Connexins 26, 32 and 43 are expressed in virgin, pregnant and lactating mammary glands

Gap junctions (GJ) are formed by a number of homologous proteins termed connexins. Here expression of connexins Cx26, Cx32 and Cx43, was evaluated by immunofluorescence (IF) in mammary glands from virgin, pregnant and lactating rats. Cx26, Cx32 and Cx43 labeling was detected in epithelial parenchymal cells at all functional stages. Cx26 and Cx32 labeling was very low in glands from virgin animals, somewhat greater in glands from pregnant animals and significantly higher (in number and size) in lactating animals. In the last ones, Cx26 and Cx32 punctate labeling was localized to the basal and lateral membranes of alveolar epithelial cells and collecting ductules. Cx43 punctate labeling was restricted to the periphery of alveoli towards the basal pole of epithelial cells at all functional stages, and it enlarged slightly during lactation. At this localization, Cx43 may form GJ between myoepithelial cells and/or between epithelial and myoepithelial cells. Cx43 was also found to be steadily expressed in the connective tissue which surrounds and invades each parenchymal lobe, at all functional stages. At this localization, Cx43 may couple fibroblasts and/or adipose cells. IF studies in sections from lactating mice showed the same distribution of connexins. Immunoblots confirmed specificity of labeling and the presence of Cx32 and Cx43 in the mammary gland. The increase in connexin expression detected during pregnancy and lactation may be important for epithelial cell differentiation and secretion in the mammary gland.     

Effect of human nasal secretions on the antiviral activity of human fibroblast and leukocyte interferon.

Many normal human nasal secretions contain an inhibitor of human fibroblast IF. This inhibitor had no effect on human leukocyte IF. The amount of inhibition of fibroblast IF increased with increasing quantities of nasal secretions. Also, the inhibition could be overcome with increasing concentrations of IF.     

Production of Mouse Interferon with High Titers in a Large-Scale Suspension Culture System1

L-MS cells, adapted to grow in suspension, were obtained by selection from a high interferon (IF)-producing line of mouse L cell monolayers. A large volume of L-MS cells (20 liters or more; 1–2 × 10¹⁰ cells) was readily grown in a spinner culture, retaining their ability to produce high yields of IF in serum-free medium following induction with Newcastle disease virus (NDV). The optimal condition for the production of IF in the suspension culture of L-MS cells was established. The system also proved itself to be susceptible to IF induction by polyinosinic-polycytidylic acid (Poly I · Poly C) and by NDV inactivated with ultraviolet light (NDV-UV). By employing the present system, large quantities of mouse IF of a high titer could be routinely prepared.     

An immortalized human fibroblast cell line is permissive for human cytomegalovirus infection.

Human foreskin fibroblasts (HFF) were immortalized via retrovirus-mediated gene transfer of the E6 and E7 genes of human papillomavirus type 16. An immortalized fibroblast (IF) cell line which was morphologically akin to the parental cell line was isolated. The IF cell line was evaluated for permissiveness to human cytomegalovirus (HCMV) infection after the IF cell line surpassed the normal passage limitation of diploid fibroblasts. Western immunoblot analysis of representative HCMV-encoded immediate-early (72-kDa), early (gB), and late (gH) gene products demonstrated that the IF cell line produced these proteins analogous to those produced by the parental HFF cells. Similar quantities of infectious virus were produced in the IF and HFF cell lines as determined in one-step growth curve experiments. Compared with the HFF cells, morphologically identical plaques were produced in the IF cell line in approximately 10 to 12 days postinfection. These findings indicate that fibroblast cell lines immortalized with transforming genes of human papillomavirus retain complete permissiveness to HCMV infection and support plaque formation. The IF cell line will be useful for future genetic analysis of HCMV.     

Colchicine-sensitive and colchicine-insensitive intermediate filament systems distinguished by a new intermediate filament-associated protein, IFAP-70/280 kD.

