Immortalized connexin43 knockout cell lines display a subset of biological properties associated with the transformed phenotype

Immortalized cells from embryonic connexin43 knockout mice (Cx43-/-) and homozygous littermates (Cx43+/+) were cloned and characterized to determine whether the absence of Cx43 function would induce observable phenotypic changes. Cells of the Cx43+/+ clones expressed Cx43 and engaged in gap junctional communication with 10-12 neighboring cells. The Cx43-/- cells were devoid of Cx43 and communicated to less than 1 cell. Electrophysiological analysis indicated that the Cx43-/- cells communicated through Cx45 channels from 8-80-fold less than did the Cx43+/+ subclones, which seemed to communicate through Cx43 and Cx45 channels. The Cx43-/- clones grew at faster rates and to higher saturation densities, had a more spindly morphology, were more refractile, and adhered less well to the substratum than did the Cx43+/+ clones. Reintroducing the Cx43 gene into the Cx43-/- clones resulted in three subclones that communicated to 3-4 cells. Partial restoration of gap junctional communication in the three subclones was accompanied by reduced growth rates and saturation densities (2-fold compared to that of parental Cx43-/- clones) but no reversions in morphology or cell-substratum adhesion. The increased growth rates and saturation densities, altered morphology, and decreased cell adhesion displayed by the Cx43-/- clones reflect a subset of the properties of transformed cells. These studies advance the hypothesis that loss of Cx43 function during development may cause cells to acquire a preneoplastic condition.     

Neurite outgrowth stimulated by neural cell adhesion molecules requires growth-associated protein-43 (GAP-43) function and is associated with GAP-43 phosphorylation in growth cones

The mechanisms whereby cell adhesion molecules (CAMs) promote axonal growth and synaptic plasticity are poorly understood. Here we show that the neurite outgrowth stimulated by NCAM-mediated fibroblast growth factor (FGF) receptor activation in cerebellar granule cells is associated with increased GAP-43 phosphorylation on serine-41. In contrast, neither NCAM nor FGF was able to stimulate neurite outgrowth in similar neurons from mice in which the GAP-43 gene had been deleted by homologous recombination. Integrin-mediated neurite outgrowth was unaffected by GAP-43 deletion. Both neurite outgrowth and rapid phosphorylation of GAP-43 in isolated growth cones required the first three Ig domains of a NCAM-Fc chimera and were stimulated maximally at 5 micrograms/ml (approximately 50 nM). Likewise, GAP-43 phosphorylation in isolated growth cones also was stimulated by an L1-Fc chimera. Both neurite outgrowth and NCAM-stimulated GAP-43 phosphorylation were inhibited by antibodies to the FGF receptor and a diacylglycerol lipase inhibitor (RHC80267) that blocks the production of arachidonic acid in response to activation of the FGF receptor. Direct activation of the FGF receptor and the arachidonic acid cascade with either basic FGF or melittin also resulted in increased GAP-43 phosphorylation. These data suggest that the stimulation of neurite outgrowth by NCAM requires GAP-43 function and that GAP-43 phosphorylation in isolated growth cones occurs via an FGF receptor-dependent increase in arachidonic acid.     

Nutritional modulation of guinea pig skin hyperproliferation by essential fatty acid deficiency is associated with selective down regulation of protein kinase C-beta

