Antiviral activity of acyclic nucleoside phosphonates PMEA, (S)-HPMPC, PMEDAP and ribavirin against Cauliflower mosaic virus in Brassica pekinensis

Antiviral effects of acyclic nucleoside phosphonates PMEA, (S)-HPMPC, PMEDAP, and ribavirin on double-stranded DNA Cauliflower mosaic virus (CaMV) were evaluated in Brassica pekinensis plants grown in vitro on liquid medium. A double-antibody sandwich ELISA was used for relative quantification of viral protein and PCR for detection of CaMV nucleic acid in plants. Ribavirin and PMEA had no significant antiviral effect. (S)-HPMPC at concentration 50?mg?l^?1 and PMEDAP at concentrations 50 and 12.5?mg?l^?1 significantly (P?<?0.05) reduced CaMV concentration in plants within 42?C63?days to levels detectable neither by ELISA nor by PCR. A phytotoxicity experiment resulted in progressive yellowing of leaves and dwarfing in plants cultured 42?days on media with concentrations 12.5, 25 and 50?mg?l^?1 of (S)-HPMPC and PMEDAP. Reduction in fresh and dry weights of plants was significant (P?<?0.05) already at 12.5?mg?l^?1 with both compounds.     

Antiviral Activity of Selected Nucleoside Analogues Against Human Herpes Virus Type 6

Abstract

HHV-6 was evaluated in vitro for its susceptibility to a broad range of nucleoside analogues. PFA and several acyclic nucleoside phophonates emerged as the most potent inhibitors of HHV-6 replication as monitored by a newly developed immunofluorescence / flow cytometric assay as well as by microscopical evaluation of their inhibitory effect on HHV-6-induced cytopathogenicity.     

Flow Cytometry Demonstrates Bacteriocin-Induced Injury to Listeria monocytogenes

Flow cytometry was used to study the effect of the bacteriocin leucocin B-TA11a on Listeria (L.) monocytogenes. Mixed proportions of dead and live control populations were analyzed by flow cytometry to determine detection limits of the Dead/Live Baclight Bacterial Viability Kit^TM. High correlations for flow cytometric detection of defined proportions of live or dead cells in mixtures between 10 and 100% of dead (r² = 0.97) or live (r² = 0.99) cells were obtained. However, mixtures containing less than 10% of either live or dead control cells gave correlations below 0.72. The growth of L. monocytogenes in the absence and presence of leucocin B-TA11a was analyzed by flow cytometry with Baclight, plate counts, and optical density measurements. Although leucocin B-TA11a initially inhibited listerial growth, the uptake of both Baclight dyes suggested that cells remained viable but became leaky, possibly indicating bacteriocin-induced pore formation in the target membranes. Received: 30 June 1997 / Accepted: 20 October 1997     

Metagenome-derived haloalkane dehalogenases with novel catalytic properties

Applied Microbiology and Biotechnology - Haloalkane dehalogenases (HLDs) are environmentally relevant enzymes cleaving a carbon-halogen bond in a wide range of halogenated pollutants. PCR with...     

Iodine in early pregnancy--is there enough?

Aims: To evaluate the iodine status of patients in early pregnancy and its dependence on level of thyroid-stimulating hormone (TSH). Methods: Between June 2005 and December 2006, 168 patients with a confirmed vital pregnancy (up to 10(th) week of pregnancy) were included in the study. The entry criteria were no prior thyroid disease, did not take any other medication, had not undergone radio-iodine therapy and did not take multivitamins containing iodine. The iodine status was measured as the amount of iodine in urine over 24 hours. The TSH level was determined from the blood using chemiluminescence. Results: The average ioduria value in patients was found to be 3.04 micromol/24 hr, with the norm 0.6-2.4 micromol/ 24 hr, median 2.9, SD 1.5. None of the patients had a value lower than 0.9 micromol/24 hr. The average TSH value was 1.98 mIU/l, median was 1.31, SD 0.98. The laboratory limits were set to 0.25-3 mIU/l for pregnant women in the first trimester. Three pregnancies ended in miscarriage by week 12, 1 miscarriage occurred in week 22 and the other pregnancies concluded in delivery between weeks 38-41. Fourteen patients had TSH levels above 3 mIU/l with normal levels od free thyroxine (T4) : 10.3-25 pmol/l. Conclusions: The results of this study did not reveal any iodine deficit in any of the patients. However 14 patients had elevated TSH levels signalling subclinical or incipiently clinical hypothyroidism. These pacients underwent levothyroxine therapy after endocrinologist's consultations.     