A monoclonal antibody was produced, using as antigen a BHK-21 cytoskeletal preparation enriched in intermediate filaments (IF) and their associated proteins. This antibody reacted exclusively with a reproducible set of 70-280 kD polypeptides present in minor quantities in this preparation, as detected by immunoblot analysis. Based upon several criteria, this immunologically related group of polypeptides was designated as IFAP-70/280 kD (IF-Associated Protein): (1) it co-isolated with IF in vitro, (2) it co-localized (by both immunofluorescence and immunoelectron microscopy) with IF in situ in all stages of cell spreading, and (3) it segregated in vitro with the 54/55 kD (desmin/vimentin) structural IF subunit proteins of BHK cells through two cycles of in vitro disassembly/assembly. Immunogold labeling further localized IFAP-70/280 kD to regions of parallel or loosely bundled IF in situ, suggesting a role in regulating the supramolecular organization of IF. When this monoclonal antibody was used for double-label immunofluorescence observations of colchicine-treated BHK cells, it demonstrated the presence of colchicine-sensitive and colchicine-insensitive IF. Anti-IFAP-70/280 kD localized entirely to the drug-induced juxtanuclear IF cap, while a polyclonal antibody directed against the desmin/vimentin structural IF subunits and the previously characterized monoclonal anti-IFAP-300 kD [Yang et al., 1985; J. Cell Biol. 100:620] localized to both the juxtanuclear IF cap and a colchicine-insensitive IF network peripheral to the cap in the same cells. The colchicine-insensitive IF pattern often exhibited similarities to that observed for the actin-based stress fiber system, suggesting that stress fiber association may be an additional factor in IF organization.     

Myometrial activity related to gap junction area in periparturient and in ovariectomized oestrogen treated sheep

Propagation of electrical (EMG) activity in the myometrium may be facilitated by the presence of gap junctions (GJ), leading to improved synchronization of contractility. EMG and mechanical (IUP) activity in relation to GJ area were studied in 9 periparturient and in 2 ovariectomized (OVX) oestradiol-17 beta (E2) treated ewes, chronically instrumented with bipolar electrodes and intrauterine sponge-tip catheters. Myometrial biopsies were taken under epidural anaesthesia at various time intervals around delivery and after intra-arterial administration of 0.1 mg of E2. In pregnant ewes we found a significant increase in the rate of rise and area of IUP cycles during labour. Both were closely related to a significant increase in GJ area. In E2 treated OVX sheep we found a significant increase in GJ area, with a maximum at 24 hours after injection. The increase in GJ area was associated with a significant increase in the rate of rise of IUP cycles. The results of our study support the hypothesis that gap junctions facilitate the spread of EMG activity across the myometrium, which may improve synchronization of uterine contractility during labour.     

Human synemin gene generates splice variants encoding two distinct intermediate filament proteins.

Intermediate filament (IF) proteins are constituents of the cytoskeleton, conferring resistance to mechanical stress, and are encoded by a dispersed multigene family. In man we have identified two isoforms (180 and 150 kDa) of the IF protein synemin. Synemin alpha and beta have a very short N-terminal domain of 10 amino acids and a long C-terminal domain consisting of 1243 amino acids for the alpha isoform and 931 amino acids for the beta isoform. An intronic sequence of the synemin beta isoform is used as a coding sequence for synemin alpha. Both mRNA isoforms (6.5 and 7.5 kb) result from alternative splicing of the same gene, which has been assigned to human chromosome 15q26.3. Analyses by Northern and Western blot revealed that isoform beta is the predominant isoform in striated muscles, whereas both isoforms (alpha and beta) are present in almost equal quantities in smooth muscles. Co-transfection and immunolabeling experiments indicate that both synemin isoforms are incorporated with desmin to form heteropolymeric IFs. Furthermore synemin and desmin are found aggregated together in certain pathological situations.     

