Brain Glucose-Sensing Mechanism and Energy Homeostasis.

The metabolic and energy state of the organism depends largely on the availability of substrates, such as glucose for ATP production, necessary for maintaining physiological functions. Deregulation in glucose levels leads to the appearance of pathological signs that result in failures in the cardiovascular system and various diseases, such as diabetes, obesity, nephropathy, and neuropathy. Particularly, the brain relies on glucose as fuel for the normal development of neuronal activity. Regions adjacent to the cerebral ventricles, such as the hypothalamus and brainstem, exercise central control in energy homeostasis. These centers house nuclei of neurons whose excitatory activity is sensitive to changes in glucose levels. Determining the different detection mechanisms, the phenotype of neurosecretion, and neural connections involving glucose-sensitive neurons is essential to understanding the response to hypoglycemia through modulation of food intake, thermogenesis, and activation of sympathetic and parasympathetic branches, inducing glucagon and epinephrine secretion and other hypothalamic-pituitary axis-dependent counterregulatory hormones, such as glucocorticoids and growth hormone. The aim of this review focuses on integrating the current understanding of various glucose-sensing mechanisms described in the brain, thereby establishing a relationship between neuroanatomy and control of physiological processes involved in both metabolic and energy balance. This will advance the understanding of increasingly prevalent diseases in the modern world, especially diabetes, and emphasize patterns that regulate and stimulate intake, thermogenesis, and the overall synergistic effect of the neuroendocrine system.

Expression of a Novel Ciliary Protein, IIIG9, During the Differentiation and Maturation of Ependymal Cells.

IIIG9 is the regulatory subunit 32 of protein phosphatase 1 (PPP1R32), a key phosphatase in the regulation of ciliary movement. IIIG9 localization is restricted to cilia in the trachea, fallopian tube, and testicle, suggesting its involvement in the polarization of ciliary epithelium. In the adult brain, IIIG9 mRNA has only been detected in ciliated ependymal cells that cover the ventricular walls. In this work, we prepared a polyclonal antibody against rat IIIG9 and used this antibody to show for the first time the ciliary localization of this protein in adult ependymal cells. We demonstrated IIIG9 localization at the apical border of the ventricular wall of 17-day-old embryonic (E17) and 1-day-old postnatal (PN1) brains and at the level of ependymal cilia at 10- and 20-day-old postnatal (PN10-20) using temporospatial distribution analysis and comparing the localization with a ciliary marker. Spectral confocal and super-resolution Structured Illumination Microscopy (SIM) analysis allowed us to demonstrate that IIIG9 shows a punctate pattern that is preferentially located at the borders of ependymal cilia in situ and in cultures of ependymocytes obtained from adult rat brains. Finally, by immunogold ultrastructural analysis, we showed that IIIG9 is preferentially located between the axoneme and the ciliary membrane. Taken together, our data allow us to conclude that IIIG9 is localized in the cilia of adult ependymal cells and that its expression is correlated with the process of ependymal differentiation and with the maturation of radial glia. Similarly, its particular localization within ependymal cilia suggests a role of this protein in the regulation of ciliary movement.

Apical Polarization of SVCT2 in Apical Radial Glial Cells and Progenitors During Brain Development.

During brain development, radial glial (RG) cells and the different progenitor subtypes are characterized by their bipolar morphology that includes an ovoid cell body and one or two radial processes that span across the developing cerebral wall. Different cells transport the reduced form of vitamin C, ascorbic acid (AA), using sodium-dependent ascorbic acid cotransporters (SVCT1 or SVCT2). SVCT2 is mainly expressed in the nervous system (CNS); however, its localization in the central nervous system during embryonic development along with the mechanism by which RG take up vitamin C and its intracellular effects is unknown. Thus, we sought to determine the expression and localization of SVCT2 during CNS development. SVCT2 is preferentially localized in the RG body at the ventricular edge of the cortex during the neurogenic stage (E12 to E17). The localization of SVCT2 overexpressed by in utero electroporation of E14 embryos is consistent with ventricular polarization. A similar distribution pattern was observed in human brain tissue sections at 9 weeks of gestation; however, SVCT2 immunoreaction was also detected in the inner and outer subventricular zone (SVZ). Finally, we used C17.2 neural stem cell line, J1ES cells and primary cell cultures derived from the brain cortex to analyze functional SVCT2 activity, AA effects in progenitor cells bipolar morphology, and SVCT2 expression levels in different culture conditions. Our results indicate that basal RG cells and apical intermediate and subapical progenitors are the main cell types expressing SVCT2 in the lissencephalic brain. SVCT2 was mainly detected in the apical region of the ventricular zone cells, contacting the cerebrospinal fluid. In gyrencephalic brains, SVCT2 was also detected in progenitor cells located in the inner and outer SVZ. Finally, we defined that AA has a strong radializing (bipolar morphology) effect in progenitor cells in culture and the differentiation condition modulates SVCT2 expression.

