ACTH and adrenocortical gap junctions

Since the initial identification of gap junctions in the adrenal gland, it has been proposed that a system involving direct cell-cell communication might be involved in adrenal cortical functions. Gap junction channels do, in fact, provide pathways for direct intercellular exchange of small molecules (<1,000 Da), many of which have the potential to influence a wide range of cellular activities. Gap junctions are composed of proteins called connexin which, in the adrenal cortex, have proven to be remarkably consistent in both type and zonal distribution with connexin 43 (Cx43) as the predominant component in mammalian adrenal glands thus far evaluated. Only the inner two zones of the cortex (zonae fasciculata and reticularis) exhibit significant amounts of Cx43 and functional coupling. Adrenocorticotropin (ACTH) has been shown to increase Cx43 protein in vivo and in vitro, and a strong correlation has been noted between the presence of gap junctions and certain adrenal cortical functions, especially steroidogenic capacity and cell proliferation. This review summarizes evidence of the Cx43 expression in adrenal cortical cells and the likely role of Cx43 in steroidogenesis and cell proliferation. It is concluded that control of gap junction expression in the adrenal gland is hormonally dependent and is functionally linked to adrenal gland zonation.     

Analysis of the morphogenesis and cell proliferation in the retina of a pleuronectiform fish,the turbot psetta maxima (Pleuronectiformes: Teleostei)

The morphogenesis and cell proliferation in the retina of turbot (Psetta maxima, Pleuronectiformes: Teleostei) from embryo through metamorphosis have been examined by using proliferating cell nuclear antigen (PCNA)‐immunohistochemistry and general histological procedures. In the embryonic retina, cell proliferation and spatial cell reorganization form the anlage of the pigment epithelium and neural retina. Neurogenesis begins around hatching in the temporal retina, dorsal to the optic nerve exit, and then a wave of cell differentiation spreads to the nasal retina to yield a laminated retina by the end of the prolarval turbot stage. Germinal zones in the differentiated retina persist as a rim at the retinal margin, as well as surrounding the optic fissure in premetamorphic and metamorphic turbot larvae. In these zones, progenitor cells with different morphologies show a similar spatial arrangement, which suggests that they have a similar retinogenic potential. During metamorphosis, asymmetric proliferative activity in turbot germinal zones is associated with a marked expansion of the retinal tissue. Scattered stem cells in the laminated retina, related to the lineage of rod photoreceptors, were also observed both in large premetamorphic larvae and metamorphic turbots. The proliferative activity of these cells increases considerably during metamorphosis, when rod photoreceptors become morphologically differentiated. Microsc. Res. Tech. 76:588–597, 2013. © 2013 Wiley Periodicals, Inc.     

High level of circPTN promotes proliferation and stemness in gastric cancer

Increasing evidence proves that circular RNAs (circRNAs) play an important role in regulating the biological behaviors of tumors. The central purpose of this research was to investigate the functions of circRNA in gastric cancer. The utilization of real-time PCR was to test circPTN expression in gastric cancer cells. Cell counting colony formation assays, CCK-8 assay, and EdU assay were used to investigate proliferation. Transwell assay was applied to investigate migration. We discovered that circPTN was highly expressed in gastric cancer cells. Low expression of circPTN inhibits gastric cancer cell proliferation and migration. Elevated expression of circPTN promotes gastric cancer cell proliferation and migration. Moreover, we discovered that circPTN could accelerate self-renewal and increase the expression of stemness markers. The results of our study suggested that a high level of circPTN expression promotes the proliferation and stemness of gastric cancer cells.     