In a previous study we demonstrated that 13-hydroxyoctadecadienoic acid (13-HODE), a 15-lipoxygenase metabolite of linoleic acid is incorporated into epidermal phosphatidyl 4,5-bisphosphate (PtdIns 4,5-P2) and released as 13-HODE-containing-diacylglycerol (13-HODE-DAG). In vitro, 13-HODE-DAG was shown to selectively inhibit epidermal total protein kinase C (PKC-beta) activity. To determine whether these observations are relevant in vivo, guinea pigs were made essential fatty acid deficient (EFAD) by feeding them a basal diet supplemented with 4% hydrogenated coconut oil for 8 wk. Tissue levels of putative 13-HODE-DAG, protein kinase C (PKC) isozymes and tissue hyperproliferation were determined in the epidermal preparations from skin of control safflower oil-fed guinea pigs, those fed EFAD diet and those fed EFAD diet followed by the control diet for 2 wk. Our data revealed that cutaneous 13-HODE and 13-HODE-DAG were significantly lower in EFAD animals than in safflower-fed controls. These reductions were associated with both elevated epidermal hyperproliferation and elevated expressions and activities of PKC-alpha and beta-isozymes. Refeeding the animals with safflower oil for 2 wk replenished tissue levels of 13-HODE-DAG, which inversely correlated with the selective down regulation of PKC-beta expression and activity and the reversal of hyperproliferation. In contrast, although, the expression and activity of PKC-alpha was elevated in the epidermis of the EFAD guinea pigs, this elevated PKC-alpha expression was not down regulated after refeeding the safflower oil diet to the animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

B-50, the growth associated protein-43: modulation of cell morphology and communication in the nervous system

The growth-associated protein B-50 (GAP-43) is a presynaptic protein. Its expression is largely restricted to the nervous system. B-50 is frequently used as a marker for sprouting, because it is located in growth cones, maximally expressed during nervous system development and re-induced in injured and regenerating neural tissues. The B-50 gene is highly conserved during evolution. The B-50 gene contains two promoters and three exons which specify functional domains of the protein. The first exon encoding the 1-10 sequence, harbors the palmitoylation site for attachment to the axolemma and the minimal domain for interaction with G0 protein. The second exon contains the "GAP module", including the calmodulin binding and the protein kinase C phosphorylation domain which is shared by the family of IQ proteins. Downstream sequences of the second and non-coding sequences in the third exon encode species variability. The third exon also contains a conserved domain for phosphorylation by casein kinase II. Functional interference experiments using antisense oligonucleotides or antibodies, have shown inhibition of neurite outgrowth and neurotransmitter release. Overexpression of B-50 in cells or transgenic mice results in excessive sprouting. The various interactions, specified by the structural domains, are thought to underlie the role of B-50 in synaptic plasticity, participating in membrane extension during neuritogenesis, in neurotransmitter release and long-term potentiation. Apparently, B-50 null-mutant mice do not display gross phenotypic changes of the nervous system, although the B-50 deletion affects neuronal pathfinding and reduces postnatal survival. The experimental evidence suggests that neuronal morphology and communication are critically modulated by, but not absolutely dependent on, (enhanced) B-50 presence.     

Observations on rat Sertoli ectoplasmic ('junctional') specializations in their association with germ cells of the rat testis

The ectoplasmic ('junctional') specialization, a subsurface modification of the Sertoli cell that is often seen facing germ cells, was studied in relation to the development and maturation of these germ cells. This structure is composed of subsurface bundles of filaments and more deeply placed endoplasmic reticulum. The data indicate that these subsurface modifications of Sertoli cells are reutilized in a cyclic fashion, being transferred from their position facing late spermatids to one opposing less mature germ cells. Ectoplasmic specializations appeared to function mechanically in grasping the heads of the spermatids which are undergoing the elongation and maturation phases of spermiogenesis rather than in actually attaching Sertoli cells to these germ cells. It is postulated that the ectoplasmic specialization imparts rigidity to that area of the Sertoli cell that surrounds the head region of the germ cell, forming a recess and a mantle by which the germ cell may be moved toward the base or toward the surface of the seminiferous epithelium. The observed linkage of microtubules to the cisternae of the complex provided a morphological basis for the changes in the cytoarchietecture of the Sertoli cell, which must accompany these movements.     