Activation of kinin B1 receptors induces chemotaxis of human neutrophils.

Kinins are biologically active peptides that are powerful mediators of cellular inflammation. They mimic the cardinal signs of inflammation by inducing vasodilatation and by increasing vascular permeability and pain. Neutrophils are chemoattracted to sites of inflammation by several stimuli. However, the evidence concerning the chemotactic effect of kinin peptides has been contradictory. We analyzed the chemotactic effect of kinin B(1) receptor agonists on neutrophils isolated from peripheral blood of human healthy subjects. Chemotaxis was performed using the migration under agarose technique. To test the effect of B(1) receptor agonists, each assay was carried out overnight at 37 degrees C in 5% CO(2)-95% air on neutrophils primed with 1 ng/ml interleukin-1beta. Simultaneous experiments were performed using unprimed cells or cells challenged with formyl-Met-Leu-Phe (fMLP). A clear chemotactic activity was observed when primed neutrophils were challenged with Lys-des[Arg(9)]-bradykinin (LDBK) or des[Arg(9)]-bradykinin at 10(-10) M but not when unprimed cells were used. A reduction in the chemotactic response was observed after priming of cells in the presence of 0.5 mM cycloheximide and 10 mug/ml brefeldin A, suggesting that some protein biosynthesis is required. Techniques such as reverse transcriptase-polymerase chain reaction and in situ hybridization confirmed the expression of the B(1) receptor mRNA, and immunocytochemistry and autoradiography demonstrated the expression of the B(1) receptor protein. In contrast to other chemoattractants such as fMLP, cytosolic intracellular calcium did not increase in response to the B(1) receptor agonist LDBK. A generation of kinin B(1) receptor agonists during the early phase of acute inflammation may favor the recruitment of neutrophils to the inflammatory site.

Early expression of glycine and GABA(A) receptors in developing spinal cord neurons. Effects on neurite outgrowth.

Using fluorometric and immunocytochemical techniques, we found that high glycine concentrations or blockade of glycine receptors increases neurite outgrowth in developing mouse spinal cord neurons. Glycine- and GABA(A)-activated currents were demonstrated during applications of glycine and GABA (50-100 microM) in 5 days in vitro (DIV) neurons. Long application (> or =10 min) of 100 microM glycine desensitized the membrane response by more than 95%. Application of glutamate in the absence of external Mg(2+), at several membrane potentials, did not produce any detectable membrane response in these cells. Immunocytochemical studies with NR1 and GluR1 antibodies showed a delayed appearance of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors respectively. Spontaneous synaptic activity was readily observed in 5 DIV neurons. The use of various receptor antagonists (strychnine, bicuculline, DL-2-amino-5-phosphonovalerate [APV], 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX]) revealed that this activity was predominantly glycinergic, and to a smaller extent, GABAergic. In the presence of bicuculline, APV and CNQX, we detected abundant spontaneous depolarizing potentials which often reached the action potential threshold. Further evidence for functional synaptic activity was provided by the detection of co-localization of gephyrin and synaptophysin at 5 DIV using confocal microscopy. Fluorometric studies with Fluo-3, a Ca(2+) indicator, in 5 DIV cultures showed the presence of spontaneous fluctuations associated with tetrodotoxin-sensitive synaptic events. The number of neurons displaying these fluctuations was significantly increased (>100%) when the cells were bathed in a strychnine-containing solution. On the other hand, these synaptically mediated Ca(2+) events were blocked by the co-application of strychnine and bicuculline. This suggests that glycine and GABA(A) receptors provide a fundamental regulation of both neuronal excitability and intracellular Ca(2+) at this early time of development.The neurotrophic effects of agonists and antagonists for glycine, GABA(A) and glutamate receptors were examined in neurons cultured for 2 or 5 DIV. From all the agonists used, only high concentrations of glycine increased neurite outgrowth in 5 DIV neurons. We found that strychnine also increased neurite outgrowth, whereas tetrodotoxin (1 microM), nimodipine (4 microM) and bicuculline (20 microM) completely blocked it. On the other hand, APV (50 microM) and CNQX (20 microM) were unable to affect neurite outgrowth. These data suggest that spinal glycine receptors depress neurite outgrowth by shunting neuronal excitability. Outgrowth induction possibly results from the enhanced activity found after the inhibition of glycinergic activity. We postulate that this resets the intracellular calcium at a concentration that favors neurite outgrowth.

