Vitamin B6 suppresses growth of the feline mammary tumor cell line FRM

Growth of FRM cells was inhibited by the addition of pyridoxine in a dose-dependent manner. Use of 5 mM pyridoxine caused an almost complete arrest of cell growth. Pyridoxal was as effective as pyridoxine, but pyridoxamine showed weak inhibitory action. Electron-microscopic examination of control cells revealed large nuclei and cellular membranes with villi, but, in pyridoxine-treated cells, condensed or degraded nuclei were observed. Many vacuoles and cholesterol crystals were widely distributed inside the cellular membrane of pyridoxine-treated cells. One of the vacuoles was identified as a lipid droplet. The DNA ladder was observed in the pyridoxine-treated cells. It is suggested that pyridoxine treatment of FRM cells causes cytolysis of cells by apoptosis.     

Pyridoxine improves hippocampal cognitive function via increases of serotonin turnover and tyrosine hydroxylase,and its association with CB1 cannabinoid receptor-interacting protein and the CB1 cannabinoid receptor pathway

BackgroundIn the present study, we investigated the effects of pyridoxine on hippocampal functions and changes in protein profiles based on the proteomic approach.MethodsEight-week-old mice received intraperitoneal injections of physiological saline (vehicle) or 350 mg/kg pyridoxine twice a day for 21 days.ResultsPhosphoglycerate mutase 1 was up-regulated, while CB1 cannabinoid receptor-interacting protein 1 (CRIP1) was down-regulated, in the pyridoxine-treated group. Additionally, the serotonin and tyrosine hydroxylase was increased in the hippocampus of the pyridoxine-treated group than in that of the vehicle-treated group. Furthermore, discrimination indices based on the novel object recognition test were significantly higher in the pyridoxine-treated group than in the vehicle-treated group. Administration of CRIP1a siRNA significantly increases the discrimination index as well as cell proliferation and neuroblast differentiation in the dentate gyrus. In addition, the administration of rimonabant, a CB1 cannabinoid receptor antagonist, for 3 weeks significantly decreased the novel object recognition memory, the tyrosine hydroxylase level, the amount of cell proliferation, and neuroblast differentiation in the dentate gyrus. Treatment with pyridoxine significantly increased novel object recognition memory, but slightly ameliorated rimonabant-induced reduction in serotonin, the tyrosine hydroxylase level, the amount of cell proliferation, and neuroblast differentiation in the dentate gyrus.ConclusionThese results suggest that pyridoxine promotes hippocampal functions by increasing serotonin and tyrosine hydroylase immunoreactivity in the hippocampus. This positive effect may be associated with CRIP1a and CB1 cannabinoid receptor function.General significanceVitamin-B₆ enhances hippocampal functions and this is closely associated with CRIP1a and CB1 cannabinoid receptors.     

Pyridoxine Enhances Cell Proliferation and Neuroblast Differentiation by Upregulating the GABAergic System in the Mouse Dentate Gyrus

We investigated the effects of pyridoxine (vitamin B₆) on cell death, cell proliferation, neuroblast differentiation, and the GABAergic system in the mouse dentate gyrus. We administered pyridoxine (350 mg/kg intraperitoneally) to 8 week old mice twice a day for 14 days and sacrificed them at 10 weeks of age. Pyridoxine treatment did not induce neuronal death or activate microglia in the dentate gyrus, while glial fibrillary acidic protein (GFAP)-positive cells were significantly increased in the subgranular zone of the dentate gyrus. The increase in GFAP-positive cells was confirmed to be due to proliferating cells based on double immunofluorescence staining. GFAP-positive cells, which were also labeled with Ki67, a marker for cell proliferation, and doublecortin, a marker for neuroblast differentiation, were significantly increased in the pyridoxine-treated group compared to those in the vehicle-treated group. Pyridoxine treatment also increased the protein levels of glutamic acid decarboxylase (GAD) 67, an enzyme for GABA synthesis, and pyridoxal 5′-phosphate (PNP) oxidase, an enzyme for pyridoxal phosphate synthesis, in the dentate gyrus. These results suggest that pyridoxine treatment distinctly increases cell proliferation, neuroblast differentiation, and upregulated the GABAergic system, as revealed by the increases of GAD67 and PNP oxidase in the mouse dentate gyrus.     

