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The structure of gap junctions in osteoblast-like cells (OBs) and the connexins (cx) that build up these structures were characterized by ultrastructural, immunocytochemical, and molecular techniques. Ultrastructural studies revealed numerous gap junctions which were mostly located on processes of neighboring cells. Immunofluorescence labeling using two different antibodies (specific to mouse live cx26 and cx32 and to a peptide-specific rat heart gap junction protein cx43) gave evidence that in OBs, gap junctions consist mainly of cx43. The presence of cx43 in cultured OB was also confirmed by Western blot analysis. Dye-coupling with Lucifer yellow led to a staining of up to 30 neighboring cells. Parallel intracellular recordings showed that membrane potential amplitude changes (4-5 mV) are typically related to those in the coupled cells. Thus, there is morphological and functional evidence for intercellular communication between OB in culture. OBs in culture express the same connexins as observed in vivo and may serve as a model to investigate electrophysiological events in response to different stimulation signals. [ABSTRACT FROM AUTHOR]

The existence of functional gap junctions between haematopoietic progenitor cells (HPCs) and stromal cells of the haematopoietic microenvironment in the human system is a controversial issue. Primary CD34+ HPCs isolated from leukapheresis products were co-incubated with the human fibroblastoid bone marrow stromal cell line L87/4 in short-term liquid culture. Using the highly sensitive double whole-cell patch-clamp technique, we found that the majority (91%) of CD34+ HPCs are electrically coupled to L87/4 cells. Importantly, efficient coupling was observed within 1 h of the attachment of CD34+ HPCs to plastic adherent L87/4 cells. By comparison, homologous cell pairs formed by L87/4 cells exhibited a significantly higher electric coupling. Analysis of single-channel conductances revealed an electric profile characteristic of connexin 43 (Cx43)-type gap junctions for both homologous and heterologous cell pairs. The Cx phenotype was confirmed using Cx43-specific monoclonal antibodies in a flow cytometric assay and reverse transcription polymerase chain reaction (RT-PCR) for the detection of Cx43 mRNA. Finally, the electrophysiological studies were complemented by dye-transfer experiments using the recently described 'parachute' technique that allows the monitoring of dye diffusion without disruption of the plasma membrane. Taken together, our data indicate that functional Cx43-type gap junctions exist between stromal cells and immature HPCs and, thus, may provide an important regulatory pathway in haematopoiesis.

To examine the role of cell-cell communication via gap junctions in controlling proliferation and differentiation we transfected the malignant trophoblast cell line Jeg-3, which exhibits extremely low cell-cell communication mediated by endogenously expressed connexin40, with connexin26, connexin40, and connexin43, respectively. In vitro growth of all cell clones transfected with connexin genes was significantly reduced compared to controls. This effect corresponded to a significant increase in total junctional conductance of all clones. Single-channel conductances for channels formed by the transfected connexins were in the range of the values published previously. Though total junctional conductance varied highly among clones and even within one clone, differentiation of the cells indicated by beta-hCG secretion was most prominent in the clones that revealed the largest amount of well-coupled cell pairs. Connexin26 channels enable cells of one clone to reduce drastically growth rate and produce significantly higher secretion of beta-hCG. Connexin43 had only moderate effects on the differentiation properties of Jeg-3 cells. These findings suggest that restoration of cell-cell communication plays a role in growth reduction and in differentiation of tumor cells and that different channel proteins might have different effects., (Copyright 1999 Academic Press.)

This report describes the clinico-pathological features of myofibroblastic tumors of the breast in six patients. Four women and one man presented with a benign myofibroblastoma. The sixth patient was a woman with myofibrosarcoma. All myofibroblastomas were composed of a fascicular arrangement of spindle cells embedded in dense bundles of collagen. Tumors differed with respect to their proportion of neoplastic cells and collagenous stroma as well as cellular pleomorphism. Based on this variation, the tumors could be subclassified as classic, collagenized, epithelioid and cellular myofibroblastoma. Immunohistological staining confirmed myofibroblastic differentiation by strong expression of either desmin or smooth muscle actin with coexpression of vimentin. In addition, numerous cells reacted with antibodies to CD68. Proliferative activity was rather low in the myofibroblastoma with an average of 0-2 mitotic figures per 10 HPF. DNA cytometric analysis was performed in two cases and showed diploid stem lines with minor S-phase fractions (1% and 3%). In the myofibrosarcoma, cells contained pleomorphic nuclei with some giant cells and numerous mitotic figures (6-7/10 HPF) and had infiltrating margins that were apparent even grossly. Immunohistochemically, tumor cells strongly expressed vimentin, smooth muscle actin and fibronectin. Ultrastructurally, neoplastic cells met the criteria of myofibroblasts, i.e. contained abundant intermediate filaments and myofilament bundles with focal densities as well as fibronexus junctions. DNA cytometric analysis exhibited again a diploid stemline but marked proliferative activity was present as indicated by an S-phase fraction of 20%. In conclusion, in benign myofibroblastoma there may be some cellular pleomorphism but mitotic activity is always low. The malignant counterpart, myofibrosarcoma, is characterized by marked cellular pleomorphism, infiltrating margins and high mitotic rate.

Little is known about the mechanisms of action of kava pyrones which are the pharmacological active compounds of the plant Piper methysticum Forst. We investigated the effects of the synthetic kava pyrone (+/-)-kavain on long-term potentiation (LTP) in the CA1-region of guinea pig hippocampal slices. (+/-)-Kavain reduced the amplitudes of extracellular field potential changes evoked by electrical stimulation in a concentration dependent manner. These effects were reversible. In experiments with LTP no changes were found in the presence of (+/-)-kavain. In conclusion, our findings suggest (+/-)-kavain to be an effective drug in modulating excitatory signals in the hippocampus of guinea pigs. Additionally, no alterations on synaptic plasticity in hippocampal neurons for this kava pyrone can be presumed.

Background: The decline in the concentration of high density lipoproteins (HDL) observed in postmenopausal women is thought to contribute to the increasing incidence of coronary artery disease (CAD) after menopause. Human serum arylesterase (EC 3.1.1.2) is exclusively associated with HDL. We therefore investigated possible differences in the decline of HDL-levels and of HDL-subfractions HDL2 and HDL3 between postmenopausal women without and with angiographically documented CAD., Patients and Methods: HDL-, HDL2-and-HDL3- concentrations were studied in postmenopausal women with angiographically documented CAD (n = 24; 51 to 72 years mean: 62 years) and compared to HDL-parameters of women without CAD (n = 22; 51 to 81 years, mean: 58 years). Arylesterase activities of HDL2-and HDL3-subfractions and HDL2-cholesterol concentrations were determined after differential precipitation with polyethylene glycol (4.7 mM PEG). Phenotyping of HDL-arylesterase was achieved in CAD patients and in women without CAD after determining hydrolysis of arylesterase substrates paraoxon (PO) and phenylacetate (PA) by calculating paraoxonase/arylesterase activity ratios R (R = [PO]/[PA] x 1000): phenotype A (n = 26) with R < 2.5, phenotype AB (n = 16) with 5.0 < R < 10.7, and phenotype B (n = 4) with R > 13.5., Results: In postmenopausal women with documented CAD, as compared to women without CAD, HDL-cholesterol (55 +/- 3 mg/dl vs. 69 +/- 3 mg/dl HDL2-arylesterase (25 +/- 1 kU/l vs. 33 +/- 2 kU/l), and HDL3-arylesterase (89 +/- 4 kU/l vs. 106 +/- 5 kU/I) were found to be significantly reduced. Analysis of the correlation of lipid parameters and age revealed in CAD patients, but not in postmenopausal women without CAD, a significant increase of total cholesterol (r = 0.42), and significant reductions of both HDL2-arylesterase (r = -0.47) and HDL3-arylesterase (r = 0.74) with increasing age. In contrast, HDL-cholesterol (r = -0.14) and HDL2-cholesterol (r = -0.06) of CAD patients showed only slight and non-significant reductions with age. Since HDL3-arylesterase was found to be age-dependently reduced in women without CAD (r = 0.17), HDL2-arylesterase of postmenopausal women, among all lipid parameters showed the most pronounced differences between women without CAD and CAD patients. The age-dependent decrease of HDL2-arylesterase in postmenopausal women with CAD does not result from an increased frequency of B-allele carriers in the subgroup of CAD patients with an age above the median (64 years)., Conclusion: Genetically determined serum HDL-arylesterase is well suited to quantify HDL in postmenopausal women without and with CAD. HDL2-arylesterase of postmenopausal women should be evaluated as a screening parameter for both primary and secondary CAD prevention.

The dependence of macroscopic gap junctional conductance (g(j)) upon transjunctional voltage (Vj) was examined in 39 paired osteoblast-like (OB) cells from primary cultures using the double whole cell patch clamp technique. OB cells were derived from calvarial explants of new-born rats. Instantaneous current-voltage (Ij-Vj) relationships of OB cell pairs (n = 6) were linear in the entire voltage range (-150 < Vj < 150 mV) examined. The steady-state Ij-Vj relationship was non-linear for V > or = +/-60 mV. The curve for the normalised steady-state junctional conductance-voltage relationship (Gss/G0-Vj) was bell-shaped, and was fitted with a two-state Boltzmann equation with a minimum conductance (Gmin) of 0.2-0.3, and a half deactivation voltage (Vo) of +/-83 mV. In two recordings unitary gap junction channel activity was observable. The linear I-V relationships revealed a single channel conductance of approximately 100 pS. Application of parathyroid hormone (10(-8) M) had no effect on the voltage dependence nor the magnitude of macroscopic currents (n = 7).