenografts was considerably reduced by ICI 182 780, an effect enhanced by concomitant treatment with 50 SSE, but reduced by the higher dosage (i.e. 100 mg/kg per day). Thrombospondin-1 (TSP-1) and kallikrein 6 (KLK6) levels were also reduced following ICI 182 780, although to a lesser degree; again, combined anti-estrogen and SSE produced a dose-dependent regulation in TSP-1 and KLK6 tumor level, with a further reduction in the mRNA gene expression at 50 SSE (compared with ICI 182 780) and a partial reversion of the drug-induced down-regulation at 100 mg/kg per day. No modulation was detected in the serum concentration of IGF-1 (a potent mitogen for estrogen receptor-positive breast cancer cell lines) either upon treatment with ICI 182 780 or concomitant administration of the anti-estrogen with SSE. In conclusion, results from this study raise concerns about the consumption of isoflavone supplements in conjunction with ICI 182 780 therapy, in postmenopausal women with estrogen-dependent breast cancer.

Decidualization induces the expression and activation of an extracellular protease neuropsin in mouse uterus.

Uterine decidualization is accompanied by the remodeling of the cell-matrix and cell-cell interactions around the endometrial stromal cells to allow an appropriate invasion of trophoblasts. This remodeling is thought to require the proteolysis of extracellular matrix proteins or cell adhesion molecules; however, the molecular mechanism remains poorly understood. In this study, decidualization induced the expression and activation of an extracellular serine protease neuropsin in the mouse uterus. Although nonpregnant uteri contained little neuropsin, the protein content and enzymatic activity increased markedly and peaked at the midgestational period in pregnant uteri. Neuropsin expression and activity was also upregulated in artificially induced deciduomata but not in nondecidualized pseudopregnant uteri. Neuropsin is the first extracellular protease to show the evident induction of expression and activity by decidualization and might contribute to the remodeling of extracellular components after decidualization.

Cloning and characterization of mouse klk27, a novel tissue kallikrein expressed in testicular Leydig cells and exhibiting chymotrypsin-like specificity.

A cDNA clone of a new mouse tissue kallikrein, designated mKlk27, was isolated from an adult mouse testis cDNA library. mKlk27 was expressed in the submaxillary glands and testis of the mouse. In testis, mKlk27 gene was expressed exclusively in the Leydig cells of the adult mouse. Active recombinant mKlk27 exhibited chymotrypsin-like cleavage specificity. A single amino-acid substitution of Gly for Asp at position 209 in mKlk27 resulted in complete loss of its chymotryptic activity but acquisition of tryptic activity. mKlk27 effectively hydrolyzed casein, gelatin and fibronectin. Insulin-like growth factor binding protein-3 was also hydrolyzed by recombinant mKlk27. These results suggest that mKlk27 plays an important role in association with the function of the adult mouse testis.

Reduced cardiac hypertrophy and altered blood pressure control in transgenic rats with the human tissue kallikrein gene.

To evaluate the cardiovascular actions of kinins, we established a transgenic rat line harboring the human tissue kallikrein gene, TGR(hKLK1). Under the control of the zinc-inducible metallothionein promoter, the transgene was expressed in most tissues including the heart, kidney, lung, and brain, and human kallikrein was detected in the urine of transgenic animals. Transgenic rats had a lower 24-h mean arterial pressure in comparison with control rats, which was further decreased when their diet was supplemented with zinc. The day/night rhythm of blood pressure was significantly diminished in TGR(hKLK1) animals, whereas the circadian rhythms of heart rate and locomotor activity were unaffected. Induction of cardiac hypertrophy by isoproterenol treatment revealed a marked protective effect of the kallikrein transgene because the cardiac weight of TGR(hKLK1) increased significantly less, and the expression of atrial natriuretic peptide and collagen III as markers for hypertrophy and fibrosis, respectively, were less enhanced. The specific kinin-B2 receptor antagonist, icatibant, abolished this cardioprotective effect. In conclusion, the kallikrein-kinin system is an important determinant in the regulation of blood pressure and its circadian rhythmicity. It also exerts antihypertrophic and antifibrotic actions in the heart.

Potassium supplement upregulates the expression of renal kallikrein and bradykinin B2 receptor in SHR.