Mucoepidermoid carcinoma of the salivary gland in pleural fluid. A case report

The cellular features of mucoepidermoid carcinoma of submandibular gland origin observed in pleural fluid are presented. The pleural fluid contained predominantly atypical spheroid cell clusters accompanied by numerous mesothelial cells. The cells had round nuclei with conspicuous nucleoli, coarsely granular chromatin and abundant cytoplasm with vacuoles. The cellular features of the malignant cells in the pleural fluid were correlated with the histology of the parent lesion.     

Isolation of Vacuoles from Root Storage Tissue of Beta vulgaris L

Morphologically intact and osmotically active vacuoles were isolated from root storage tissue of the red beet Beta vulgaris L., and the factors influencing both yield and stability of the vacuoles were determined. Successful isolation depended upon slicing the tissue in an apparatus specifically designed to cut open plant cells without the use of high shear forces and to liberate cellular organelles into an undisturbed reservoir of osmoticum. The resulting brei was centrifuged at 2,000g for 10 min to yield a pellet which contained many vacuoles but which also contained tissue fragments, nuclei, mitochondria, and plastids. The vacuoles were further purified by accelerated flotation through a Metrizamide step gradient. Biochemical assays, light microscopy, and electron microscopy confirmed that there was only trace contamination of the final vacuole preparation by other organelles. Isolated vacuoles were intact and retained their in vivo coloration.     

A peptide from the first fibronectin domain of NCAM acts as an inverse agonist and stimulates FGF receptor activation, neurite outgrowth and survival

Neural cell adhesion molecule (NCAM) contributes to axon growth and guidance during development and learning and memory in adulthood. Although the Ig domains mediate homophilic binding, outgrowth activity localizes to two membrane proximal fibronectin-like domains. The first of these contains a site identified as a potential FGF receptor (FGFR) activation motif (FRM) important for NCAM stimulation of neurite outgrowth, but its activity has hitherto remained hypothetical. Here, we have tested the effects of a domain-specific antibody and peptides corresponding to the FRM in cellular assays in vitro. The first fibronectin domain antibody inhibited NCAM-stimulated outgrowth, indicating the importance of the domain for NCAM function. Monomeric FRM peptide behaved as an inverse agonist; low concentrations specifically inhibited neurite outgrowth stimulated by NCAM and cellular responses to FGF2, while saturating concentrations stimulated FGFR-dependent neurite outgrowth equivalent to NCAM itself. Dendrimeric FRM peptide was 125-fold more active and stimulated FGFR activation, FGFR-dependent and FGF-mimetic neurite outgrowth and cell survival (but not proliferation). We conclude that the FRM peptide contains NCAM-mimetic bioactivity accounted for by stimulation of FGF signalling pathways at the level of or upstream from FGF receptors, and discuss the possibility that FRM comprises part of an FGFR activation site on NCAM.     

Chronic Effects of Pyridoxine in the Gerbil Hippocampal CA1 Region after Transient Forebrain Ischemia

In a previous study, we reported that the administration of pyridoxine (vitamin B₆) to mice for 3 weeks significantly increased cell proliferation and neuroblast differentiation in the dentate gyrus without any neuronal damage. In the present study, we investigated the restorative potentials of pyridoxine on ischemic damage in the hippocampal CA1 region of Mongolian gerbils. Gerbils were subjected to 5 min of transient ischemia, and surgical operation success was assessed by ophthalmoscope during occlusion of common carotid arteries and spontaneous motor activity at 1 day after ischemia/reperfusion. Pyridoxine (350 mg/kg) or its vehicle (physiological saline) was intraperineally administered to ischemic gerbils twice a day starting 4 days after ischemia/reperfusion for 30 or 60 days. The repeated administration of pyridoxine for 30 and 60 days significantly increased doublecortin-immunoreactive neuroblasts in the dentate gyrus and increased NeuN-immunoreactive mature neurons and βIII-tubulin-immunoreactive dendrites in the hippocampal CA1 region. Furthermore, brain-derived neurotrophic factor (BDNF) protein levels were significantly increased in pyridoxine-treated groups compared to those in the vehicle-treated groups. These results suggest that chronic administration of pyridoxine enhances neuroblast differentiation in the dentate gyrus and induces new mature neurons in the hippocampal CA1 region by up-regulating BDNF expression in hippocampal homogenates.     