Ovarian acetylcholine and muscarinic receptors: hints of a novel intrinsic ovarian regulatory system

More than two decades ago, the degrading enzyme of the neurotransmitter acetylcholine (ACH) was reported in nerve fibers of the rat ovary. Subsequently, it was assumed that ACH is a neurotransmitter of ovarian nerves, although the sole presence of the degrading enzyme, ACH-esterase, does not allow such a conclusion. That ACH may be involved in the complex regulation of ovarian functions, including hormone production, was indicated by studies using, for example, granulosa cells (GCs). The lack of detailed information about both source(s) and functions of ACH in the ovary prompted us to examine sites of ovarian ACH-synthesis and ACH-receptor-bearing target cells. We also started to identify functions of ACH in cultured human GCs. While ovarian innervation and recently described neuron-like cells of the ovary were not immunoreactive for the ACH-synthesizing enzyme, choline-acetyl transferase (CHAT), we found immunoreactivity in GCs of rodents and primates. Isolated human and rat GCs produced ACH and contained the vesicular ACH transporter (VACHT). These results indicate that endocrine GCs are an unexpected non-neuronal source of ACH in the ovary. Moreover, these cells and GCs in vivo contain ACH-receptors of the muscarinic subtype (MR), namely M1R and M5R. In contrast, oocytes express M3R. MR of human GCs are functional and cholinergic stimulation is linked to rapid increases in intracellular Ca(++) levels. M1/5R activation also led to increased cell proliferation of human GCs in vitro and this stimulatory effect was found to be associated with rapid disruption of gap junction communication. Ongoing studies begin to identify regulation of ion channels and altered gene expression as consequences of MR stimulation. Thus, our results outline first details of an unexpected intraovarian, non-neuronal cholinergic system, and suggest that it may be involved in the regulation of cell proliferation in the ovary.     

Histological,transmission electron microscopic,and immunohistochemical study of the adrenal gland in the Persian squirrel (Sciurus anomalus)

This research was aimed to present the histological and ultrastructure properties of the adrenal gland in the Persian squirrel. Two male and female animals were included in the study. The adrenal gland was bean-shaped and located on the cranial pole of kidney. The enveloping capsule was dense connective tissue that reacted positively with Periodic-Acid Schiff (PAS) and Masson trichrome stainings. The parenchyma of the gland consisted of two-part, namely cortex and medulla; the cortex had three layers: zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR). The cells of the ZG were mainly spherical and ovoid with circular arrangement and few lipid droplets in TEM micrographs. The cells of the ZF were columnar and spherical that were arranged in cord-like rows. Transmission electron microscopy (TEM) indicated conspicuous lipid droplets and mitochondria in this zone. The cells of the ZR were arranged in a tangled networks and were almost similar to those in the ZF. TEM images showed fewer lipid vesicles in the ZR compared to the ZF and ZG. Chromaffin cells were located in the medulla of the adrenal gland in two layers. TEM images showed that some of them were smaller and contained fewer secretory granules; other cells were larger and contained more electron-dense secretory granules. Immunofluorescence staining showed that steroidogenic factor 1 (SF1) expressed from cortex to the corticomedullary junction (CMJ) and tyrosine hydroxylase (TH) expressed in the medulla. In conclusion, the results indicated both similarities and differences between the adrenal gland of the Persian squirrel and other animals such as mammals and rodents.     

Searching for specific binding sites of the secretory glycoproteins of the subcommissural organ

The molecular organization of Reissner's fiber (RF), the structure of its proteins, and the permanent turnover of these proteins are all facts supporting the possibility that RF may perform multiple functions. There is evidence that CSF-soluble RF-glycoproteins may occur under physiological conditions. The present investigation was designed to investigate the probable existence within the CNS of specific binding sites for RF-glycoproteins. Three experimental protocols were used: (1) immunocytochemistry of the CNS of bovine fetuses using anti-idiotypic antibodies, raised against monoclonal antibodies developed against bovine RF-glycoproteins; (2) in vivo binding of the RF glycoproteins, perfusing into the rat CSF 125I-labeled RF-glycoproteins, or grafting SCO into a lateral ventricle of the rat; (3) in vitro binding of unlabeled RF-glycoproteins to rat and bovine choroid plexuses maintained in culture. One of the anti-idiotypic antibody generated by a Mab raised against RF-glycoproteins binds to choroidal cells. Furthermore, binding of RF-glycoproteins to the rat choroid plexus was obtained when: (1) the choroid plexus was cultured in the presence of unlabeled RF-glycoproteins; (2) the concentration of soluble RF-glycoproteins in the CSF was increased by isografting SCOs into a lateral ventricle; (3) radiolabeled glycoproteins were perfused into the ventricular CSF. This evidence suggests that the apical plasma membrane of the ependymal cells of the choroid plexus has specific binding sites for RF-glycoproteins, of unknown functional significance. The radiolabeled RF-glycoproteins perfused into the rat CSF also bound to the paraventricular thalamic nucleus, the floor of the Sylvian aqueduct and of the rostral half of the fourth ventricle, and the meninges of the brain and spinal cord. The labeling of the paraventricular thalamic nucleus points to a functional relationship between this nucleus and the SCO. The possibility that the SCO may be a component of the circadian timing system is discussed.     

