Conjugates of a Photoactivated Rhodamine with Biopolymers for Cell Staining

Conjugates of the photoactivated rhodamine dyes with biopolymers (proteins, polysaccharides, and nucleic acids) are important tools for microscopic investigation of biological tissue. In this study, a precursor of the photoactivated fluorescent dye (PFD) has been successfully used for staining of numerous mammalian cells lines and for conjugate formation with chitosan (“Chitosan-PFD”) and histone H1 (“Histone H1.3-PFD”). The intensive fluorescence has been observed after photoactivation of these conjugates inside cells (A431, HaCaT, HEK239, HBL-100, and MDCK). Developed procedures and obtained data are important for further application of novel precursors of fluorescent dyes (“caged” dyes) for microscopic probing of biological objects. Thus, the synthesized “Chitosan-PFD” and “Histone H1-PFD” have been successfully applied in this study for intracellular transport visualization by fluorescent microscopy

Urokinase-type Plasminogen Activator (uPA) Promotes Angiogenesis by Attenuating Proline-rich Homeodomain Protein (PRH) Transcription Factor Activity and De-repressing Vascular Endothelial Growth Factor (VEGF) Receptor Expression

Urokinase-type plasminogen activator (uPA) regulates angiogenesis and vascular permeability through proteolytic degradation of extracellular matrix and intracellular signaling initiated upon its binding to uPAR/CD87 and other cell surface receptors. Here, we describe an additional mechanism by which uPA regulates angiogenesis. Ex vivo VEGF-induced vascular sprouting from Matrigel-embedded aortic rings isolated from uPA knock-out (uPA(−/−)) mice was impaired compared with vessels emanating from wild-type mice. Endothelial cells isolated from uPA(−/−) mice show less proliferation and migration in response to VEGF than their wild type counterparts or uPA(−/−) endothelial cells in which expression of wild type uPA had been restored. We reported previously that uPA is transported from cell surface receptors to nuclei through a mechanism that requires its kringle domain. Intranuclear uPA modulates gene transcription by binding to a subset of transcription factors. Here we report that wild type single-chain uPA, but not uPA variants incapable of nuclear transport, increases the expression of cell surface VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2) by translocating to the nuclei of ECs. Intranuclear single-chain uPA binds directly to and interferes with the function of the transcription factor hematopoietically expressed homeodomain protein or proline-rich homeodomain protein (HHEX/PRH), which thereby lose their physiologic capacity to repress the activity of vehgr1 and vegfr2 gene promoters. These studies identify uPA-dependent de-repression of vegfr1 and vegfr2 gene transcription through binding to HHEX/PRH as a novel mechanism by which uPA mediates the pro-angiogenic effects of VEGF and identifies a potential new target for control of pathologic angiogenesis

Interaction of Lipase with Lipid Model Systems

The aim of this work was to study the interaction of lipases (as an important biopolymer) with models of biomembranes based on the phospholipid and cholesterol. Lipases (triacylglycerolacyl hydrolases) are widely distributed enzymes and well-known by their hydrolytic activity. The study of the lipase interactions with lipid vesicles in aqueous dispersions is of fundamental and practical interest. The pure phosphatidylcholine from egg yolk (ePC) and cholesterol (Chol) were obtained from Sigma-Aldrich. Lipase was obtained from hog pancreas. Measurements of the current and equilibrium surface tension (ST and eST) values were carried out using a BPA-1P device and ADSA program. The particle sizes in the prepared colloidal solutions were determined by the method of dynamic light scattering. An addition of lipase led to some decrease both, of ST and eST for the samples of ePC:Chol (in the ratios from19:1 to 1:1). The mean particle diameter (MPD) and effective particle diameter (EPD) values for the samples of ePC:Chol changed drastically by lipase addition. The EPD/MPD ratios increased from 1.7 to 2.0, from 1.8 to 2.6, from 2.3 to 6.5, from 1.5 to 2.9 for the samples of ePC:Chol at the ratios of 19:1, 14:1, 9:1, 7:1, respectively by lipase concentration increase. This general tendency can be explained by strong interaction of lipase with lipid membrane that leads to the formation of the mixed particles ePC:Chol:lipase with more narrow particle size distribution as compared to the initial EPD/MPD ratio (for the ePC:Chol mixture without lipase

Insulinotropic compounds decrease endothelial cell survival

Hyperglycemia induces damage of vascular endothelial cells leading to diabetic complications. We investigated the effects of insulinotropic compounds and elevated glucose on endothelial cells in the absence or presence of vascular endothelial growth factor (VEGF). Human umbilical vein endothelial cells (HUVECs) were treated with glibenclamide, repaglinide and insulinotropic imidazolines at high glucose concentration in the presence or absence of VEGF and viability, proliferation and nitric oxide production were measured. Hyperglycemia inhibited pro-survival effects of VEGF on endothelial cells. Glibenclamide and repaglinide decreased HUVEC viability at elevated glucose concentration in the absence but not in the presence of VEGF, without affecting HUVEC proliferation. Repaglinide also had some positive influence on HUVEC function elevating NO production in the presence of VEGF. Imidazolines showed different activities on endothelial cell survival. Efaroxan diminished HUVEC viability at elevated glucose concentration in the presence, however not in the absence of VEGF, while RX871024 decreased HUVEC survival regardless of the presence of VEGF. Our data demonstrate an important interplay between the actual insulinotropic compounds, VEGF and ambient glucose concentration affecting the survival of the vascular endothelial cells. Consequently, this interplay needs to be taken into consideration when designing novel oral antidiabetic compounds. •Oral insulinotropic compounds decrease endothelial cell survival.•Sulfonylurea, glinide and imidazolines differently affect endothelial cell survival.•Repaglinide has some positive effect on endothelial function elevating NO production

