[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.

Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick, type A fibroblasts.

We examined the metabolism and intracellular transport of a fluorescent sphingomyelin analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl])- sphingosylphosphorylcholine (C6-NBD-SM), in both normal and Niemann-Pick, type A (NP-A) human skin fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 3 min of warming to 37 degrees C, both normal and NP-A fibroblasts had internalized C6-NBD-SM from the plasma membrane, resulting in a punctate pattern of intracellular fluorescence. Rates for C6-NBD-SM internalization and transport from intracellular compartments to the plasma membrane (recycling) were similar for normal and NP-A cells. With increasing time at 37 degrees C, internalized C6-NBD-SM accumulated in the lysosomes of NP-A fibroblasts, while normal fibroblasts showed increasing Golgi apparatus fluorescence with no observable lysosomal labeling. Since NP-A fibroblasts lack lysosomal (acid) sphingomyelinase (A-SMase), this result suggested that hydrolysis of C6-NBD-SM prevented its accumulation in the lysosomes of normal fibroblasts during its transport along the degradative pathway. We used the amount of C6-NBD-SM hydrolysis by A-SMase in normal cells as a measure of C6-NBD-SM transported from the cell surface to the lysosomes. After a lag period, C6-NBD-SM was delivered to the lysosomes at a rate of approximately 8%/h. This rate was approximately 18-19 fold slower than the rate of C6-NBD-SM recycling from intracellular compartments to the plasma membrane. Thus, small amounts of C6-NBD-SM were transported along the degradative pathway, while most endocytosed C6-NBD-SM was sorted for transport along the plasma membrane recycling pathway.

Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts.

We examined the metabolism and intracellular transport of the D-erythro and L-threo stereoisomers of a fluorescent analogue of sphingomyelin, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl])-sphingosylphosphorylcholine (C6-NBD-SM), in Chinese hamster ovary (CHO-K1) fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 10-15 min of being warmed to 37 degrees C, C6-NBD-SM was internalized from the plasma membrane to a perinuclear location that colocalized with the centriole and was distinct from the lysosomes and the Golgi apparatus. This perinuclear region was also labeled by internalized rhodamine-conjugated transferrin. C6-NBD-SM endocytosis was not inhibited when the microtubules were disrupted with nocodazole; rather, the fluorescent lipid was distributed in vesicles throughout the cell periphery instead of being internalized to the perinuclear region of the cell. The metabolism of C6-NBD-SM to other fluorescent sphingolipids at 37 degrees C and its effect on C6-NBD-SM transport was also examined. To study plasma membrane lipid recycling, C6-NBD-SM was first inserted into the plasma membrane of CHO-K1 cells and then allowed to be internalized by the cells at 37 degrees C. Any C6-NBD-SM remaining at the plasma membrane was then removed by incubation with nonfluorescent liposomes at 7 degrees C, leaving cells containing only internalized fluorescent lipid. The return of C6-NBD-SM to the plasma membrane from intracellular compartments upon further 37 degrees C incubation was then observed. The half-time for a complete round C6-NBD-SM recycling between the plasma membrane and intracellular compartments was approximately 40 min. Pretreatment of cells with either monensin or nocodazole did not inhibit C6-NBD-SM recycling.

The dynamics of chromosome movement in the budding yeast Saccharomyces cerevisiae.

Nuclear DNA movement in the yeast, Saccharomyces cerevisiae, was analyzed in live cells using digital imaging microscopy and corroborated by the analysis of nuclear DNA position in fixed cells. During anaphase, the replicated nuclear genomes initially separated at a rate of 1 micron/min. As the genomes separated, the rate of movement became discontinuous. In addition, the axis defined by the segregating genomes rotated relative to the cell surface. The similarity between these results and those previously obtained in higher eukaryotes suggest that the mechanism of anaphase movement may be highly conserved. Before chromosome separation, novel nuclear DNA movements were observed in cdc13, cdc16, and cdc23 cells but not in wild-type or cdc20 cells. These novel nuclear DNA movements correlated with variability in spindle position and length in cdc16 cells. Models for the mechanism of these movements and their induction by certain cdc mutants are discussed.

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.

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.

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.

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.

Intracellular transport and metabolism of sphingomyelin.

SM is unique among the phospholipids because it is restricted to the lumenal aspect of organelles involved in the secretory and endocytic pathways. Given the intracellular sites of SM biosynthesis and hydrolysis, and the interconnections between these sites by vesicle-mediated transport pathways, the basic mechanism for maintaining the intracellular distribution of SM seems clear. It remains to be determined how SM metabolism and transport are coordinated to maintain the SM content of each organelle. For example, the size of the SM pool at the cell surface is maintained by regulation of at least five processes: transport of newly synthesized SM from the Golgi apparatus, plasma membrane lipid recycling, local SM synthesis, local SM hydrolysis, and SM transport from the cell surface to lysosomes. Although SM cannot undergo spontaneous transbilayer movement, SM metabolism generates both DAG, Cer and (indirectly) SPhB which can rapidly 'flip-flop', and thus gain access to the cytoplasmic leaflet of a membrane. It is of particular interest that these lipid species may be involved in the regulation of PK-C, suggesting that SM metabolism could play a role in signal transduction. However, physiological effects of endogenous Cer and SPhB remain elusive, even though the pharmacological effect of SPhB on PK-C is well established. Aside from the direct generation of second messengers, stimulation of SM hydrolysis has also been shown to induce cholesterol movement from the cell surface to intracellular membranes. It is not known whether this reflects the possibility that cholesterol may act as a second messenger. Alternatively, this phenomenon suggests that SM metabolism may cause rapid changes in the physical properties of the cell surface. For example, erythrocytes extensively treated with exogenously-added SMase will undergo endovesiculation It is tempting to speculate that any involvement of SM in the regulation of intracellular processes requires a combination of both the generation of biochemical second messengers and the alteration of membrane biophysical properties that can result from SM metabolism.

Endosomal density shift is related to a decrease in fusion capacity.

Dinitrophenol (DNP)-beta-glucuronidase and mannosylated anti-DNP IgG, which are endocytosed by the mannose receptor and delivered to lysosomes, were previously developed as probes for examination of fusion between early endosomes in a cell-free system. In this study, these probes were found to be transported by intact cells to endocytic vesicles with heavy buoyant density at different rates, as determined by Percoll gradient fractionation of cell homogenates. There was a concomitant loss of in vitro fusion activity as the ligands moved to dense compartments. In monensin-treated cells, DNP-beta-glucuronidase was retained in a light compartment corresponding to intracellular vesicles capable of fusion in vitro. Pulse-chase studies using a DNP-derivatized transferrin-alkaline phosphatase conjugate showed that a recycling ligand was always found in light intracellular vesicles that were capable of fusion to early endosomes in vitro. In contrast to cell-free systems, intact cells sequentially labeled with DNP-beta-glucuronidase and then mannosylated anti-DNP IgG showed ligand mixing in both early and late endocytic compartments. Treatment with nocodazole or colchicine did not affect the rate of DNP-beta-glucuronidase transport to heavy vesicles in intact cells, however, the extent of ligand mixing in late endosomes was decreased by microtubule disruption. Using sequentially labeled cells split into two groups, we directly compared ligand mixing in vitro to mixing by intact cells. Fusion alone does not mediate increases in vesicle density, since DNP-beta-glucuronidase/anti-DNP IgG complexes formed in vitro were found in light vesicles, while intact cells showed immune complexes predominantly in heavy vesicles. These results suggest that the density shift is an initial step in targeting to lysosomes.

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.

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.

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.