Junctional complexes and cell polarity in the urinary tubule

In this review, we demonstrate how differentiated membrane domains can be detected in epithelial cells using conventional light and electron microscopy, freeze-fracture electron microscopy and the immunoand cytochemical detection of membrane components. Using specific examples from the kidney, we show how the polarized insertion of these components into either apical or basolateral plasma membrane regions on either side of the tight junction barrier is related to specific functions of principal and intercalated cells in the collecting duct. In addition, distinct basal and lateral membrane domains have been revealed in some cells that are maintained in the absence of a tight junctional barrier in the plane of the membrane. This suggests that other factors, possibly related to cytoskeletal elements, may be involved in the functional segregation of these membrane areas. We propose that epithelial cell plasma membranes should be subdivided into apical, lateral and basal regions, and that the term “basolateral” may be an oversimplification.     

Interaction between carbon nanotubes and human cell

Carbon nanotubes (CNTs) have been attracting increasing attention for the possibilities of their widespread applications and many studies have been conducted for their development. On the other hand, the research on the influence of CNTs on human organisms is still scarce and some show concerns about genotoxicity or cytotoxicity. Therefore, in this research, we are proposing a new way of biocompatibility evaluation for CNTs by examining the relation between biocompatibility and structure of CNTs. In our cytotoxicity tests, neither chromosome abnormality nor cell cycle dysregulation occurred, evaluated by fluorescence activated cell sorting (FACS) analysis. However, significances were assessed for cell proliferation and viability for semiconducting CNTs by the paired t-test. It is also clear that CNTs showed adherence to cell, since efficiency of cell ablation by trypsin decreased dramatically in CNTs dishes; this phenomenon was observed especially in semiconducting CNTs. Since semiconducting CNTs do not include armchair CNTs while metallic CNTs do, it indicates that armchair CNTs have high possibility of affection toward biocompatibility. It is expected that armchair CNTs change the behavior of cells such as proliferation and viability.     

Fine structural cytology of the adenohypophysis in rat and man

The present review deals with the use of electron microscopy in the identification of pituitary cell types as well as the assessment of their functional state, in rat and man. Application of immunoelectron microscopy, especially immunogold techniques, utilizing multiple labeling in establishing differentiation and hormone content of cell types, is emphasized. Recent evidence of plurihormonality in various pituitary cell types indicates that the once axiomatic one cell-one hormone theory is untenable and that the present perception of pituitary cell types and their function requires modification. Detection of hormonal and nonhormonal substances in pituitary cell types, not associated with their known endocrine function, suggests that hypophysial cells may have yet unknown roles, possibly in the realm of paracrine and autocrine regulation.     

Force spectroscopy of hepatocytic extracellular matrix components

We present atomic force microscopy and force spectroscopy data of live hepatocytes (HEPG2/C3A liver cell line) grown in Eagle's Minimum Essential Medium, a complex solution of salts and amino acids commonly used for cell culture. Contact-mode imaging and force spectroscopy of this system allowed correlation of cell morphology and extracellular matrix (ECM) properties with substrate properties. Force spectroscopy analysis of cellular “footprints” indicated that the cells secrete large polymers (e.g., 3.5 μm contour length and estimated MW 1000 kDa) onto their substrate surface. Although definitive identification of the polymers has not yet been achieved, fluorescent-labeled antibody staining has specified the presence of ECM proteins such as collagen and laminin in the cellular footprints. The stretched polymers appear to be much larger than single molecules of known ECM components, such as collagen and heparan sulfate proteoglycan, thus suggesting that the cells create larger entangled, macromolecular structures from smaller components. There is strong evidence which suggests that the composition of the ECM is greatly influenced by the hydrophobicity of the substrate surface, with preferential production and/or adsorption of larger macromolecules on hydrophobic surfaces.     