Temperature-mediated germ cell loss in the testis is associated with altered expression of the cell-cycle regulator p53

The dependence of spermatogenesis on the relatively cool environment of the scrotum is well known, and recent work has shown that germ cells undergo apoptosis upon exposure to abdominal temperature. p53 is a potent inducer of apoptosis and regulator of cell growth, and is found in high concentrations in the testis. The purpose of this study was to determine whether exposure of the testes to suprascrotal temperature was associated with alterations in testicular p53 expression. Male adult CD-1 mice were rendered unilaterally cryptorchid by surgically fixing one testis to the anterior abdominal wall while leaving the contralateral tests in the scrotum to serve as the euthermic control. p53 expression was evaluated in the cytoplasmic, soluble nuclear, and insoluble fractions by Western blot analysis with the monoclonal p53 antibody pAb240. The weights of the scrotal testes were unchanged over the 15 day study period. The weights of the cryptorchid testes remained stable for 7 days and then decreased by approximately 40% over the next two days. Histological evidence of germ cell loss was evident only after day 7. Altered expression of p53 protein in the cryptorchid testis was noted beginning on day 7, and consisted of the expression of a new 47 kD isoform of p53 in the cytosolic form and a 30 kD isoform in the soluble nuclear fraction. Scrotal testes showed no changes at any time point. These results demonstrate altered expression of the regulatory protein p53 beginning 1-2 days prior to the onset of germ cell loss following experimental unilateral cryptorchidism. Given the known function of p53 as an inducer of apoptotic cell death, these observations suggest a significant role for p53 in temperature-mediated germ cell loss.     

Testin secreted by Sertoli cells is associated with the cell surface, and its expression correlates with the disruption of Sertoli-germ cell junctions but not the inter-Sertoli tight junction

Testin is a testosterone-responsive Sertoli cell secretory product. In the present study, we demonstrated that the amount of testin secreted by Sertoli cells in vitro was comparable with several other Sertoli cell secretory products. However, virtually no testin was found in the luminal fluid and cytosols of the testis and epididymis when the intercellular junctions were not previously disrupted, suggesting that secreted testin may be reabsorbed by testicular cells in vivo. Studies using Sertoli cells with and without a cell surface cross-linker and radioiodination in conjunction with immunoprecipitation illustrated the presence of two polypeptides of 28 and 45 kDa, which constitute a binding protein complex that anchors testin onto the cell surface. The 28- and 45-kDa peptide appear to be residing on and inside the cell surface, respectively. Immunogold EM studies illustrated testin was abundantly localized on the Sertoli cell side of the ectoplasmic specialization (a modified adherens junction) surrounding developing spermatids. In contrast, very few testin gold particles were found at the site of inter-Sertoli tight junctions. When the inter-Sertoli tight junctions were formed or disrupted, no significant change in testin expression was noted. This is in sharp contrast to the disruption of Sertoli-germ cell junctions, which is accompanied by a surge in testin expression. These results demonstrate the usefulness of testin in examining Sertoli-germ cell interactions.     

Blood barriers of the epididymis and vas deferens act asynchronously with the blood barrier of the testis in the mink (Mustela vison)

The purpose of the present study was to determine whether the blood barrier of the epididymis and vas deferens acted synchronously or not with the blood barrier of the testis. The permeability of the blood-epididymis and blood-vas deferens barrier was tested in neonatal kit mink up to puberty and monthly in adult minks throughout the annual seasonal reproductive cycle. Attention was focused particularly on time intervals when the blood barrier of the testis has been documented to be permeable, namely, before puberty and during testicular regression in the adult. One of two electron-opaque permeability tracers was perfused into the blood stream: horseradish peroxidase (HRP) or lanthanum nitrate. The convoluted tube of the epididymis was divided into three anatomical regions: the caput, corpus, and cauda. The vas deferens was divided into proximal and distal regions. At birth and throughout puberty, the three regions of the epididymis and the two of the vas deferens showed a lumen and a competent blood barrier. In the adult, a lumen persisted in the epididymis and vas deferens throughout the annual seasonal reproductive cycle, and the blood barrier of the excurrent duct remained impermeable even when the blood barrier in the testis became momentarily permeable during testicular regression. When HRP was used to test the permeability of the blood-tissue barrier of the excurrent ducts, no tracer deposits were observed on the lumenal surface of the epithelium. Conversely, when lanthanum served as the tracer, deposits of the probe were associated with microvilli and intracellular membranes despite impermeability of tight junctions. The data show that the lanthanum technique can yield false-positive results. The findings also indicate that 1) a blood-excurrent duct barrier is established before the blood-testis barrier and 2) the two barriers act asynchronously. It is therefore plausible that they are modulated by distinct factors.     