High-affinity sodium-vitamin C co-transporters (SVCT) expression in embryonic mouse neurons.

The sodium-vitamin C co-transporters SVCT1 and SVCT2 transport the reduced form of vitamin C, ascorbic acid. High expression of the SVCT2 has been demonstrated in adult neurons and choroid plexus cells by in situ hybridization. Additionally, embryonic mesencephalic dopaminergic neurons express the SVCT2 transporter. However, there have not been molecular and kinetic analyses addressing the expression of SVCTs in cortical embryonic neurons. In this work, we confirmed the expression of a SVCT2-like transporter in different regions of the fetal mouse brain and in primary cultures of neurons by RT-PCR. Kinetic analysis of the ascorbic acid uptake demonstrated the presence of two affinity constants, 103 microM and 8 microM. A K(m) of 103 microM corresponds to a similar affinity constant reported for SVCT2, while the K(m) of 8 microM might suggest the expression of a very high affinity transporter for ascorbic acid. Our uptake analyses also suggest that neurons take up dehydroascorbic acid, the oxidized form of vitamin C, through the glucose transporters. We consider that the early expression of SVCTs transporters in neurons is important in the uptake of vitamin C, an essential molecule for the fetal brain physiology. Vitamin C that is found at high concentration in fetal brain may function in preventing oxidative free radical damage, because antioxidant radical enzymes mature only late in the developing brain.

Effect of protein kinase C activation on the glycine evoked Cl(-) current in spinal cord neurons.

We investigated whether the effect of phorbol-12-myristate-13-acetate (PMA) was altered by a kinase inhibitor and by down-regulation of protein kinase C (PKC) in order to determine if glycine receptors in mouse spinal neurons, unlike those in hippocampal and trigeminal neurons, can be inhibited by PKC. To examine the above, electrophysiological and immunofluorescence studies were carried out in mouse spinal neurons kept in culture for up to 3 weeks. The inhibition of the glycine activated current by PMA (1 microM) increased from 12+/-3% during week 1 to 27+/-6% during week 3. The effect of PMA was completely blocked by the PKC selective inhibitor RO 31-8220 (1 microM). After culturing the cells with 1 microM PMA for 24 h, the inhibitory effect of acute application of PMA disappeared altogether, suggesting that the effect of PMA was via PKC. Immunofluorescence studies showed that a short stimulation with PMA translocated the enzyme to the periphery whereas longer term stimulation (24 h) down regulated the PKC signal. These results indicate that activation of PKC by PMA inhibits the glycine receptor in cultured spinal neurons and that its sensitivity changes during neuronal development.

Biosynthesis and molecular biology of the secretory proteins of the subcommissural organ.

