A női nemi hormonok koncentrációja és a testzsírosság változókorú nőknél

The relationship between sex hormonal levels and body fatness in relation to reproductive ageing in women. The number of postmenopausal women in our ageing societies is increasing, thus the importance of studies on menopause and the risk factors of early and late menopause is increasing. The age at the final menstrual period holds clinical and public health interest because the age at menopause can be a marker of aging and health. In general the menopausal transition increases the risk of many diseases, in addition to this health consequence of menopause it is also evidenced that women undergoing early menopause have a higher incidence of osteoporotic fractures, cardiovascular diseases, neurological diseases, psychiatric diseases, while women with late menopause are at higher risk for breast and endometrial cancer. The purpose of the research was to study the relationship between the menopausal status and the body fatness in Hungarian women. A random sample of 1932 Hungarian women (aged 35+ years) was studied between 2012 and 2014. Body composition was estimated by body impedance analysis. Subjects were divided into pre-, peri- and postmenopausal subgroups on the basis of their menstrual history and in a subsample by considering the estradiol and progesterone levels in saliva. Women who had any diseases or were taking any medications known to affect body composition, or who had been hysterectomized or ovariectomized were not included in the present analysis. The level of studied sex hormones showed a decrease from the early 40s. The body fat mass had an increase until the late 50 years and then had a significant decrease through senescence. Premenopausal women who were much older than the median age at menopause had significantly higher amount of fat than their postmenopausal age-peers, while postmenopausal women, whose permanent cessation of menstruation occurred much earlier than the median age at menopause, had significantly less fat amount than their premenopausal age-peers. The body fat mass in women, who were divided into the postmenopausal subgroup by their sex hormone levels but experienced regular menstruations, was always above the age median value of their menopausal status subgroups, while in women, who were considered premenopausal according to their sex hormone levels but were amenorrheic at least for the past 12 months, below the age medians

Real-time kinetic method to monitor isopeptidase activity of transglutaminase 2 on protein substrate

Transglutaminase 2 (TG2) is a ubiquitously expressed multifunctional protein with Ca2+-dependent transamidase activity forming protease resistant Nε-(γ-glutamyl)lysine crosslinks between proteins. It can also function as an isopeptidase cleaving the previously formed crosslinks. The biological significance of this activity has not been revealed yet mainly because of the lack of protein based method for its characterization. Here we report development of a novel kinetic method for measuring isopeptidase activity of human TG2 by monitoring decrease in the fluorescence polarisation of a protein substrate previously formed by crosslinking fluorescently labelled glutamine donor FLpepT26 to S100A4 at a specific lysine residue. The developed method could be applied to test mutant enzymes and compounds which influence isopeptidase activity of TG2

Tailoring Uptake Efficacy of HSV-1 gD Tailoring Uptake Efficacy of Hsv-1 GD Derived Carrier Peptides

Regions of the Herpes simplex virus-1 (HSV-1) glycoprotein D (gD) were chosen to design carrier peptides based on the known tertiary structure of the virus entry receptor complexes. These complexes consist of the following: HSV-1 gD–nectin-1 and HSV-1 gD–herpesvirus entry mediator (HVEM). Three sets of peptides were synthesised with sequences covering the (i) N-terminal HVEM- and nectin-1 binding region -5–42, (ii) the 181–216 medium region containing nectin-1 binding sequences and (iii) the C-terminal nectin-1 binding region 214–255. The carrier candidates were prepared with acetylated and 5(6)-carboxyfluorescein labelled N-termini. The peptides were chemically characterised and their conformational features in solution were also determined. In vitro internalisation profile and intracellular localisation were evaluated on SH-SY5Y neuroblastoma cells. Peptide originated from the C-terminal region 224–247 of the HSV-1 gD showed remarkable internalisation compared to the other peptides with low to moderate entry. Electronic circular dichroism secondary structure studies of the peptides revealed that the most effectively internalised peptides exhibit high helical propensity at increasing TFE concentrations. We proved that oligopeptides derived from the nectin-1 binding region are promising candidates—with possibility of Lys237Arg and/or Trp241Phe substitutions—for side-reaction free conjugation of bioactive compounds—drugs or gene therapy agents—as cargos

Drug conjugation induced modulation of structural and membrane interaction features of cationic cell-permeable peptides

Cell-penetrating peptides might have great potential for enhancing the therapeutic effect of drug molecules against such dangerous pathogens as Mycobacterium tuberculosis (Mtb), which causes a major health problem worldwide. A set of cationic cell-penetration peptides with various hydrophobicity were selected and synthesized as drug carrier of isoniazid (INH), a first-line antibacterial agent against tuberculosis. Molecular interactions between the peptides and their INH-conjugates with cell-membrane-forming lipid layers composed of DPPC and mycolic acid (a characteristic component of Mtb cell wall) were evaluated, using the Langmuir balance technique. Secondary structure of the INH conjugates was analyzed and compared to that of the native peptides by circular dichroism spectroscopic experiments performed in aqueous and membrane mimetic environment. A correlation was found between the conjugation induced conformational and membrane affinity changes of the INH–peptide conjugates. The degree and mode of interaction were also characterized by AFM imaging of penetrated lipid layers. In vitro biological evaluation was performed with Penetratin and Transportan conjugates. Results showed similar internalization rate into EBC-1 human squamous cell carcinoma, but markedly different subcellular localization and activity on intracellular Mtb

Synthesis and in vitro biochemical evaluation of oxime bond-linked daunorubicin-GnRH-III conjugates developed for targeted drug delivery