Ulfrastructure of the histological zones in growing vegetative buds of Salix spp.

Ultrastructural differentiation in the shoot apex of growing vegetative buds of Salix was studied, and some micrographs analysed morphometrically. The distribution of inorganic phospahte (P;) was analysed cytochemically. A distinct histological zona–tion was observed in the apex. The relative volumes of nuclei and plastids were significantly higher in the central tunica zone than in the peripheral one. The corpus differed from the central tunica zone by significantly lower volume density of nuclei and higher of vacuoles and mitochondria. During differentiation of the rib meristem vacuole volume increased significantly, while the relative volumes of nuclei, mitochondria, nucleoli, and heterochromatin decreased. It was not possible to decide whether the vacuoles originate from ER or GERL. Morphogenesis of chloroplasts with large starch grains and grana from proplastids was evident in the rib meristem; dedifferentiation to S–plastids was found in the protophloem. Prolamellar bodies were observed in the procambium plastids. The protophloem was characterized by P–protein and spiny vesicles. P_i was found in the nucleoli of most epidermis cells, several procambium cells, and a few chlorencyma cells, but never in the tunica of the growing apical and developing lateral buds. P_i also occurred in some plasmalemma–somes and occasionally in the walls in connection with plasmodesmata.     

Fluorescence of neurotransmitters and their reception in plant cell

The use of fluorescent reagents for the histochemical detection of catecholamines or histamine, as well as luminescent antagonists of the intracellular neurotransmitters revealed that they can bind to certain cellular compartments. After the treatment with glyoxylic acid (a reagent used for the detection of catecholamines), blue fluorescence with maximum at 460–475 nm was visualized in nuclei and chloroplasts (in control preparations no emission in this spectral region was recorded), as well as an intense fluorescence, exceeding the control level, in the vacuoles. After the exposure to ortho-phthalic aldehyde (a reagent used for the histamine detection), blue emission was more noticeable in nuclei and chloroplasts, which correlates with previously observed effects on intact cells, such as pollen and vegetative microspores. A comparison of the intensities of the biogenic amine-related emission in various organelles showed that the greatest emission was in vacuoles and the weakest, in chloroplasts. Thus, on the surface, and possibly within the organelles, fluorescence could demonstrate the presence of biogenic amines. Antagonists of the neurotransmitters (dtubocurarine for acetylcholine; yohimbine for dopamine; norepinephrine and inmecarb for serotonin), which fluoresce in the blue and blue-green region and usually bind with the plasmalemma of intact cells, also interacted with the membranes of the organelles studied. Fluorescence intensity depended on the object; most prominent it was for yohimbine in the outer membrane of the nucleus, vacuoles, and chloroplasts.     

Observations on the Neocytoplasm and Proteid Vacuoles During the Fertilization of Keteleeria evelyniana

Distributions and dynamics of the neocytoplasm and proteid vacuoles during the fertilization of Keteleeria evelyniana were studied by histochemical methods. Before fertilization cytoplasmic sheath surrounding the male and female gametes was indistinct. After fertilization, the dense neocytoplasm appeared around the zygote. Part of the neocytoplasm is invaded by mitochondria of maternal origin which had collected in large numbers in the perinuclear zone. The mitochondria contain electron compact little body which looks like a nucleus in the cytoplasm, but not observed in the rosette tier cell of proembryo and jacket cells. Hence, it was showed that the neocytoplasm participated in the development of embryo by all these observations. By using Feulgen reaction, the staining reaction of neocytoplasm was positive, the egg nucleus or zygote nucleus was weaker in positive reaction, while the proteid vacuoles were negative. When the proembryo developed, there were a few starch grains accumulated in the other three tiers except the upper tier. The Feulgen reaction was in- creased in intensity in the suspensor tier and embryonal cell tier nuclei. When the young embryo developed, Feulgen reaction became normal in the nuclei of the embryo initials. The embryo initials and Suspensor cells showed very weak Feulgen positive reaetion in the proembryo and young embryo. The development of the large proteid vacuoles was from plastid. During the early stage of egg nucleus, contents of large proteid vacuoles were less. When the zygote was formed, they reached the highest. However, after the zygote produced, the proteid vacuoles and egg cytoplasm were getting disintegrated following the course of fission of free nuclei. After the proembryo formed, the proteid vacuoles were wholly disintegrated.     