Generation of lipopolysaccharide-induced interferon in spleen cell cultures

Incubation of murine spleen-cell cultures with lipopolysaccharide (LPS) induces interferon (IF) production. Maximal IF levels are obtained after incubation with 100 g/ml for 10 h. Two inbred mouse strains differing in their ability to generate LPS-induced IF in spleen-cell cultures were used: C3H/eB, which generates high levels of IF (about 60 units/ml), and C3H/HeJ, which fails to generate detectable quantities of IF. In a genetic analysis these strains were hybridized and IF production was determined in spleen-cell cultures from F₁ and F₂ generations, and from backcrosses of F₁ hybrids to parent strains. The results indicate that, in parent strains, a single dominant autosomal gene is responsible for differences in IF production in spleen cultures. LPS-induced IF in spleen-cell cultures resists pH 2 for as long as 48 h, but is labile to heating at 56° C for 30 min. Both macrophages and lymphocytes must be present in cultures for generation of LPS-induced IF. By using mixed cultures of macrophages and lymphocytes from C3H/eB and C3H/HeJ mice, it was shown that macrophages have to interact directly with LPS to enable IF production in the cultures.     

Organization of intermediate filaments and their association with collagen-containing phagosomes in mouse decidual cells

We have analyzed the distribution of intermediate filaments (IF) in the cytoplasm of mature decidual cells of mice. IF were scattered throughout the cytoplasm of these cells although there was a preferential accumulation around the nuclei. In many cells a large area of the cytoplasm was occupied by a rich network of IF that extended from the perinuclear region toward the cell surface. Thin bundles of IF crossed the cytoplasm without a preferential orientation. IF were also seen in close association with nuclear pore complexes, gap junctions, mitochondria, and lysosomes. A very developed network of IF surrounded phagosomes that contained collagen fibrils. Longitudinal and cross sections of these phagosomes showed a very close association of IF with the phagosome membrane.     

Final Energy Requirements of Steam for Use in Environmental Life Cycle Assessment

Steam is an important utility that is required in nearly all industrial process chains and hence needs to be modeled in life cycle assessment studies. Industrial steam systems are often very complex, with different steam flows varying in pressure and temperature and being transported over different distances. This should be accounted for when calculating the energy requirements related to steam supply. In this article, we constructed a generic model that allows estimating final energy requirements (i.e., gate‐to‐gate energy required to generate the steam) of various types of single‐fuel steam systems without turbines (i.e., open and closed cycles) with or without flash steam and expressed per tonne (t) of steam supplied to a process (before heat exchange) or per gigajoule (GJ) heat delivered within the process (after heat exchange, i.e., as useful energy). The model focuses on steam provided for covering process heat requirements and hence excludes cogeneration schemes with steam turbines. Based on the final energy requirements estimated with our generic model, primary energy requirements and environmental impacts can be calculated for various circumstances. Depending on the conditions chosen, final energy requirements for natural gas–fueled systems, as estimated in this study, are 2.71 to 3.44 GJ/t produced steam or 1.33 to 1.78 GJ/GJ delivered heat.     

Identification of the agent from Lactobacillus plantarum KFRI464 that enhances bacteriocin production by Leuconostoc citreum GJ7

