[Non-hormonal correction of menopausal syndrome: potential of xenon therapy]. / Negormonal'naya korrektsiya klimaktericheskogo sindroma: vozmozhnosti ksenonoterapii.

The menopausal syndrome is associated with a combination of neuropsychic, autonomic, vascular, and metabolic disorders. Sex steroids regulate neurotransmitter metabolism, activate neuronal plasticity, improve cerebral blood flow, maintain a stable mood, and have an antidepressant effect. In Russia, only 1% of women use menopausal hormone therapy (MHT). The reason for the low adherence to MHT is avoidance of using hormones because of the possible risks of cancer. OBJECTIVE: To evaluate the effect of subnarcotic doses of xenon on the menopausal syndrome signs in patients during the menopausal transition. MATERIAL AND METHODS: A comparative study including 32 randomly selected female patients with menopausal syndrome during the menopausal transition was conducted. Group 1 (the study treatment group) included 16 patients who refused to use MHT. They received a course of xenon therapy, consisting of 5 procedures every other day. Group 2 (control group) included 16 patients receiving MHT. Menopausal symptoms were assessed using the Greene Menopausal Scale. Psychoemotional status was determined using the Spielberger-Hanin neuropsychological test. Estradiol and progesterone concentrations were measured in a morning saliva sample to determine the steroid profile. The parameters were assessed and compared at baseline and 1 month after the start of therapy. RESULTS: In assessing the severity of the menopausal syndrome in women in both groups, the significantly decreased mean final Green's scale score was observed: from 17.12±3.28 to 6.12±4.34 points in group 1 and from 16.01±4.12 to 4.02±3.12 points in group 2. Also a significant decrease in state and trait anxiety compared with baseline data was demonstrated. In the study treatment group, the trait anxiety score decreased from 53.1 [35.1; 66.0] to 27.2 [25.3; 30.0] points, and in the control group, from 55.6 [38.2; 70.4] to 22.0 [20.2; 25.0] points. Similar change was shown for the state anxiety score in the study groups. A decrease from 40.1 [35.3; 45.0] to 21.0 [23.2; 27.3] points in group 1 and from 46.1 [45.2; 52.0] to 20.1 [16.3; 23.0] points in group 2 was observed. At one month, the significant increase of estradiol (from 1.1 [0.5; 2.1] to 12.2 [10.3; 14.4] pg/mL) and progesterone (from 14.0 [4.4; 20.1] to 100.2 [60.6; 130.0] pg/mL) was observed in the MHT group of patients. No significant changes in hormone levels were recorded in the xenon therapy group. CONCLUSION: The xenon inhalations in subnarcotic doses are an effective method to control the vasomotor and psychoemotional signs and symptoms of the menopausal syndrome in patients who refuse to use MHT or have contraindications to this type of therapy.

ANALYSIS OF DIAGNOSTICS AND NEWEST PATHOGENESIS ASPECTS OF TEMPOROMANDIBULAR DYSFUNCTION (REVIEW).

The research was predisposed for a progressing increase in incidence of the temporomandibular joint dysfunction, low effectiveness of traditional treatment as well as the necessity of implementation of high-effective etiopathogenetic therapy methods. The necessity of systematizing diagnostics and treatment planning is evident, considering the cause-effect mechanisms of the disease initiation and complex individual approach, required for improving the effectiveness of prevention and pathogenetic therapy in early rehabilitation of the patients with the temporomandibular dysfunction, The visualization model will provide for planning certain treatment stages using a virtual system, synchronizing and combining them with the modern methods of the prosthetic construction fabrication, which will represent a new quality stage of stomatological treatment. The chosen theme is not only of multidisciplinary medical importance, but of the social one as well. The wide coverage of the theme in stomatological sources evidences about urgency of searching for the new diagnostic sources and treatment methods and proves actuality of the topic. The article presents urgent aspects of the temporomandibular dysfunction development pathogenetic mechanisms, depicts new methods of diagnostics and early clinical manifestations, reviewed by the Ukrainian and foreign scientists over the previous 10 years.

Association with ZO-1 correlates with plasma membrane partitioning in truncated connexin45 mutants.