云南油杉受精过程中新细胞质及蛋白泡的动态观察

云南油杉(Keteleeria evelyniana Mast)在受精前,精核与卵核周围的细胞质鞘不明显。受精后,合子核周围出现细密的新细胞质。应用孚尔根核染色法,可以较清晰地将新细胞质染出,呈现较弱的正反应,而合子的核质及受精前的精核与卵核染色极弱。卵细胞质及其中的蛋白泡均为负反应。原胚形成后,除上层外,其余几层细胞质内开始积累淀粉粒。此时胚原细胞核的孚尔根染色深度有所增加。幼胚形成后,在顶端的胚原细胞群中核的孚尔根染色反应已恢复正常。在原胚及幼胚胚原细胞质中也呈现很弱的正反应。在电镜下,胚原层细胞质及新细胞质中均含有核样电子致密小体或称作染色质小体,而原胚莲座层细胞质及四周套细胞质中的线粒体则不含这种核样小体。因此,大蛋白泡在卵核形成的早期数量不多,当合子形成时含量最高,而随着游离核的分裂进程,蛋白泡以及原卵质均逐渐地解体,在原胚形成后全部消解。     

Understanding Saline and Osmotic Tolerance of Populus euphratica Suspended Cells

Tolerance of Populus euphratica suspended cells to ionic and osmotic stresses implemented respectively by NaCl and PEG (6000) was characterized by monitoring cell growth, morphological features, ion compartmentation and polypeptide patterns. The cells grew and proliferated when submitted to stresses of 137 mM NaCl or 250 g l⁻¹ PEG, and survived at 308 mM of NaCl, showing tolerance to saline and particularly osmotic stress. They were resistant to plasmolysis and had dense cytoplasms, large nuclei and nucleoli, and evident cytoplasmic strands under high saline and osmotic stress. The sequestration of Cl⁻ into the vacuoles was observed in the cells stressed with 137 and 223 mM NaCl. The cellular protein profile was modified by high salt and osmotic stress and showed 28 kDa polypeptides up-regulated by both NaCl and PEG, and 66 and 25 kDa polypeptides up-regulated only by high NaCl stress. The salt tolerance of P. euphratica cells might be related to their capacity of adapting to higher osmotic stress by maintaining cell integrity, sequestrating Cl⁻ into vacuoles and modulating polypeptides that reflect cellular metabolic adaptations.     

The effects of salicylate on the activity of rat liver tyrosine–2-oxoglutarate aminotransferase in vitro and in vivo

1. Salicylate, in concentrations of 0.25mm and above, enhances the basal activity of tyrosine–2-oxoglutarate aminotransferase in homogenates of rat liver incubated in the absence of added pyridoxal 5′-phosphate (endogenous activity). The effect is decreased by increasing the concentration of the cofactor. 2. The intraperitoneal administration of sodium salicylate enhances the activity of rat liver tyrosine aminotransferase; the major effect during the first hour being on the enzyme in the absence of added pyridoxal phosphate. Actinomycin D prevents the induction of the enzyme by cortisol and tryptophan. Induction by pyridoxine or salicylate is 50% inhibited by actinomycin D. The effects of the injections of various combinations of cortisol, pyridoxine and salicylate were also studied in the absence or presence of actinomycin D. 3. It is suggested that salicylate induces rat liver tyrosine aminotransferase by displacing its protein-bound cofactor and that a cofactor-type induction of the hepatic enzyme occurs in pyridoxine-treated rats.     

The Harderian gland of the Cheesman's gerbil (Gerbillus cheesmani ) of the Kuwaiti desert

The Harderian gland is a large orbital structure. Several functions have been ascribed to the gland such as lubrication of the eye, a source of pheromones, thermoregulartory lipids and photoprotective secretions and a part of the retinal-pineal axis. In the present study, the Harderian gland of the Cheesman's gerbil, Gerbillus cheesmani, is described for the first time. The gland is located around the posterior portion of the eyeball. The gland is compound tubular, surrounded by a thin connective tissue capsule. Only one secretory epithelial cell type was recognized, characterized by the presence of lipid vacuoles and cytoplasmic slashes in high numbers; the former being more concentrated towards the apical part while the latter being more concentrated towards the central and basal parts. Some of the cytoplasmic slashes contained electron dense filamentous structures. Similar structures were observed in the lipid vacuoles. Thus, a functional relationship between the cytoplasmic slashes and the lipid vacuoles is suggested. A unique structure was observed, termed dome-like cells, located between the epithelial cells and the basement membrane. These cells were characterized by the extensive presence of pleomorphic mitochondria and compact lamellae of granular endoplasmic reticulum (GER) in the form of finger prints. The gland was found to be actively secreting porphyrins as well as lipids. Cellular debris was also seen in the tubular lumina. Myoepithelial cells with their spindle shape and elongated nuclei were evident between the basement membrane and the secretory epithelium. Sparse interstitial tissue was observed in-between the gland tubules of both male and female gerbils. Macrophages, dendritic melanocytes and lymphocytes are the most represented cellular components of the interstitium. Further studies are required to investigate the function of the dome-like cells as well as the role of lymphocytes in the rodents Harderian gland.     

Pyridoxine uptake by colonocytes: a specific and regulated carrier-mediated process

The water-soluble vitamin B6 (pyridoxine) is important for normal cellular functions, growth, and development. The vitamin is obtained from two exogenous sources: a dietary source, which is absorbed in the small intestine, and a bacterial source, where the vitamin is synthesized in significant quantities by the normal microflora of the large intestine. Evidence exists to suggest the bioavailability of the latter source of the vitamin, but nothing is known about the mechanism involved and its regulation. In this study, we addressed these issues using young adult mouse colonic epithelial (YAMC) cells and human colonic apical membrane vesicles (AMV) as models and using [3H]pyridoxine as the uptake substrate. The results showed the initial rate of [3H]pyridoxine uptake by YAMC cells to be 1) energy- and temperature- (but not Na-) dependent and to occur without metabolic alteration in the transported substrate; 2) saturable as a function of concentration with an apparent Km and Vmax of 2.1 +/- 0.5 muM and 53.4 +/- 4.3 pmol.mg protein(-1).3 min(-1), respectively; 3) cis-inhibited by unlabeled pyridoxine and its structural analogs, but not by the unrelated compounds theophylline, penicillamine, and isoniazid; 4) trans-stimulated by unlabeled pyridoxine; 5) amiloride sensitive; and 6) regulated by extracellular and intracellular factors. Uptake of pyridoxine by native human colonic AMV was also found to involve a carrier-mediated process. These studies demonstrate, for the first time, the functional existence of a specific and regulatable carrier-mediated process for pyridoxine uptake by mammalian colonocytes.     

Diurnal rhythm of hypophyseal cells in mich. II. Pars intermedia.