Identification of amino acid residues associated with modulation of flavin-containing monooxygenase (FMO) activity by imipramine: structure/function studies with FMO1 from pig and rabbit

The activity of the flavin-containing monooxygenase (FMO) can be modulated by a number of nitrogen-containing compounds in a manner that is both isoform and modulator-dependent. We now show that the direction (activation or inhibition) and extent of modulation can also be dependent on substrate concentration. Imipramine activates methimazole metabolism catalyzed by rabbit FMO1 or FMO2 at methimazole concentrations greater than 50 or 100 microM, respectively, and inhibits at lower methimazole concentrations. The extent of the activation increases as the substrate concentration increases, and the extent of inhibition increases as the substrate concentration decreases. With either inhibition or activation, the magnitude of the effect shows a similar, direct dependency on imipramine concentration. In contrast, imipramine inhibits the metabolism of methimazole catalyzed by pig FMO1 at all substrate concentrations. The structural basis for this unique ortholog difference between the responses of rabbit and pig FMO1 to imipramine was studied by random chimeragenesis and site-directed mutagenesis. Results with chimeras indicated that modulation of FMO1 activity by imipramine is controlled to a great extent by two areas of the FMO primary structure (residues 381-432 and 433-465). Four amino acids in these regions (positions 381, 400, 420 and 433) and one additional residue (position 186) were identified by site-directed mutagenesis as primary determinants of the imipramine response. When the residues at these positions in rabbit FMO1 are exchanged for the corresponding residues of pig FMO1, a mutant with the functional properties of pig FMO1 is produced. Our results suggest that the response of FMO1 to imipramine involves a distribution between two sites that is regulated by structural features that do not alter the overall binding. The inhibition observed, although it appears to be competitive, likely does not involve competition for a binding site since alteration of imipramine metabolism has no effect on the parameters of methimazole metabolism.     

p21WAF1/Cip1 expression is associated with cell differentiation but not with p53 mutations in squamous cell carcinomas of the larynx

The anti-metastatic effect of Z-100, an immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis, was investigated in mice bearing B16 melanoma cells. Treatment of BF10 mice implanted with high metastatic B16F10 melanoma cells with a 10 mg/kg dose of Z-100 resulted in the reduction of experimental pulmonary metastasis as compared with that of BF10 mice treated with saline. The number of pulmonary metastatic colonies in BF1 mice (mice implanted with low metastatic B16F1 melanoma cells) was greatly increased after the inoculation of CD4+ CD11b+ CD281+ TCR alphabeta+ type 2 T cells (F10-Th2 cells) derived from BF10 mice, while only a few metastatic colonies were demonstrated in lungs of BF1 mice inoculated with naive CD4+ T cells. However, the numbers of metastatic colonies in BF1 mice were not increased when they were inoculated with the F10-Th2 cell fraction derived from Z-100-treated BF10 mice and the generation of F10-Th2 cells in BF10 mice was effectively suppressed by the Z-100 treatment. These results suggest that Z-100 inhibits pulmonary metastasis of B16 melanoma through the regulation of tumor-associated Th2 cells, which are a key cell in the acceleration of tumor metastasis.     