High potassium intake is known to attenuate hypertension, glomerular lesion, ischemic damage, and stroke-associated death. Our recent studies showed that expression of recombinant kallikrein by somatic gene delivery reduced high blood pressure, cardiac hypertrophy, and renal injury in hypertensive animal models. The aim of this study is to explore the potential role of the tissue kallikrein-kinin system in blood pressure reduction and renal protection in spontaneously hypertensive rats (SHR) on a high-potassium diet. Young SHR were given drinking water with or without 1% potassium chloride for 6 wk. Systolic blood pressure was significantly reduced beginning at 1 wk, and the effect lasted for 6 wk in the potassium-supplemented group compared with that in the control group. Potassium supplement induced 70 and 40% increases in urinary kallikrein levels and renal bradykinin B2 receptor density, respectively (P < 0.05), but did not change serum kininogen levels. Similarly, Northern blot analysis showed that renal kallikrein mRNA levels increased 2.7-fold, whereas hepatic kininogen mRNA levels remained unchanged in rats with high potassium intake. No difference was observed in beta-actin mRNA levels in the kidney or liver of either group. Competitive RT-PCR showed a 1.7-fold increase in renal bradykinin B2 receptor mRNA levels in rats with high potassium intake. Potassium supplement significantly increased water intake, urine excretion, urinary kinin, cAMP, and cGMP levels. This study suggests that upregulation of the tissue kallikrein-kinin system may be attributed, in part, to blood pressure-lowering and diuretic effects of high potassium intake.

Kallikrein-binding protein levels are reduced in the retinas of streptozotocin-induced diabetic rats.

PURPOSE: To determine the involvement of rat kallikrein-binding protein (RKBP) in the development of diabetic retinopathy. METHODS: Diabetes was induced by streptozotocin (STZ) (55 mg/kg body weight in 0.05 M citrate buffer, pH 4.5) in male Sprague-Dawley rats (150 to 175 g, 6 weeks old) as confirmed by hyperglycemia and reduced body weight. Retinas were dissected from animals at 1, 2, and 4 months of diabetes. The functional activity of RKBP in retinal homogenates was determined by its complex formation with tissue kallikrein. Immunoreactive RKBP levels were measured by enzyme-linked immunosorbent assay. The RKBP messenger RNA (mRNA) levels in the retina were measured by Northern blot analysis using the RKBP complementary DNA probe. The activity of total Na+,K(+)-ATPase was determined by a radioassay. Total protein concentration was determined by a protein assay. RESULTS: The kallikrein-binding activity was reduced in the retinas of STZ-diabetic rats at 1 (59%), 2 (50%), and 4 (38%) months of diabetes compared to those of age-matched control subjects. Levels of immunoreactive RKBP were significantly lower in the diabetic animals at each time point examined compared to those of control subjects. At 1 and 2 months of diabetes, RKBP levels (nanogram/milligram protein) were decreased significantly to 6.9 +/- 0.7 (n = 8) and 10.6 +/- 1.0 (n = 8), respectively, compared to those of age-matched control subjects (14.1 +/- 0.7, n = 8, P < 0.001, and 14.1 +/- 1.2, n = 8, P < 0.01). At 4 months of diabetes, retinal RKBP levels were lower in both control and diabetic groups, but RKBP levels in diabetic groups were significantly lower (5.8 +/- 0.6, n = 8) than those of the age-matched control subjects (8.4 +/- 0.9, n = 8, P < 0.01). Similarly, Northern blot analysis showed that RKBP mRNA levels were reduced in the retina of each group of STZ-diabetic rats, suggesting that the decrease in RKBP occurred at the level of transcription. CONCLUSIONS: The results show that STZ-induced diabetic rats have decreased retinal RKBP; moreover, this suggests that RKBP may contribute to diabetic retinopathy.

Expression of the renal kallikrein gene in mineralocorticoid-treated and genetically hypertensive rats.

On the basis of both clinical observations and experimental studies it has been proposed that renal kallikrein is a mineralocorticoid regulated protein. In other studies, changes in renal kallikrein activity have been implicated in the genesis of, and/or response to, hypertension. Using a cloned complementary DNA (cDNA) to rat pancreatic kallikrein (pcXP39) for hybridization histochemistry, and both Northern and dot blot analysis, we studied expression of the kallikrein gene in steroid-treated control animals, and in three strains of genetically hypertensive rats. No differences in renal kallikrein messenger RNA (mRNA) levels were found between adrenalectomized rats and those treated for 5-14 days with 9 alpha-fludrocortisone, corticosterone or dexamethasone, or between hypertensive rats and their appropriate controls. Since mRNA levels appear essentially invariant under such circumstances, the change in renal kallikrein activity/immunoreactivity after chronic mineralocorticoid elevation, or in hypertensive rats, presumably reflects modulation at the post-transcriptional level.