Zonula occludens-1 (ZO-1), the most abundant known connexin-interacting protein in osteoblastic cells, associates with the carboxyl termini of both Cx43 and Cx45. To learn more about the role of the cormexin-ZO-1 interaction, we analyzed connexin trafficking and function in ROS 17/2.8 cells that were stably transfected either with full length Cx45 or with Cx45 lacking 34 or 37 amino acids on the carboxyl terminus (Cx45t34 or Cx45t37). All three proteins were transported to appositional membranes in the transfected cells: Cx45 and Cx45t34 displayed a punctate appositional membrane-staining pattern, while Cx45t37 staining at appositional membranes was more linear. Expression of Cx45 decreased gap junction communication as assayed by dye transfer, while expression of Cx45t34 or Cx45t37 increased the amount of dye transfer seen in these cells. We found that Cx43, Cx45 and Cx45t34 co-precipitated with ZO-1 in these cells, while Cx45t37 did not. We also found that Cx45t37 was much more soluble in 1% Triton X-100 than the other connexins examined. In addition, Cx45t37 migrated to a fraction of lighter buoyant density on sucrose flotation gradients than Cx43, Cx45, ZO-1 and Cx45t34. As ZO-1 is an actin-binding protein, this suggested that the differences in Cx45t37 solubility might be due to a difference between the interaction of gap junctions and the actin cytoskeleton in the ROS/Cx45t37 and in the other transfected ROS cells. To examine this possibility, the transfected ROS cells were stained with fluorescently labeled phalloidin and demonstrated that there was a notable loss of actin stress fibers in the ROS/Cx45t37 cells. These findings suggest that association with ZO-1 alters the plasma membrane localization of Cx45 by removing it from a lipid raft compartment and rendering it Triton-insoluble, presumably by promoting an interaction with the actin cytoskeleton; they also suggest that Cx45 has a complex binding interaction with ZO-1 that involves either an extended carboxyl terminal domain or two distinct binding sites.

Multimeric connexin interactions prior to the trans-Golgi network.

Cells that express multiple connexins have the capacity to form heteromeric (mixed) gap junction hemichannels. We used a dominant negative connexin construct, consisting of bacterial beta-galactosidase fused to the C terminus of connexin43 (Cx43/beta-gal), to examine connexin compatibility in NIH 3T3 cells. Cx43/beta-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool induced by Cx43/beta-gal colocalized with a medial Golgi apparatus marker and was readily disassembled by treatment with brefeldin A. This was unexpected, since previous studies indicated that Cx43 assembly into hexameric hemichannels occurs in the trans-Golgi network (TGN) and is sensitive to brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/beta-gal were assembled into a subhexameric complex. Cx43/beta-gal also specifically interacted with Cx46, but not Cx32, consistent with the ability of Cx43/beta-gal to simultaneously inhibit multiple connexins. We confirmed that interactions between Cx43/beta-gal and Cx46 reflect the ability of Cx43 and Cx46 to form heteromeric complexes, using HeLa and alveolar epithelial cells, which express both connexins. In contrast, ROS osteoblastic cells, which differentially sort Cx43 and Cx46, did not form Cx43/Cx46 heteromers. Thus, cells have the capacity to regulate whether or not compatible connexins intermix.

Heterocellular gap junctional communication between alveolar epithelial cells.

We analyzed the pattern of gap junction protein (connexin) expression in vivo by indirect immunofluorescence. In normal rat lung sections, connexin (Cx)32 was expressed by type II cells, whereas Cx43 was more ubiquitously expressed and Cx46 was expressed by occasional alveolar epithelial cells. In response to bleomycin-induced lung injury, Cx46 was upregulated by alveolar epithelial cells, whereas Cx32 and Cx43 expression were largely unchanged. Given that Cx46 may form gap junction channels with either Cx43 or Cx32, we examined the ability of primary alveolar epithelial cells cultured for 6 days, which express Cx43 and Cx46, to form heterocellular gap junctions with cells expressing other connexins. Day 6 alveolar epithelial cells formed functional gap junctions with other day 6 cells or with HeLa cells transfected with Cx43 (HeLa/Cx43), but they did not communicate with HeLa/Cx32 cells. Furthermore, day 6 alveolar epithelial cells formed functional gap junction channels with freshly isolated type II cells. Taken together, these data are consistent with the notion that type I and type II alveolar epithelial cells communicate through gap junctions compatible with Cx43.