By the use of Mac Conaill's lead hematoxylin, periodic acid and Schiff's reagent (PAS, PbH-positive and PAS-positive cells were distinguished in the pars intermedia of the hypophysis in mice. Nuclear volume in PbH-positive and PAS-positive cells in the pars intermedia of the hypophysis in male and female mice under conditions of 12 h light and 12 h darkness shows distinct diurnal rhythmicity. Maximum nuclear volume in PbH-positive cells of the pars intermedia in both sexes was observed at 1800 h, and minimum at 2400 h. In the Pas-positive cells in females maximum nuclear volume was observed at 600 h, and minimum at 2400 h. In males maximal nuclear volume in these cells appears at 2400 h, and minimum at 1800 h. Maximum number of vacuoles in the nuclei of PbH-positive cells in the pars intermedia in both sexes appeared at 1800 h, and minimum at 2400 h. Maximum numbers of vacuoles in the nuclei of PAS-positive cells in females was noted at 1200 h, and minimum at 2400 h. In males the maximum number of vacuoles appeared at 600 h, and minimum at 1200 h. Differences in the number of vacuoles in the nuclei of PAS-positive cells between males and females were also noted.     

Pancreatic damage produced by injecting excess lysine in rats

Intraperitoneal injection of lysine (400 mg/100 g body weight) in rats caused necrosis of pancreatic acinar cells with fat necrosis and a significant increase in serum amylase and lipase. The early morphological changes in the pancreas were investigated. At 3 to 6 h, marked swelling of mitochondria was observed throughout the cytoplasm followed later by dilation of the endoplasmic reticulum and the formation of autophagic vacuoles, indicative of rapid cellular degeneration. These results suggest that transient disturbance of energy formation following mitochondrial swelling resulted in disorders of protein metabolism, with disorganization of the endoplasmic reticulum and pyknosis of the nuclei as later events.     

ELECTRON MICROSCOPY OF A CONTRACTILE-VACUOLE MUTANT OF CHLAMYDOMONAS MOEWUSII (CHLOROPHYTA) DEFECTIVE IN THE LATE STAGES OF DIASTOLE1

Electron microscopy of a “vacuole-less” mutant of Chlamydomonas moewusii Gerloff revealed the presence of small anterior vacuoles. These vacuoles behaved like contractile vacuoles in wild-type cells, but they were apparently unable to complete diastole and discharge their contents. When wild-type and mutant cells were incubated in hypertonic medium, small coated vacuoles persisted in the region where contractile vacuoles form. When these cells were transferred to hypotonic medium, the vacuoles appeared to fill and fuse to form larger vacuoles Shortly after the appearance of full expanded contractile vacuoles, collapsed vacuoles were observed in wild-type cells suggesting the completion of diastole and the onset of systole. In mutant cells, the initial steps of filling and fusion to form larger vacuoles apparent interactions of vacuoles with the plasma membrane were not observed. New contractile vacuoles accumulated around the nucleus. When fusion of the contractile vacuole with the plasma membrane was blocked by EGTA, a similar accumulation of large vacuoles occurred. Our observations suggest that the contractile-vacuole mutant of C. Moewusii produces vacuoles which can accumulate excess water as part of the mechanism of osmoregulation but which cannot complete diastole.     

Regeneration of static-load-degenerated articular cartilage extracellular matrix by vitamin C supplementation

The effect of a physiological dose of vitamin C (100 mug/ml) on goat articular cartilage chondrocytes cultured in an alginate matrix and subjected to static pressurization of 2.4 MPa was investigated. Biochemical analyses of DNA, glycosaminoglycan (GAG), collagen and protease activity were carried out in various matrix fractions, i.e. cellular matrix (CM) and further removed matrix (FRM), and in culture medium. The treatment of chondrocytes with vitamin C after static pressure increased the GAG content in both CM and FRM (P < 0.03) as compared with control or vitamin C/ static load alone. The collagen content of chondrocytes treated with vitamin C alone and vitamin C after static load also increased significantly in FRM (P < 0.003) as compared with control and static load alone. The specific activity of protease in CM and FRM decreased after vitamin C supplementation both with and without static pressure relative to control (P < 0.003). Transmission electron-microscopic images showed a mixed population of spherical and elliptical chondrocytes when vitamin C was added after static load as compared with static load alone where only elliptical cells were seen. Abundant pericellular and collagen fibrils were seen in this group of chondrocytes as compared with all other groups and the control. The results thus show that, in vitro, vitamin C supplementation of chondrocytes after static loading has the potential to reduce the morphological and biochemical degeneration of chondrocytes caused by static loading, thereby improving the cellular health and functioning of articular cartilage.