AIM: To provide evidence that the production of bacteriocin by lactic acid bacteria can be enhanced by the presence of a bacteriocin-sensitive strain and identify the agent that is responsible for enhancing bacteriocin production. METHODS AND RESULTS: One bacteriocin-producing lactic acid bacterium was isolated from kimchi. The strain GJ7 was designated as Leuconostoc citreum GJ7 based on Gram staining, biochemical properties, and 16S rRNA gene sequencing. The isolate produced a heat- and pH-stable bacteriocin (kimchicin GJ7), which has antagonistic activity against a broad spectrum of micro-organisms. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified kimchicin GJ7 showed a single band of molecular weight c. 3500 Da. Cultures of Leuc. citreum GJ7 in the presence of thermally inactivated kimchicin GJ7-sensitive strains, Lactobacillus plantarum KFRI 464, Lactobacillus delbrueckii KFRI 347, or Leuconostoc mesenteroides KCTC 1628, increased bacteriocin production. This inducing factor was characterized and purified from Lact. plantarum KFRI 464, which showed the greatest enhancement of kimchicin GJ7 activity. The inducing factor was purified using a DEAE (diethyl aminoethyl)-Sephacel column and high-performance liquid chromatography, and yielded a single band of c. 6500 Da. N-terminal sequencing of the inducing factor identified 16 amino acids. The N-terminal sequence of the inducing factor was synthesized and examined for the induction of kimchicin GJ7 activity, and was found to induce activity, but at a level about 10% lower than that of the entire molecule. CONCLUSIONS: The presence of a bacteriocin-sensitive strain, Lact. plantarum KFRI 464, acts as an environmental stimulus to activate the production of kimchicin GJ7 by Leuc. citreum GJ7. The inducing factor from Lact. plantarum KFRI 464 is highly homologous to the 30S ribosomal protein S16 from various micro-organisms. The N-terminal sequence of the inducing factor examined in this study is a very important sequence related to the inducing activity. Nevertheless, the inducing factor may not be part of the ribosomal protein S16 itself. SIGNIFICANCE AND IMPACT OF THE STUDY: We believe that the present study is the first to identify an agent that is produced by one micro-organism and influences bacteriocin production in another. The bacteriocin-enhancing system described in this study could be effectively used to control the growth of other micro-organisms (sensitive cells) in food systems. Moreover, this enhancement of bacteriocin production can be applied usefully in industrial production of natural food preservatives.     

Characterization of mutations in the GTP-binding domain of IF2 resulting in cold-sensitive growth of Escherichia coli

The infB gene encodes translation initiation factor IF2. We have determined the entire sequence of infB from two cold-sensitive Escherichia coli strains IQ489 and IQ490. These two strains have been isolated as suppressor strains for the temperature-sensitive secretion mutation secY24. The mutations causing the suppression phenotype are located within infB. The only variations from the wild-type (wt) infB found in the two mutant strains are a replacement of Asp409 with Glu in strain IQ489 and an insertion of Gly between Ala421 and Gly422 in strain IQ490. Both positions are located in the GTP-binding G-domain of IF2. A model of the G-domain of E.coli IF2 is presented in. Physiological quantities of the recombinant mutant proteins were expressed in vivo in E.coli strains from which the chromosomal infB gene has been inactivated. At 42 degrees C, the mutants sustained normal cell growth, whereas a significant decrease in growth rate was found at 25 degrees C for both mutants as compared to wt IF2 expressed in the control strain. Circular dichroism spectra were recorded of the wt and the two mutant proteins to investigate the structural properties of the proteins. The spectra are characteristic of alpha-helix dominated structure, and reveal a significant different behavior between the wt and mutant IF2s with respect to temperature-induced conformational changes. The temperature-induced conformational change of the wt IF2 is a two-state process. In a ribosome-dependent GTPase assay in vitro the two mutants showed practically no activity at temperatures below 10 degrees C and a reduced activity at all temperatures up to 45 degrees C, as compared to wt IF2. The results indicate that the amino acid residues, Asp409 and Gly422, are located in important regions of the IF2 G-domain and demonstrate the importance of GTP hydrolysis in translation initiation for optimal cell growth.     

Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules

The role of cytoskeletal elements in gap junction (GJ) assembly has been studied using Novikoff hepatoma cells treated with cytochalasin B (CB) to disrupt actin filaments or with colchicine or nocodazole to disrupt microtubules. After 60 min of cell reaggregation, freeze-fracture was used to evaluate quantitatively the "initiation," "maturation," and "growth" phases of GJ assembly. The development of junctional permeability to fluorescent dyes was also analyzed. The only effects of CB on the structure or permeability of the developing junctions involved an elongation of GJ aggregates and a small decrease in formation plaque areas. Colchicine (but not the inactive form, lumicolchicine) prevented the enhancement of GJ growth by cholesterol, but its effect on basal growth was equivocal. Nocodazole inhibited the growth of GJ, even under basal conditions, without an effect on initiation. Nocodazole also blocked the forskolin-enhanced increase in the growth of GJs and, in living MDCK cells, reduced the movement of transport intermediates containing green fluorescent protein-tagged connexin43. Thus, neither actin filaments nor microtubules appear to restrict GJ assembly by anchoring intramembrane GJ proteins, nor are they absolutely required for functional GJs to form. However, microtubules are necessary for enhanced GJ growth and likely for facilitating connexin trafficking under basal conditions.     