Connexin45 interacts with zonula occludens-1 and connexin43 in osteoblastic cells.

The relative expression of connexin43 and connexin45 modulates gap junctional communication and production of bone matrix proteins in osteoblastic cells. It is likely that changes in gap junction permeability are determined by the interaction between these two proteins. Cx43 interacts with ZO-1, which may be involved in trafficking of Cx43 or facilitating interactions between Cx43 and other proteins. In this study we sought to identify proteins that associate with Cx45 by coprecipitation in non-denaturing conditions. Cx45 was isolated with a 220-kDa protein that we identified as ZO-1. Under the same conditions, Cx43 also was isolated with anti-Cx45 antiserum from Cx45-transfected ROS cells (ROS/Cx45 cells). Cx43 antiserum could also coprecipitate ZO-1 in the transfected and untransfected ROS cells. Double label immunofluorescence studies showed that ZO-1, Cx43, and Cx45 colocalized at appositional membranes in ROS/Cx45 cells suggesting that all three proteins are normally associated in the cells. Additionally, we found that in vitro translated ZO-1 binds to the carboxyl-terminal of Cx45 indicating that there is a direct interaction between the carboxyl-terminal of Cx45 and ZO-1. These studies demonstrate that ZO-1 interacts with Cx45 as well as with Cx43, and suggest that the interaction of connexins with ZO-1 may play a role in regulating the composition of the gap junction and may modulate connexin-connexin interactions.

Connexin45 interacts with zonula occludens-1 in osteoblastic cells.

Connexin43 (Cx43) and Cx45 are co-expressed in a number of different tissues. Studies demonstrated that Cx45 transfected ROS (ROS/Cx45) cells, were less permeable to low molecular weight dyes than untransfected ROS cells, that have gap junctions made of Cx43. This suggests that there may be a functionally important interaction between Cx43 and Cx45 in these cells. One way in which these proteins may interact is by associating with the same set of proteins. In order to isolate connexin interacting proteins, we isolated Cx45 from Cx45 transfected ROS cells (ROS/Cx45 cells) under mild detergent conditions. These studies showed that Cx45 co-purified with the tight junction protein, ZO-1. Immunofluorescence studies of ROS/Cx45 cells simultaneously stained with polyclonal Cx45 antibody and a monoclonal ZO-1 antibody showed that Cx45 and ZO-1 colocalized in ROS/Cx45 cells. Furthermore we found that ZO-1 could bind to peptides derived from the carboxyl terminal of Cx45 that had been covalently bound to an agarose resin. These data suggests that Cx45 and ZO-1 directly interact in ROS/Cx45 cells.

Cx43/beta-gal inhibits Cx43 transport in the Golgi apparatus.

A connexin construct consisting of bacterial beta-galactosidase fused to the C-terminus of connexin43 (Cx43/beta-gal) was used to examine Cx43 assembly in NIH 3T3 cells. Cx43/beta-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool trapped by Cx43/beta-gal was retained in a compartment that co-localized with a medial Golgi apparatus marker by immunofluorescence microscopy and that was readily disassembled by treatment with brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/beta-gal were assembled into a sub-hexameric complex, and that Cx43/beta-gal expression also inhibited Cx43 assembly into hemichannels. While this is consistent with Cx43 hemichannel assembly in the trans Golgi network (TGN), these data also suggest that the dominant negative effect of Cx43/beta-gal on Cx43 trafficking may reflect a putative sub-hexameric assembly intermediate formed in the Golgi apparatus.

Phenotypic control of gap junctional communication by cultured alveolar epithelial cells.