Neurotrophic control of ovarian development

Substantial evidence now exists indicating that the neurotrophins, a family of growth factors required for the survival, development, and differentiation of various neuronal populations of the nervous system, are also important for the development of nonneuronal tissues. Such a function was first suggested by studies showing the presence of high-affinity neurotrophin receptors in a variety of nonneuronal tissues including those of the cardiovascular, endocrine, immune, and reproductive systems. Within the latter, the gonads appear to be a preferential site of neurotrophin action as suggested by the presence in the mammalian ovary of at least four of the five known neurotrophins and all of the neurotrophin receptors thus far identified. While the various functions that the neurotrophins may have in the ovary are still being elucidated, it is now clear that in addition to recruiting the ovarian innervation, they play a direct role in the regulation of two different maturational periods that are critical for the acquisition of female reproductive function: early follicular development and ovulation. Neurotrophins facilitate the development of newly formed follicles by promoting the initial differentiation and the subsequent growth of primordial follicles. These actions appear to be related to the ability of neurotrophins to sustain the proliferation of both mesenchymal and granulosa cells, and to induce the synthesis of follicle stimulating hormone (FSH) receptors. At the time of the first ovulation, neurotrophins contribute to the ovulatory cascade by increasing prostaglandin E(2) release, reducing gap junction communication, and inducing cell proliferation within the thecal compartment of preovulatory follicles.     

Tachykinins and tachykinin receptors in bone

Adrenomedullin (AM) was originally characterized in extracts of an adrenal medullary tumor. Since this original finding the peptide and its mRNA have also been found in the adrenal cortex, specifically, in the cells of the aldosterone-secreting zona glomerulosa. It is clear that the synthesis of AM is actively regulated in both cortex and medulla. Much research effort has been focused on identifying a role for AM in the adrenal gland. To date, no consistent effect on medullary catecholamine biosynthesis has been demonstrated. In the cortex the actions of AM are controversial and appear to depend on both the tissue preparation used and on the specific receptor population expressed in the individual gland. The results of further studies on the long-term actions of AM on adrenal growth and differentiation are awaited with interest.     

Investigation of morphological and functional changes during neuronal differentiation of PC12 cells by combined hopping probe ion conductance microscopy and patch-clamp technique

PC12 cells derived from rat pheochromocytoma can differentiate into sympathetic-neuron-like cells in response to nerve growth factor (NGF). These cells have been proved to be a useful cell model to study neuronal differentiation. NGF induces rapid changes in membrane morphology, neurite outgrowth, and electrical excitability. However, the relationship between the 3D morphological changes of NGF-differentiated PC12 cells and their electrophysiological functions remains poorly understood.In this study, we combined a recently developed Hopping Probe Ion Conductance Microscopy (HPICM) with patch-clamp technique to investigate the high-resolution morphological changes and functional ion-channel development during the NGF-induced neuronal differentiation of PC12 cells. NGF enlarged TTX-sensitive sodium currents of PC12 cells, which associated with cell volume, membrane surface area, surface roughness of the membrane, and neurite outgrowth. These results demonstrate that the combination of HPICM and patch-clamp technique can provide detailed information of membrane microstructures and ion-channel functions during the differentiation of PC12 cells, and has the potential to become a powerful tool for neuronal research.     