Primary structure of bovine Hageman factor (blood coagulation factor XII): comparison with human and guinea pig molecules

An understanding of the consequences of autologous vein grafting reveals both the reasons why cryopreserved allogenic veins are being used clinically and how they are most likely to be expected to fail. Autologous vein bypass grafts are characterized by a series of distinct biological properties that influences their in vivo patency. Current surgical practice ensures that the endothelium of vein grafts is preserved at the time of implantation and that there is minimal damage to the smooth muscle cells. After implantation, the endothelial cells show varying degrees of morphological changes that are maximal within the first 3 days after grafting. In autografts, extensive endothelial denudation does not appear to occur. During the initial grafting period, the smooth muscle cells change from a contractile phenotype to a synthetic phenotype, migrate from the media, proliferate in the intima, and lay down connective tissue. Thereafter, endothelial cell changes regress and the smooth muscle cells return to their contractile phenotype. Perioperative manipulation of vein grafts results in decreased endothelial cell function but preservation of smooth muscle cell responsiveness. Postoperatively endothelial cell-mediated relaxation to acetylcholine is lost and smooth muscle cell contractility is decreased. Within 7 days after implantation, smooth muscle cell contractility returns and, with time, becomes markedly greater than that of the control vein. Endothelium-mediated relaxation to acetylcholine never returns in vein grafts and this loss of endothelial cell function appears to be related to receptor-coupled G-protein defects. Smooth muscle cell contractility remains abnormal. Many of the intimal hyperplastic lesions in vein grafts progress to stenosis or become sites of accelerated atherosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the development of the fatty acid content and composition of the brain of a precocial species (guinea pig) and a non-precocial species (rat)

The fatty acid compositions of the brains of a precocial (guinea pig) and a non-precocial (rat) species have been studied as a function of development. In the rat brain the total fatty acid content expressed as mg g wet wt.-1 increased more than fourfold during the period from 5 days after birth to adulthood. However, the percentage composition of this total fatty acid content when expressed per individual fatty acid remained fairly constant, with the exception of nervonic acid (C24:1) which also increased fourfold on a percentage basis. In the guinea pig brain, however, at birth the total fatty acid content, expressed as mg g wet wt-1, is the same as that of the adult, the concentration doubling during the period from 25 days before birth until birth. Again, if the fatty acid content is analysed and expressed on a percentage basis, the relative concentrations of the individual fatty acids remain fairly constant over the period from 25 days before birth until adulthood, with the exception of nervonic (C24:1) acid which increases about fivefold from 25 days before birth to birth and only marginally (20%) from birth to adulthood. These results are discussed in relationship to the onset of neurological competence in the two species. It is concluded that the increase in fatty acid content (both total and individually) of the brains of these species as a function of the foetal and neonatal development follows a pattern which is similar to the pattern of development of certain key enzymes of energy metabolism and of neurological competence.     

Ultrastructural localization of prostaglandin endoperoxide synthase (cyclooxygenase) to isolated, purified fractions of guinea pig peritoneal macrophage and line 10 hepatocarcinoma cell lipid bodies

The majority of equations used to predict values for basal metabolic rates (BMRs) are the result of indirect calorimetry measurements performed in the 1930s and 1950s. To assess the reliability of these equations in predicting the resting energy expenditure (REE) of the children in our community, indirect calorimetry was performed on 92 male and 107 female healthy children 2-3 h postprandial. Each individual was measured for a duration of 15-20 min. The data for analysis were obtained from 5-15 min steady-state periods. Subjects ranged in age from 5 to 16 years. The results were compared with BMRs calculated from the Harris-Benedict equation (Harris J, Benedict F. A biometric study of basal metabolism in man. Washington, DC: Carnegie Institute of Washington, publication no. 279, 1919.), the Food and Agriculture Organization/World Health Organization/United Nations University (FAO/WHO/UNU) equations, and the equations proposed by Schofield for use by the 1985 FAO/WHO/UNU Nutrition Committee. The values predicted by the FAO/WHO/UNU and Schofield equations were consistent with the measured resting values for all the children in the study population. Ninety-two children weighed between 90-110% of their ideal body weight. When the measured REE and estimated BMR were compared by gender and age in these children, the Schofield equations provided the best estimates. Ninety-four of the study subjects weighed > 110% of their ideal body weight. The predicted estimates by all equations were consistent with the measured values in this subgroup of the population. We conclude that the FAO/WHO/UNU and Schofield equations are reliable estimates of metabolic rate in healthy children when measurement of REE is not possible.     