We examined phenotype-specific changes in gap junction protein [connexin (Cx)] expression and function by cultured rat alveolar type II cells. Type II cells cultured on extracellular matrix in medium containing keratinocyte growth factor (KGF) and 2% fetal bovine serum (FBS; KGF/2) retained expression of surfactant protein C and the 180-kDa lamellar body membrane protein (lbm180). These markers were lost when cells were cultured in medium containing 10% FBS (MEM/10). With RT-PCR, cells cultured in MEM/10 showed transient increases in Cx43 and Cx46 mRNA expression, whereas Cx32 and Cx26 decreased and Cx30.3 and Cx37 were unchanged. Transient changes in Cx32, Cx43, and Cx46 protein expression were confirmed by immunoblot. In contrast, cells cultured in KGF/2 retained expression of Cx32 and showed increased expression of Cx30.3 and Cx46 mRNAs, compared with that in day 0 cells. With immunofluorescence microscopy, Cx32 and Cx43 were at the plasma membrane of cells grown in KGF/2, whereas Cx46 was exclusively intracellular. Type II cells cultured in MEM/10 showed approximately 3- to 4-fold more intercellular transfer of microinjected lucifer yellow through gap junctions than cells grown in 2% FBS. Thus type II cells dynamically alter gap junctional communication, and distinct alveolar epithelial cell phenotypes express different connexins.

Gap junctional communication modulates gene expression in osteoblastic cells.

Bone-forming cells are organized in a multicellular network interconnected by gap junctions. In these cells, gap junctions are formed by connexin43 (Cx43) and connexin45 (Cx45). Cx43 gap junctions form pores that are more permeable to negatively charged dyes such as Lucifer yellow and calcein than are Cx45 pores. We studied whether altering gap junctional communication by manipulating the relative expression of Cx43 and Cx45 affects the osteoblast phenotype. Transfection of Cx45 in cells that express primarily Cx43 (ROS 17/2.8 and MC3T3-E1) decreased both dye transfer and expression of osteocalcin (OC) and bone sialoprotein (BSP), genes pivotal to bone matrix formation and calcification. Conversely, transfection of Cx43 into cells that express predominantly Cx45 (UMR 106-01) increased both cell coupling and expression of OC and BSP. Transient cotransfection of promoter-luciferase constructs and connexin expression vectors demonstrated that OC and BSP gene transcription was down-regulated by Cx45 cotransfection in ROS 17/2. 8 and MC3T3-E1 cells, in association with a decrease in dye coupling. Conversely, cotransfection of Cx43 in UMR 106-01 cells up-regulated OC and BSP gene transcription. Activity of other less specific osteoblast promoters, such as osteopontin and osteonectin, was less sensitive to changes in gap junctional communication. Thus, altering gap junctional permeability by manipulating the expression of Cx43 and Cx45 in osteoblastic cells alters transcriptional activity of osteoblast-specific promoters, presumably via modulation of signals that can diffuse from cell to cell. A communicating intercellular network is required for the full elaboration of a differentiated osteoblastic phenotype.

Size of IgG-opsonized particles determines macrophage response during internalization.

It is generally assumed that particles > 1 micron elicit a phagocytic response. To determine whether this is the case, we examined the uptake and transport of IgG-opsonized polystyrene beads of defined size, ranging from 0.2 to 3 microns, by mouse bone marrow-derived macrophages. The kinetics of opsonized bead internalization were comparable for each of the different beads examined. We used rhodamine phalloidin to examine particle-induced assembly of F-actin phagocytic cups by fluorescence microscopy. Phagocytic cup formation was size dependent in a nonlinear fashion. Less than 30% of 0.2- to 0.75-micron particles and greater than 80% of 2- and 3-micron particles were associated with F-actin. Cells treated with 0.25 micron cytochalasin D showed decreased phagocytic cup formation and a linear decrease in bead uptake as a function of particle surface area. In contrast, potassium depletion, which preferentially inhibits clathrin-mediated endocytosis, was more effective at inhibiting the uptake of smaller beads. Thus, with increasing particle size, IgG-opsonized particle uptake became less clathrin dependent and more actin dependent. The kinetics of ligand delivery to lysosomes was measured using an immunoprecipitation assay based on the intermixing of internalized anti-dinitrophenol (DNP) IgG with DNP-derivitized beta-glucuronidase (DNP-beta-glu) incorporated into lysosomes. Soluble mannosylated anti-DNP IgG was delivered to lysosomes after an 8-min lag period. The kinetics of anti-DNP IgG-opsonized beads showed a size-dependent response, where beads sized 0.2, 0.5, and 0.75 micron showed a lag period prior to delivery to lysosomes. In contrast, beads 1.0 micron or larger showed no lag in delivery to lysosomes. Since beads that had no lag in delivery to lysosomes also showed high levels of phagocytic cup formation, this suggests that phagocytic cups may be important in the rapid delivery of internalized particles to lysosomes.