Statistical analysis of labelling patterns of mammary carcinoma cell nuclei on histological sections

It is of central interest for tumour biology to explore the mechanisms of tumour cell proliferation. In this study, methods of spatial statistics were used to study the spatial distribution of proliferating cells within tumour tissue quantitatively and objectively. Mammary cancer tissue was studied as an example. It was attempted to clarify whether cell division occurs entirely at random (random labelling), i.e. the process of division occurs at random, independently from the state of the neighbouring nuclei, or whether the spatial distribution of proliferation is more complex, e.g. in the form of actively proliferating clusters alternating with relatively silent zones. In the case of random labelling, the reduced second moment functions K(r) of the labelled and the unlabelled nuclei would be identical. The same would hold for the pair correlation functions g(r) . The alternative hypothesis is that the second‐order properties of the processes of the labelled and the unlabelled nuclei are different. Twenty cases of invasive ductal mammary carcinomas were studied. The nuclei of proliferating cells were stained immunohistochemically with the monoclonal antibody MIB‐1, which detects specifically the proliferation‐associated nuclear antigen Ki 67. The planar coordinates of the tumor cell nucleus profiles from two rectangular visual fields per case were recorded. For each visual field, the following investigations were performed: estimation of the explorative summary characteristics K(r) and g(r) , fitting of the parameters of a stationary Strauss hard‐core model to the observed point patterns, estimation of two distance‐dependent Simpson indices and Monte Carlo tests of all individual patterns on the null hypothesis of random labelling. Significant differences between the mean K‐functions and the mean g‐functions of the labelled and the unlabelled nuclei were found. Moreover, the mean interaction parameter γ of the stationary Strauss hard‐core model was significantly higher for the labelled nuclei than for the unlabelled nuclei. The estimates of the two distance‐dependent Simpson indices showed a tendency of points with the same label towards a positive spatial correlation. In the Monte Carlo tests, the null hypothesis of random labelling was rejected for the majority of the visual fields. These four lines of investigation led to the concordant conclusion that the labelling of mammary carcinoma nuclei by MIB‐1 is not simply random. The data suggest that the second‐order properties of the point process of the labelled nuclei are significantly different from those of the unlabelled nuclei. In particular, the process of the labelled nuclei shows a higher degree of clustering (increased strength of interaction) than the process of the unlabelled points.     

Vasotocin and mesotocin in the brains of amphibians: state of the art.

Immunohistochemical studies during the last decade have revealed elaborate systems of vasotocinergic (AVT) and mesotocinergic (MST) neuronal elements in the brain of a variety of amphibians including anurans, urodeles, and gymnophionans. Apart from a well-developed hypothalamo-hypophysial system, the antibodies demonstrated the existence of extrahypothalamic AVT- and MST-immunoreactive cell groups as well as extensive extrahypothalamic networks of immunoreactive fibers. The wide distribution of AVT- and MST-immunoreactive fibers throughout the brains of amphibians suggests that the two neuropeptidergic systems are involved not only in hypothalamo-hypophysial interactions, but also in a variety of other brain functions. Moreover, there is now evidence that sex-related differences occur in amphibians as previously shown for amniotes. It should be noted, however, that substantial variation occurs in the relative densities of AVT- and MST-immunoreactive fibers and number of cells between species, even within a single order of amphibians. Similar observations have been made in other classes of vertebrates and prompt us, therefore, to critically evaluate conclusions with respect to specific functions of AVT and MST in the central nervous system of vertebrates.     

Proteomics analysis of tumor microenvironment: Implications of metabolic and oxidative stresses in tumorigenesis

Tumorigenesis is always concomitant with microenvironmental alterations. The tumor microenvironment is a heterogeneous and complex milieu, which exerts a variety of stresses on tumor cells for proliferation, survival, or death. Recently, accumulated evidence revealed that metabolic and oxidative stresses both play significant roles in tumor development and progression that converge on a common autophagic pathway. Tumor cells display increased metabolic autonomy, and the hallmark is the exploitation of aerobic glycolysis (termed Warburg effect), which increased glucose consumption and decreased oxidative phosphorylation to support growth and proliferation. This characteristic renders cancer cells more aggressive; they devour tremendous amounts of nutrients from microenvironment to result in an ever‐growing appetite for new tumor vessel formation and the release of more “waste,” including key determinants of cell fate like lactate and reactive oxygen species (ROS). The intracellular ROS level of cancer cells can also be modulated by a variety of stimuli in the tumor microenvironment, such as pro‐growth and pro‐inflammatory factors. The intracellular redox state serves as a double‐edged sword in tumor development and progression: ROS overproduction results in cytotoxic effects and might lead to apoptotic cell death, whereas certain level of ROS can act as a second‐messenger for regulation of such cellular processes as cell survival, proliferation, and metastasis. The molecular mechanisms for cancer cell responses to metabolic and oxidative stresses are complex and are likely to involve multiple molecules or signaling pathways. In addition, the expression and modification of these proteins after metabolic or oxidative stress challenge are diverse in different cancer cells and endow them with different functions. Therefore, MS‐based high‐throughput platforms, such as proteomics, are indispensable in the global analysis of cancer cell responses to metabolic and oxidative stress. Herein, we highlight recent advances in the understanding of the metabolic and oxidative stresses associated with tumor progression with proteomics‐based systems biology approaches. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:267–311, 2013     