Antibody microarray analyses of signal transduction protein expression and phosphorylation during porcine oocyte maturation

Kinex antibody microarray analyses was used to investigate the regulation of 188 protein kinases, 24 protein phosphatases, and 170 other regulatory proteins during meiotic maturation of immature germinal vesicle (GV+) pig oocytes to maturing oocytes that had completed meiosis I (MI), and fully mature oocytes arrested at metaphase of meiosis II (MII). Increases in apparent protein levels of protein kinases accounted for most of the detected changes during the GV to MI transition, whereas reduced protein kinase levels and increased protein phosphorylation characterized the MI to MII transition. During the MI to MII period, many of the MI-associated increased levels of the proteins and phosphosites were completely or partially reversed. The regulation of these proteins were also examined in parallel during the meiotic maturation of bovine, frog, and sea star oocytes with the Kinex antibody microarray. Western blotting analyses confirmed altered expression levels of Bub1A, IRAK4, MST2, PP4C, and Rsk2, and the phosphorylation site changes in the kinases Erk5 (T218 + Y220), FAK (S722), GSK3-beta (Y216), MEK1 (S217 + S221) and PKR1 (T451), and nucleophosmin/B23 (S4) during pig oocyte maturation.     

Multiple luteinizing hormone receptor (LHR) protein variants, interspecies reactivity of anti-LHR mAb clone 3B5, subcellular localization of LHR in human placenta, pelvic floor and brain, and possible role for LHR in the development of abnormal pregnancy, pelvic floor disorders and Alzheimer's disease

Distinct luteinizing hormone receptor (LHR) protein variants exist due to the posttranslational modifications. Besides ovaries, LHR immunoreactivity (LHRI) was also found in other tissues, such as the brain, fallopian tube, endometrium, trophoblast and resident tissue macrophages. The 3B5 mouse monoclonal antibody was raised against purified rat LHR. In rat, porcine and human ovaries, the 3B5 identified six distinct LHR bands migrating at ~92, 80, 68, 59, 52 and 48 kDa. Characteristic LHRI was detected in rat, human and porcine corpora lutea. During cellular differentiation, subcellular LHR distribution changed from none to granular cytoplasmic, perinuclear, surface, nuclear and no staining. There were also differences in vascular LHR expression – lack of LHRI in ovarian vessels and strong staining of vessels in other tissues investigated. In normal human term placentae, villous LHRI was associated with blood sinusoids and cytotrophoblast cells, and rarely detected in trophoblastic syncytium. In all abnormal placentae, the LHRI of sinusoids was absent, and syncytium showed either enhanced (immature placental phenotypes) or no LHRI (aged placental phenotype). LHRI in human brain was identified in microglial cells (CD68+ resident macrophages). Protein extracts from human vaginal wall and levator ani muscle and fascia showed strong ~92 and 68 kDa species, and LHRI was detected in smooth muscle cells, fibroblasts, resident macrophages and nuclei of skeletal muscle fibers. Our observations indicate that, in contrast to the theory on the role of vascular hormone receptors in preferential pick up of circulating hormones, there is no need to enhance selective pick up rather only prevent LH/CG transport to inappropriate sites. Abnormal placental LHR expression may play a role in the development of abnormal pregnancy. Expression of LHR in the pelvic floor compartments suggests that high LH levels in postmenopausal women may contribute to the pelvic floor relaxation and increased incidence of pelvic floor disorders. Since chorionic gonadotropin increases secretion of a variety of cytokines by monocytes, and induces their inflammatory reaction and phagocytic activity, high LH levels in aging individuals may also activate microglia (mononuclear phagocyte system in the central nervous system) and contribute to the development of Alzheimer's disease and other inflammation-mediated neurodegenerative diseases.     

From cellular to chemical approach for acute neural and alternative options for age-induced functional diseases

Endogenous “stem cell niche” (SCN) accompanying vessels contains immune system components which in vivo determine differentiation of multi potent stem cells toward proper cell types in given tissue. Combinations of sex steroids may represent novel chemical approach for neuronal areas of regenerative medicine, since they cause transformation of vascular smooth muscle stem cells into differentiating neuronal cells. Circulating sex steroids are present during pregnancy and can be utilized where needed, when various embryonic/fetal tissues develop from their stem cells. Utilization of induced regeneration of tissues (regenerative medicine) is expected being more effective in sudden failures of younger individuals carrying intact SCN, as compared to established chronic disorders caused by SCN alteration. An essential component of SCN are monocyte-derived cells exhibiting tissue-specific “stop effect” (SE) preventing, for instance, an aging of neuronal cells. Its alteration causes that implantation of neuronal stem cells will also result in their differentiation toward aging cells. When we repair the SE by supply of circulating mononuclear cells from young healthy individuals, we may be able to provide novel regenerative treatments of age-induced neural diseases by sex steroid combinations. Questions regarding some age-induced body alterations are also addressed.     