CD45 regulates Src family member kinase activity associated with macrophage integrin-mediated adhesion.

BACKGROUND: Adhesion of leukocytes to the extracellular matrix and to other cells is mediated by members of the integrin family of adhesion molecules. Src family kinases are activated upon integrin-mediated adhesion. In lymphocytes, CD45 is a leukocyte-specific transmembrane protein tyrosine phosphatase that activates Src family kinases associated with B-cell and T-cell antigen receptor signaling by constitutive dephosphorylation of the inhibitory carboxy-terminal tyrosine phosphorylation site. Here, we show that CD45 is also important in downregulating the kinase activity of Src family members during integrin-mediated adhesion in macrophages. RESULTS: We found that CD45 colocalized with beta2 integrin and the Src family kinase p53/56(lyn) to adhesion sites in bone marrow-derived macrophages. Macrophages from CD45(-/-) mice were unable to maintain integrin-mediated adhesion. In adherent macrophages, absence of CD45 led to the hyperphosphorylation and hyperactivation of p56/59(hck) and p53/56(lyn), but not of p58(c-fgr). CD45 directly inactivated p59(hck) but not p56(lck) in transient transfection assays. Furthermore, coexpression of CD45 with p59(hck) or p56(lyn) containing a tyrosine to phenylalanine mutation at the carboxy-terminal negative regulatory site resulted in decreased tyrosine phosphorylation of the Src family member kinases due to dephosphorylation of the potentiating tyrosine phosphorylation site within the kinase domain. CONCLUSIONS: Using primary bone marrow macrophages, these studies demonstrate that CD45 regulates Src family kinases and is required to maintain macrophage adhesion. CD45 decreases Src family kinase activity by dephosphorylating the tyrosine residue located within the kinase domain.

Connexin46 is retained as monomers in a trans-Golgi compartment of osteoblastic cells.

Connexins are gap junction proteins that form aqueous channels to interconnect adjacent cells. Rat osteoblasts express connexin43 (Cx43), which forms functional gap junctions at the cell surface. We have found that ROS 17/2.8 osteosarcoma cells, UMR 106-01 osteosarcoma cells, and primary rat calvarial osteoblastic cells also express another gap junction protein, Cx46. Cx46 is a major component of plasma membrane gap junctions in lens. In contrast, Cx46 expressed by osteoblastic cells was predominantly localized to an intracellular perinuclear compartment, which appeared to be an aspect of the TGN as determined by immunofluorescence colocalization. Hela cells transfected with rat Cx46 cDNA (Hela/Cx46) assembled Cx46 into functional gap junction channels at the cell surface. Both rat lens and Hela/Cx46 cells expressed 53-kD (nonphosphorylated) and 68-kD (phosphorylated) forms of Cx46; however, only the 53-kD form was produced by osteoblasts. To examine connexin assembly, monomers were resolved from oligomers by sucrose gradient velocity sedimentation analysis of 1% Triton X-100-solubilized extracts. While Cx43 was assembled into multimeric complexes, ROS cells contained only the monomer form of Cx46. In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers. These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell. Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.

Why do the elderly seek or avoid care? A qualitative analysis.

BACKGROUND AND OBJECTIVES: This study attempts to understand why the elderly seek or choose not to seek health care. Most studies on barriers to health care have measured obstacles defined by the researchers. We attempt to define variables that are relevant to the elderly but have not yet been articulated. METHODS: Using grounded theory, open-ended interviews of 15 non-housebound elderly were conducted and coded. The data obtained were analyzed to discover and characterize the subjects' perceptions of barriers. RESULTS AND CONCLUSIONS: The major theme that emerged involved the interactions among autonomy, self-esteem, and the degree of illness or health. The study generated two hypotheses: 1) Self-esteem is directly correlated with the willingness of the elderly to seek care, especially as illness increases and autonomy decreases. 2) The individual's perception of health status, the perceived roles of the physician and the patient, the physician-patient relationship, and systems issues contribute to the dynamic paradigm that positions the elderly patient to seek or avoid seeking health care.

Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43.

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin.