The application of pH-sensitive spin labels to studies of surface potential and polarity of phospholipid membranes and proteins.

The effects of pH titration on the EPR spectra of imidazolidine nitroxides located at the surface of mixed bilayers composed of dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylcholine (DMPC), and at the surface of the protein, human serum albumin (HSA), have been investigated. It is found that the shift in pKa of the amino group of the imidazolidine radical from its value of 4.6 in water depends both on the interfacial polarity (delta pKapol) and on the electrostatic surface potential (delta pKael) when it is positioned at the bilayer/water interface by an anchoring hydrocarbon tail. The polarity shift is determined to be: delta pKapol = -1.3 units at the surface of DMPC bilayers at 17 degrees C, corresponding to an effective interfacial dielectric constant of epsilon approximately 37, and depends on the temperature with a coefficient of d delta pKapol/dT approximately -0.01 per degree. The electrostatic shift at the surface of DMPG bilayers is delta pKael = +1.6 units in 0.1 M KCl, which corresponds to an electrostatic surface potential of -95 mV. This electrostatic shift depends strongly both on ionic strength and on the fraction of charged lipid in the DMPC/DMPG mixtures, in a manner that agrees with the predictions of electrostatic double-layer theory. It is found that the shift in pKa of an imidazolidine radical covalently bound at the surface of HSA is determined mainly by the surface electrostatics (delta pKapol approximately 0) and corresponds to an electrostatic potential of +33 mV in 0.01 M KCl at a pH below the isoelectric point of the protein.     

Cytotoxic effects of dynorphins through nonopioid intracellular mechanisms

We previously found that fibronectin (FN) had a functional site (YTIYVIAL sequence in the 14th type III module) suppressing the integrin-mediated cell adhesion to extracellular matrix. FN-derived peptides containing this antiadhesive site were also shown to regulate cellular processes such as proliferation, differentiation, and apoptosis. The present study shows that the FN-derived antiadhesive peptides suppress the myofibroblastic conversion of rat hepatic stellate cells (HSC). Freshly isolated HSC underwent myofibroblastic conversion during culture in the presence of FBS, as evaluated by indices representing the phenotypic activation of HSC, including increased proliferation, consumption of vitamin A-enriched lipid droplets, and expression of alpha-smooth muscle actin. However, appearance of these myofibroblastic characters was suppressed by coculturing HSC with the FN-derived antiadhesive peptides. On the other hand, the activated HSC, which had already acquired the myofibroblastic phenotype through repeated subculture, secreted FN and then stimulated matrix assembly of ED-A (+) cellular FN as well as plasma FN, while the FN-derived antiadhesive peptides inhibited them. Furthermore, the FN-derived antiadhesive peptides suppressed the integrin-mediated adhesion of the primary HSC to plasma FN and ED-A (+) cellular FN substrates. These results suggested that the FN-derived antiadhesive peptides down-regulated the myofibroblastic conversion of HSC in an indirect manner by inhibiting the integrin-mediated adhesive interaction of HSC with ED-A (+) cellular FN.     

Utilization of endoscopic inoculation in a mouse model of intrauterine infection-induced preterm birth: role of interleukin 1beta

A novel murine model of intrauterine infection/inflammation-induced preterm birth based on direct endoscopic intracervical inoculation is described. Using this model, we investigated infection-induced premature pregnancy loss in normal and interleukin (IL) 1beta-deficient mice. Seventy-four CD-1, HS, C57BL/6J wild type (IL-1beta+/+), and C57BL/6J IL-1beta-deficient (IL-1beta-/-) mice were inoculated intracervically using a micro-endoscope, at a time corresponding to 70% of average gestation. Intracervical injection of lipopolysaccharide (LPS) or Escherichia coli reliably induced premature birth: 100% of mice intracervically injected with LPS and 92% of mice with a positive endometrial E. coli culture delivered prematurely within 36 h after inoculation. No losses were observed in mice inoculated with saline. Pregnancy loss was associated with increased uterine tissue cyclooxygenase-2 gene expression and uterine content of IL-1beta, tumor necrosis factor alpha, macrophage inflammatory protein-1alpha, and IL-6, as well as elevation of nuclear factor-kappaB activity in uterine tissues. Although IL-1beta-/- mice exhibited decreased uterine cytokine production in response to bacteria and LPS, IL-1beta deficiency did not affect the rate of pregnancy loss. This model using direct intracervical bacterial or LPS inoculation is useful for studying preterm pregnancy loss in genetically altered mice in order to develop novel interventions for infection-associated preterm labor.     

Redox-sensitive mechanism of no scavenging by nitronyl nitroxides

Nitronyl nitroxides, NN., have been increasingly used in the field of NO-related studies as specific antagonists of NO. . We employed a combination of EPR and NMR spin trapping to study the mechanisms of the reaction of NN. with NO. in reducing environments. EPR allowed observation of NO-induced transformation of the paramagnetic trap, NN., to the corresponding iminonitroxide, IN. . In a complementary way, corresponding EPR-invisible diamagnetic products (the hydroxylamines NN-H and IN-H) were detected by 19F-NMR using newly synthesized fluorinated traps. Addition of reducing agents to a solution of NN. resulted in fast disappearance of its EPR spectra and appearance of a 19F-NMR peak of the corresponding hydroxylamine, NN-H. Addition of NO. as a bolus, or NO. generated on sodium nitroprusside photolysis, resulted in 19F-NMR-detectable accumulation of the hydroxylamine, IN-H. Upon high rates of NO. generation in ascorbate-containing solutions, partial recovery of NN. was observed, which undergoes further reactions with NO. and ascorbate in a competitive manner. Using 19F-NMR and a fluorinated trap, NO-induced conversion of NN-H into IN-H was also observed in vivo in hypertensive ISIAH rats compared with normotensive WAG rats. The results provide insight into a new potential redox-sensitive mechanism of the antagonistic action of NN. against NO., which may provide insight into previously unexplained behavior of this category of NO-reacting compounds.     

Sexual Differentiation of the Brain as a Manifestation of Its Plasticity

This review analyzes the published data and authors' own results on the roles of hormones and neurotransmitters in the formation of structural and functional sex-related dimorphism of the brain within the early ontogenesis period. Proof is presented in favor of the concept that classic neurotransmitters function as inductors of nerve cell differentiation in the process of individual development of the brain. Neurochemical mechanisms of the hormone–transmitter interaction in the process of sexual differentiation of the brain within pre- and/or post-natal periods are considered.     

Localization of disulfide bonds in the alpha 1 acidic glycoprotein molecule and study of their effect on ability of the protein to interact with ethidium bromide

alpha 1-Acid glycoprotein (orosomucoid) was purified from the human and murine blood sera using phenol deproteinization. As opposed to the murine protein, the human orosomucoid bound the fluorescent dye ethidium bromide but lost this ability after treatment with beta-mercaptoethanol, which breaks disulfide bonds. Disulfide bonds between the Cys23 and Cys165 residues of the human orosomucoid and between the Cys91 and Cys184 residues of the murine orosomucoid were identified.