Connexin hemichannels and gap junction channels are differentially influenced by lipopolysaccharide and basic fibroblast growth factor

Gap junction (GJ) channels are formed by two hemichannels (connexons), each contributed by the cells taking part in this direct cell-cell communication conduit. Hemichannels that do not interact with their counterparts on neighboring cells feature as a release pathway for small paracrine messengers such as nucleotides, glutamate, and prostaglandins. Connexins are phosphorylated by various kinases, and we compared the effect of various kinase-activating stimuli on GJ channels and hemichannels. Using peptides identical to a short connexin (Cx) amino acid sequence to specifically block hemichannels, we found that protein kinase C, Src, and lysophosphatidic acid (LPA) inhibited GJs and hemichannel-mediated ATP release in Cx43-expressing C6 glioma cells (C6-Cx43). Lipopolysaccharide (LPS) and basic fibroblast growth factor (bFGF) inhibited GJs, but they stimulated ATP release via hemichannels in C6-Cx43. LPS and bFGF inhibited hemichannel-mediated ATP release in HeLa-Cx43 cells, but they stimulated it in HeLa-Cx43 with a truncated carboxy-terminal (CT) domain or in HeLa-Cx26, which has a very short CT. Hemichannel potentiation by LPS was inhibited by blockers of the arachidonic acid metabolism, and arachidonic acid had a potentiating effect like LPS and bFGF. We conclude that GJ channels and hemichannels display similar or oppositely directed responses to modulatory influences, depending on the balance between kinase activity and the activity of the arachidonic acid pathway. Distinctive hemichannel responses to pathological stimulation with LPS or bFGF may serve to optimize the cell response, directed at strictly controlling cellular ATP release, switching from direct GJ communication to indirect paracrine signaling, or maximizing cell-protective strategies.     

Complementary roles of initiation factor 1 and ribosome recycling factor in 70S ribosome splitting

We demonstrate that ribosomes containing a messenger RNA (mRNA) with a strong Shine-Dalgarno sequence are rapidly split into subunits by initiation factors 1 (IF1) and 3 (IF3), but slowly split by ribosome recycling factor (RRF) and elongation factor G (EF-G). Post-termination-like (PTL) ribosomes containing mRNA and a P-site-bound deacylated transfer RNA (tRNA) are split very rapidly by RRF and EF-G, but extremely slowly by IF1 and IF3. Vacant ribosomes are split by RRF/EF-G much more slowly than PTL ribosomes and by IF1/IF3 much more slowly than mRNA-containing ribosomes. These observations reveal complementary splitting of different ribosomal complexes by IF1/IF3 and RRF/EF-G, and suggest the existence of two major pathways for ribosome splitting into subunits in the living cell. We show that the identity of the deacylated tRNA in the PTL ribosome strongly affects the rate by which it is split by RRF/EF-G and that IF3 is involved in the mechanism of ribosome splitting by IF1/IF3 but not by RRF/EF-G. With support from our experimental data, we discuss the principally different mechanisms of ribosome splitting by IF1/IF3 and by RRF/EF-G.     