Mitochondrial metabolism directs stemness and differentiation in P19 embryonal carcinoma stem cells

The relationship between mitochondrial metabolism and cell viability and differentiation in stem cells (SCs) remains poorly understood. In the present study, we compared mitochondrial physiology and metabolism between P19SCs before/after differentiation and present a unique fingerprint of the association between mitochondrial activity, cell differentiation and stemness. In comparison with their differentiated counterparts, pluripotency of P19SCs was correlated with a strong glycolytic profile and decreased mitochondrial biogenesis and complexity: round, low-polarized and inactive mitochondria with a closed permeability transition pore. This decreased mitochondrial capacity increased their resistance against dichloroacetate. Thus, stimulation of mitochondrial function by growing P19SCs in glutamine/pyruvate-containing medium reduced their glycolytic phenotype, induced loss of pluripotent potential, compromised differentiation and became P19SCs sensitive to dichloroacetate. Because of the central role of this type of SCs in teratocarcinoma development, our findings highlight the importance of mitochondrial metabolism in stemness, proliferation, differentiation and chemoresistance. In addition, the present work suggests the regulation of mitochondrial metabolism as a tool for inducing cell differentiation in stem line therapies.Embryonal carcinoma cells, including the P19 cell line, are pluripotent cancer stem cells (CSCs) derived from pluripotent germ cell tumors called teratocarcinomas. These have been described as the malignant counterparts of embryonic stem cells (ESCs) and are considered a good model to study stem cell (SC) differentiation. The P19 cell line can be maintained as undifferentiated cells (P19SCs) or differentiated (P19dCs) to any cell type of the three germ layers. Similar to ESCs, P19 cells differentiate with retinoic acid (RA) in a dose-dependent manner and depending on growth conditions.¹ Although differentiation generally yields a mixed population of differentiated cells, P19 cells grown in monolayer and treated with 1 μM RA primarily differentiate in endoderm or mesoderm, while retaining their immortality.^{2, 3}Although some therapeutic approaches for regenerative medicine and to targeting CSCs are based on differentiation⁴ and mitochondrial-targeted therapies,^{5, 6} very little is known about the role of mitochondrial metabolism in SC maintenance and differentiation.⁷ Several mitochondrial characteristics that distinguish transformed cells from healthy cells have been described,⁸ including increased mitochondrial transmembrane electric potential (Δψm), which may result from decreased mitochondrial ATP production under normoxia.⁹ Similarly, normal SCs primarily rely on glycolysis for energy supply, although the exact mechanism how this occurs in the presence of oxygen and the relationship between SC metabolism and cell fate control is not yet completely understood.¹⁰Given the mitochondrial involvement in stemness and differentiation,¹¹ one can ask whether manipulation of mitochondrial physiology results in an improvement of therapy efficacy. Therefore, characterizing the metabolic and mitochondrial profiles of both SCs and differentiated cells holds promise in order to explain the resistance of cancer cells expressing an embryonic signature to mitochondrial-targeted therapies. In the present work, we have two tandem hypotheses: (a) metabolic and mitochondrial remodeling accompanies P19SC differentiation and (b) P19SC differentiation results in a higher susceptibility to mitochondrial-directed therapies.     

Role of histamine receptors in the effects of histamine on the production of reactive oxygen species by whole blood phagocytes

Aims

The diverse physiological functions of histamine are mediated through distinct histamine receptors. In this study we investigated the role of H₂R and H₄R in the effects of histamine on the production of reactive oxygen species by phagocytes in whole blood.

Main methods

Changes in reactive oxygen species (ROS) production by whole blood phagocytes after treatment with histamine, H₄R agonists (4-methylhistamine, VUF8430), H₂R agonist (dimaprit) and their combinations with H₄R antagonist (JNJ10191584) and H₂R antagonist (ranitidine) were determined using the chemiluminescence (CL) assay. To exclude the direct scavenging effects of the studied compounds on the CL response, the antioxidant properties of all compounds were measured using several methods (TRAP, ORAC, and luminol–HRP–H₂O₂ based CL).

Key findings

Histamine, 4-methylhistamine, VUF8430 and dimaprit inhibited the spontaneous and OZP-activated whole blood CL in a dose-dependent manner. On the other hand, only VUF8430 was able to inhibit PMA-activated whole blood CL. Ranitidine, but not JNJ10191584, completely reduced the effects of histamine, 4-methylhistamine and dimaprit. The direct scavenging ability of tested compounds was negligible.

Significance

Our results demonstrate that the inhibitory effects of histamine on ROS production in whole blood phagocytes were caused by H₂R. Our results also suggest that H₄R agonists in concentrations higher than 10^− 6 M may also influence ROS production via binding to H₂R.