Ultrastructure of Paneth cells in the intestine of various mammals

Paneth cells in the following species were observed under an electron microscope: human, rhesus monkey, hare, guinea pig, rat, nude rat, mouse, golden hamster, and insect feeder bat. Secretory granules containing homogeneous electron-dense materials were observed in the Paneth cells of humans, monkeys, hares, guinea pigs, and bats; mouse Paneth-cell granules were bipartite (central core and peripheral halo), and the Paneth cells in rats and golden hamsters had secretory granules showing various electron densities. In humans, monkeys, and bats, immature granules near the Golgi apparatus sometimes showed bipartite substructure. The number and size of secretory granules were also diverse among various animal species. Some lysosome-like bodies were commonly observed in peri- or supranuclear regions, though the size and shape of the bodies differed from cell to cell. In apical cytoplasm, small clear vesicles (100–200 nm diameter) were more-or-less observed in all species examined, and it was especially note that rat Paneth cells contained many clear vesicles. Small dense-cored vesicles (150–200 nm diameter) were rare. It is unlikely that the various ultrastructural features of Paneth cells correlate with the phylogenetical classification.     

Ultrastructural changes in colonic epithelial cells in a rat model of inflammatory bowel disease

Inflammatory bowel disease (IBD) is a global, chronic intractable disease. The functions of drugs and food components have been evaluated in models of IBD induced by 2,4,6‐trinitrobenzene sulfonic acid (TNBS). Here, we used transmission (TEM) and osmium‐maceration scanning (SEM) electron microscopy to evaluate the ultrastructure of colonic epithelial cells in rat models of IBD induced by TNBS. Histological evaluation revealed that the intestinal crypts in the most regions of the IBD‐model colons were deformed and we classified them as having high cell migration rates (HMIG). The remaining regions in the intestinal crypts retained a relatively normal structure and we classified them as having low cell migration rates (LMIG). Osmium‐maceration SEM revealed the mucosal fluid flowing in spaces without secretory granules in crypt goblet cells of both HMIG and LMIG regions, indicating the depletion of goblet cell mucin that is found in patients with IBD. The Golgi apparatus in absorptive cells was stacked and curled in both regions. Osmium‐maceration SEM showed membrane network structures resembling endoplasmic reticulum that were large and expanded in absorptive cells with HMIG rather than with LMIG regions in IBD‐model colons. These findings indicated that endoplasmic reticulum stress is associated with susceptibility to IBD and that the effects of various agents can be evaluated according to endoplasmic reticulum stress revealed by using electron microscopy in models of IBD induced by TNBS.     

Secretome-microRNA and anti-proliferative APRO family proteins as cancer prevention and stem cell research strategies

Stemness of cancer cells contains limitless self-renewal proliferation. For the purpose of proliferation, secretome might exert its effects via the paracrine signaling. Specific microRNAs enclosed in the secretome of cancer stem cells could regulate the expression of anti-proliferative APRO family proteins. The biological functions of APRO family proteins seems to be quite intricate, however, which might be a key modulator of microRNAs, then could regulate the proliferation of cancer cells. In addition to affecting proliferation/differentiation during cellular development, APRO family proteins might also play an imperious role on keeping homeostasis in healthy stem cells under a physiological condition. Therefore, relationship between the microRNAs and the APRO family proteins has attracted much attention in the field of cancer research and regenerative medicine. Here, we have described the molecular mechanism behind this interplay that have a potential role in the future promising tools with targeting APRO family proteins for the medical applications.