Heterocellular interaction enhances recruitment of alpha and beta-catenins and ZO-2 into functional gap-junction complexes and induces gap junction-dependant differentiation of mammary epithelial cells

Gap junctions (GJ) are required for mammary epithelial differentiation. Using epithelial (SCp2) and myoepithelial-like (SCg6) mouse-derived mammary cells, the role of heterocellular interaction in assembly of GJ complexes and functional differentiation (β-casein expression) was evaluated. Heterocellular interaction is critical for β-casein expression, independent of exogenous basement membrane or cell anchoring substrata. Functional differentiation of SCp2, co-cultured with SCg6, is more sensitive to GJ inhibition relative to homocellular SCp2 cultures differentiated by exogenous basement membrane. Connexin (Cx)32 and Cx43 levels were not regulated across culture conditions; however, GJ functionality was enhanced under differentiation-permissive conditions. Immunoprecipitation studies demonstrated association of junctional complex components (α-catenin, β-catenin and ZO−2) with Cx32 and Cx43, in differentiation conditions, and additionally with Cx30 in heterocellular cultures. Although β-catenin did not shuttle between cadherin and GJ complexes, increased association between connexins and β-catenin in heterocellular cultures was observed. This was concomitant with reduced nuclear β-catenin, suggesting that differentiation in heterocellular cultures involves sequestration of β-catenin in GJ complexes.     

Metabolic requirements for the in vitro augmentation of mouse natural killer activity by interferon

The metabolic processes involved in the in vitro augmentation of mouse natural killer (NK) activity by interferon (IF) were studied. Augmentation occurred after a very brief (5–10 min), temperature-independent exposure of spleen cells to IF. This binding of, or triggering by, IF was independent of protein synthesis, since treatment with puromycin before or during contact with IF failed to block augmentation. Spleen cells, however, required new RNA and protein synthesis in the first few hours after contact with IF to develop the boosted reactivity, as shown by their susceptibility to inhibition by actinomycin D, emetine, pactamycin, and puromycin. Mitomycin C did not interfere with the boosting, suggesting that a pool of NK cells exists which rapidly responds to IF without cell proliferation. The need for new RNA and protein synthesis may be for the differentiation of pre-NK cells to functionally active cells or for the increase in lytic efficiency of already active NK cells.     

原代培养的大鼠心房肌细胞间隙连接的超微结构研究

We have studied the ultrastructure of gap junction of rat atrial myocytes of primary culture in situ embedded by ultramicrotomy. We observed a kind of gap junction-associated vesicles (GJAV), a kind of cluster of particles which are surrounded by plasma membrane, and a concatenate GJAV complexes (CGJAVC) in some big clusters. We found that both of GJAV and CGJAVC are very near the plasma membrane at the intercellular space, and at the same time they are usually adjacent the assembled GJ. So we infered that they are the pre-body or midproduct of the assembled GJ probably. This article analysed these observes and probed into the process about how to assemble the gap junction.     

Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks

We previously demonstrated that truncated desmoplakin I (DP I) molecules containing the carboxyl terminus specifically coalign with and disrupt both keratin and vimentin intermediate filament (IF) networks when overexpressed in tissue culture cells (Stappenbeck, T. S., and K. J. Green. J. Cell Biol. 116:1197-1209). These experiments suggested that the DP carboxyl-terminal domain is involved either directly or indirectly in linking IF with the desmosome. Using a similar approach, we have now investigated the behavior of ectopically expressed full-length DP I in cultured cells. In addition, we have further dissected the functional sequences in the carboxyl terminus of DP I that facilitate the interaction with IF networks. Transient transfection of a clone encoding full-length DP I into COS-7 cells produced protein that appeared in some cells to associate with desmosomes and in others to coalign with and disrupt IF. Deletion of the carboxyl terminus from this clone resulted in protein that still appeared capable of associating with desmosomes but not interacting with IF networks. As the amino terminus appeared to be dispensable for IF interaction, we made finer deletions in the carboxyl terminus of DP based on blocks of sequence similarity with the related molecules bullous pemphigoid antigen and plectin. We found a sequence at the very carboxyl terminus of DP that was necessary for coalignment with and disruption of keratin IF but not vimentin IF. Furthermore, the coalignment of specific DP proteins along keratin IF but not vimentin IF was correlated with resistance to extraction by Triton. The striking uncoupling resulting from the deletion of specific DP sequences suggests that the carboxyl terminus of DP interacts differentially with keratin and vimentin IF networks.