Creep after loading in the relaxed and contracted smooth muscle (taenia coli of the guinea pig) under various osmotic conditions

Skin reactivity to tuberculin has been studied during the course of experimental leptospirosis in guinea pigs. A depression of the delayed hypersensitivity to tuberculin was demonstrated in the infected animals. The depression was most pronounced when icterus had developed. The depression was not correlated with the amount of infectious units administered or with the demonstration of live leptospirae in the peritoneal cavity. In the infected animals there was no correlation between the initial and the final skin tests which is in contrast to findings in the control group.     

Poly(3-hydroxybutyrate) is associated with specific proteins in the cytoplasm and membranes of Escherichia coli

Poly(3-hydroxybutyrate) (PHB) is well-known as a high molecular weight homopolymer of R-3-hydroxybutyrate which accumulates in storage granules within the cytosols of certain bacteria. Escherichia coli does not amass these granules; however, small amounts of low molecular weight PHB (<0.02% of dry weight) have been found in the plasma membranes in complexes with calcium polyphosphate; the complexes serve as voltage-activated calcium channels. Here we report that polyphosphate-complexed PHB is only a minor fraction of the polyester in E. coli. PHB comprises 0.36 to 0. 55% of the dry weight of log-phase cells, depending on culture medium, and this amount increases by 15 to 20% when the cells are made genetically competent. The PHB is widely distributed throughout the cell, wherein it is primarily associated with proteins. The identity of protein-associated PHB was established by antibody reaction, chemical assay, and 1H NMR spectroscopy. As expected, the physical and chemical properties of protein-associated PHB were found to be considerably different from those of the bulk polymer or granule PHB, e.g. protein-PHB complexes are normally insoluble in chloroform, soluble in water and alkaline hypochlorite, and are converted to crotonic acid more slowly on heating in concentrated sulfuric acid. Our studies indicate that the majority of cellular PHB (over 80%) is located in cytoplasmic proteins, especially proteins of the ribosomal fraction. Western immunoblots, probed with polyclonal anti-PHB IgG, revealed a number of PHB-polypeptides having a wide range of molecular weights in all cell fractions. These results suggest that PHB is a fundamental constituent of cells that may have physiological functions in addition to facilitating ion transmembrane transport or serving as a carbon reserve.     

Familial distal renal tubular acidosis is associated with mutations in the red cell anion exchanger (Band 3, AE1) gene

All affected patients in four families with autosomal dominant familial renal tubular acidosis (dRTA) were heterozygous for mutations in their red cell HCO3-/Cl- exchanger, band 3 (AE1, SLC4A1) genes, and these mutations were not found in any of the nine normal family members studied. The mutation Arg589--> His was present in two families, while Arg589--> Cys and Ser613--> Phe changes were found in the other families. Linkage studies confirmed the co-segregation of the disease with a genetic marker close to AE1. The affected individuals with the Arg589 mutations had reduced red cell sulfate transport and altered glycosylation of the red cell band 3 N-glycan chain. The red cells of individuals with the Ser613--> Phe mutation had markedly increased red cell sulfate transport but almost normal red cell iodide transport. The erythroid and kidney isoforms of the mutant band 3 proteins were expressed in Xenopus oocytes and all showed significant chloride transport activity. We conclude that dominantly inherited dRTA is associated with mutations in band 3; but both the disease and its autosomal dominant inheritance are not related simply to the anion transport activity of the mutant proteins.