The imidazoline compound RX871024 promotes insulinoma cell death independent of AMP-activated protein kinase inhibition

We have previously shown that the insulinotropic imidazoline compound RX871024 induces death of insulinoma MIN6 cells, an effect involving stimulation of c-Jun N-terminal kinase (JNK) and caspase 3. It has also been reported that AMP-activated protein kinase (AMPK) activates JNK and induces β-cell death. Here we show that RX871024, but not another insulinotropic imidazoline compound (BL11282), suppressed AMPK activity in MIN6 cells. The inhibitory effect of RX871024 on AMPK was supported by the observation that the imidazoline induced lipid droplet formation in the cytoplasm of MIN6 cells. This reflects stimulation of anabolic pathways and inhibition of catabolic pathways in the cell that happen under conditions when AMPK is inhibited. Activation of AMPK by 5-aminoimidazole-4-carboxamide riboside (AICAR) elevated basal and cytokine-induced death in primary β-cells and in insulinoma MIN6 cells. RX871024 aggravated AICAR-induced insulinoma MIN6 cell death regardless of the presence of pro-inflammatory cytokines. The specific cytotoxic effect of imidazoline compound RX871024 on insulinoma cell death but not primary β-cell death is independent of its action on AMPK and may suggest the possibility of using this type of compound in the treatment of insulinomas

Inositol hexakisphosphate stimulates non-Ca(2+)-mediated and primes Ca(2+)-mediated exocytosis of insulin by activation of protein kinase C

d-myo-inositol 1,2,3,4,5,6-hexakisphosphate (InsP(6)), formed via complex pathways of inositol phosphate metabolism, composes the main bulk of inositol polyphosphates in the cell. Relatively little is known regarding possible biological functions for InsP(6). We now show that InsP(6) can modulate insulin exocytosis in permeabilized insulin-secreting cells. Concentrations of InsP(6) above 20 μM stimulated insulin secretion at basal Ca(2+)-concentration (30 nM) and primed Ca(2+)-induced exocytosis (10 μM), both effects being due to activation of protein kinase C. Our results suggest that InsP(6) can play an important modulatory role in the regulation of processes such as exocytosis in insulin-secreting cells. The specific role for InsP(6) can then be to recruit secretory granules to the site of exocytosis

Biochemical parameters, dynamic tensiometry and circulating nucleic acids for cattle blood analysis: a review

The animal’s blood is the most complicated and important biological liquid for veterinary medicine. In addition to standard methods that are always in use, recent technologies such as dynamic tensiometry (DT) of blood serum and PCR analysis of particular markers are in progress. The standard and modern biochemical tests are commonly used for general screening and, finally, complete diagnosis of animal health. Interpretation of major biochemical parameters is similar across animal species, but there are a few peculiarities in each case, especially well-known for cattle. The following directions are discussed here: hematological indicators; “total protein” and its fractions; some enzymes; major low-molecular metabolites (glucose, lipids, bilirubin, etc.); cations and anions. As example, the numerous correlations between DT data and biochemical parameters of cattle serum have been obtained and discussed. Changes in the cell-free nucleic acids (cfDNA) circulating in the blood have been studied and analyzed in a variety of conditions; for example, pregnancy, infectious and chronic diseases, and cancer. CfDNA can easily be detected using standard molecular biological techniques like DNA amplification and next-generation sequencing. The application of digital PCR even allows exact quantification of copy number variations which are for example important in prenatal diagnosis of chromosomal aberrations

Two Generations of Insulinotropic Imidazoline Compounds

The imidazoline RX871024 increased basal- and glucose-stimulated insulin release in vitro and in vivo. The compound inhibited activity of ATP-sensitive K+ channels as well as voltage-gated K+ channels, which led to membrane depolarization, an increase in the cytosolic Ca2+ concentration ([Ca2+]i), and insulin release. Importantly, RX871024 also enhanced the insulinotropic effect of glucose in cells with clamped [Ca2+]i but in the presence of high ATP and Ca2+concentration inside the cell. We believe that the latter effect on insulin exocytosis was at least in part mediated by a rise in diacylglycerol, which then activated protein kinase C (PKC) and increased the generation of arachidonic acid (AA) metabolites. Activation of both the PKC and AA pathways resulted in potentiation of glucose effects on insulin secretion. Unlike RX871024, the novel imidazoline BL11282 did not block ATP-dependent K+ channels, but similarly to RX871024, it stimulated insulin secretion in depolarized or permeabilized islets. Accordingly, BL11282 did not influence glucose and insulin levels under basal conditions either in vitro or in vivo, but it markedly enhanced the insulinotropic effects of glucose. BL11282 restored the impaired insulin response to glucose in islets from spontaneously diabetic GK rats. We conclude that BL11282 belongs to a new class of insulinotropic compounds that demonstrate a strong glucose-dependent effect on insulin exocytosis