Gonadotropin releasing hormone-III (GnRH-III), a native isoform of the human GnRH isolated from sea lamprey, specifically binds to GnRH receptors on cancer cells enabling its application as targeting moieties for anticancer drugs. Recently, we reported on the identification of a novel daunorubicin–GnRH-III conjugate (GnRH-III–[4Lys(Bu), 8Lys(Dau=Aoa)] with efficient in vitro and in vivo antitumor activity. To get a deeper insight into the mechanism of action of our lead compound, the cellular uptake was followed by confocal laser scanning microscopy. Hereby, the drug daunorubicin could be visualized in different subcellular compartments by following the localization of the drug in a time-dependent manner. Colocalization studies were carried out to prove the presence of the drug in lysosomes (early stage) and on its site of action (nuclei after 10 min). Additional flow cytometry studies demonstrated that the cellular uptake of the bioconjugate was inhibited in the presence of the competitive ligand triptorelin indicating a receptor-mediated pathway. For comparative purpose, six novel daunorubicin–GnRH-III bioconjugates have been synthesized and biochemically characterized in which 6Asp was replaced by D-Asp, D-Glu and D-Trp. In addition to the analysis of the in vitro cytostatic effect and cellular uptake, receptor binding studies with 125I-triptorelin as radiotracer and degradation of the GnRH-III conjugates in the presence of rat liver lysosomal homogenate have been performed. All derivatives showed high binding affinities to GnRH receptors and displayed in vitro cytostatic effects on HT-29 and MCF-7 cancer cells with IC50 values in a low micromolar range. Moreover, we found that the release of the active drug metabolite and the cellular uptake of the bioconjugates were strongly affected by the amino acid exchange which in turn had an impact on the antitumor activity of the bioconjugates

Ezrin interacts with S100A4 via both its N- and C-terminal domains

Ezrin belongs to the ERM (ezrin, radixin, moesin) protein family that has a role in cell morphology changes, adhesion and migration as an organizer of the cortical cytoskeleton by linking actin filaments to the apical membrane of epithelial cells. It is highly expressed in a variety of human cancers and promotes metastasis. Members of the Ca2+-binding EF-hand containing S100 proteins have similar pathological properties; they are overexpressed in cancer cells and involved in metastatic processes. In this study, using tryptophan fluorescence and stopped-flow kinetics, we show that S100A4 binds to the N-terminal ERM domain (N-ERMAD) of ezrin with a micromolar affinity. The binding involves the F2 lobe of the N-ERMAD and follows an induced fit kinetic mechanism. Interestingly, S100A4 binds also to the unstructured C-terminal actin binding domain (C-ERMAD) with similar affinity. Using NMR spectroscopy, we characterized the complex of S100A4 with the C-ERMAD and demonstrate that no ternary complex is simultaneously formed with the two ezrin domains. Furthermore, we show that S100A4 co-localizes with ezrin in HEK-293T cells. However, S100A4 very weakly binds to full-length ezrin in vitro indicating that the interaction of S100A4 with ezrin requires other regulatory events such as protein phosphorylation and/or membrane binding, shifting the conformational equilibrium of ezrin towards the open state. As both proteins play an important role in promoting metastasis, the characterization of their interaction could shed more light on the molecular events contributing to this pathological process

Matrigel patterning reflects multicellular contractility

Non-muscle myosin II (NMII)-induced multicellular contractility is essential for development, maintenance and remodeling of tissue morphologies. Dysregulation of the cytoskeleton can lead to birth defects or enable cancer progression. We demonstrate that the Matrigel patterning assay, widely used to characterize endothelial cells, is a highly sensitive tool to evaluate cell contractility within a soft extracellular matrix (ECM) environment. We propose a computational model to explore how cellexerted contractile forces can tear up the cell-Matrigel composite material and gradually remodel it into a network structure. We identify measures that are characteristic for cellular contractility and can be obtained from image analysis of the recorded patterning process. The assay was calibrated by inhibition of NMII activity in A431 epithelial carcinoma cells either directly with blebbistatin or indirectly with Y27632 Rho kinase inhibitor. Using Matrigel patterning as a bioassay, we provide the first functional demonstration that overexpression of S100A4, a calcium-binding protein that is frequently overexpressed in metastatic tumors and inhibits NMIIA activity by inducing filament disassembly, effectively reduces cell contractility

3D cell segregation geometry and dynamics are governed by tissue surface tension regulation

Tissue morphogenesis and patterning during development involve the segregation of cell types. Segregation is driven by differential tissue surface tensions generated by cell types through controlling cell-cell contact formation by regulating adhesion and actomyosin contractility-based cellular cortical tensions. We use vertebrate tissue cell types and zebrafish germ layer progenitors as in vitro models of 3-dimensional heterotypic segregation and developed a quantitative analysis of their dynamics based on 3D time-lapse microscopy. We show that general inhibition of actomyosin contractility by the Rho kinase inhibitor Y27632 delays segregation. Cell type-specific inhibition of non-muscle myosin2 activity by overexpression of myosin assembly inhibitor S100A4 reduces tissue surface tension, manifested in decreased compaction during aggregation and inverted geometry observed during segregation. The same is observed when we express a constitutively active Rho kinase isoform to ubiquitously keep actomyosin contractility high at cell-cell and cell-medium interfaces and thus overriding the interface-specific regulation of cortical tensions. Tissue surface tension regulation can become an effective tool in tissue engineering