Ependymal cells are specialized in the synthesis and release of different factors into the cerebrospinal fluid (CSF). The subcommissural organ (SCO) is one of the most active areas of the ventricular walls secreting into the CSF. This gland is localized in the roof of the third ventricle covering the posterior commissure. Glycoproteins synthesized in SCO cells are released into the ventricular CSF where they aggregate, in a highly ordered fashion, forming an elongated supramacromolecular structure known as the Reissner's fiber (RF). RF grows caudally and extends along the brain aqueduct, the fourth ventricle, and the whole length of the central canal of the spinal cord. The SCO cells synthesize glycoproteins of high molecular weight. A precursor form of 540 kDa is synthesized in bovine and chick SCO cells, and a transcript of 10--14 kb is expressed selectively in the bovine SCO cells. The processing of this molecule generates at least one protein of about 450 kDa (RF-Gly-I), which, after being released, is involved in the formation of RF. Additionally, biochemical data indicate that bovine SCO cells synthesize a second precursor compound of 320 kDa, which is also detected in rat, rabbit, and dog. We postulate that RF is formed by two different complexes, one of which has a very high molecular mass (700 kDa or more) and is made up of at least six polypeptides, with the polypeptide of 450 kDa being its main component. The molecules that form RF in different species have different primary structures but they express common epitopes associated to the existence of cysteine bridges, which are probably crucial for polymerization of RF. Molecular procedures involving the use of anti-RF antibodies have led to the isolation of cDNA clones encoding two proteins known as RF-GLY-I and SCO-spondin. In the last 3 years, five partial cDNA sequences encoding SCO-spondin-like proteins have been obtained (Y08560, Y08561, AJ132107, AJ132106, AJ133488). These clones along with RF-GLY-I and SCO-spondin were computer-assembled generating a cDNA consensus sequence of 14.4 kb. Analyses of the long consensus sequence revealed an extended open reading frame (ORF-1) spanning from base 1,634 to 14,400 that encodes for a putative protein of 4,256 amino acids (approximately 450 kDa). The Mr of the predicted protein is consistent with the observed Mr of the largest protein recognized with anti-RF antibodies in SCO and RF extracts. However, the absence of consensus sequences typically present near the 5J'-end of the translation initiation site suggests the existence of a second open reading frame (ORF-2) extending from base 1 to base 14,400 in frame with the ORF-1 and probably encoding for the largest protein precursor (540 kDa). An antibody raised against a peptide sequence, deduced from the open reading frame encoded by a SCO cDNA, reacted specifically with the bovine and rat SCO-RF complex, thus indicating that the protein encoded by the cloned cDNA is part of RF. Immunoblots of bovine SCO extracts using the anti-peptide serum revealed bands of 540 kDa and 450 kDa, but it did not react with the proteins of 320 and 190 kDa. These data support the existence of two precursors for the bovine RF-glycoproteins (540 and 320 kDa) with the 450-kDa protein being a processed form of the 540-kDa precursor. We postulate that the cloned cDNAs encode for a protein that corresponds to the 540-kDa precursor and that at least part of this sequence is present in the processed form of 450 kDa that is secreted to form the RF.

Spatial distribution of Reissner's fiber glycoproteins in the filum terminale of the rat and rabbit.

The subcommissural organ secretes into the third ventricle glycoproteins that condense to form the Reissner's fiber (RF). At the distal end of the central canal of the spinal cord, the RF-glycoproteins accumulate in the form of an irregular mass known as massa caudalis. Antibodies against RF-glycoproteins and a set of lectins were used at the light and electron microscopic level to investigate the spatial distribution of the massa caudalis material in the rat and rabbit filum terminale. In the sacral region of the rat, the central canal presents gaps between the ependymal cells through which RF-glycoproteins spread out. The bulk of massa caudalis material, however, escapes through openings in the dorsal wall of the terminal ventricle. In the rabbit, the massa caudalis is formed within the ependymal canal, at the level of the second coccygeal vertebra, it accumulates within preterminal and terminal dilatations of the central canal, and it escapes out through gaps in the dorsal ependymal wall of the terminal ventricle. The existence of wide intercellular spaces and a large orifice (neuroporous) in the dorsal ependymal wall of the terminal ventricle, and the passage of RF-material through them, appear to be conserved evolutionary features. After leaving the terminal ventricle of the rat and rabbit, RF-glycoproteins establish a close spatial association with the numerous blood vessels irrigating the filum terminale, suggesting that in these species the blood vessels are the site of destination of the RF-glycoproteins escaping from the central canal, thus resembling the situation found in lower vertebrates. When passing from the RF stage to the massa caudalis stage, the rabbit RF-glycoproteins lose their sialic acid residues, exposing galactose as the terminal residue. Since this sialic acid-galactose modification of RF-glycoproteins had also been described in lamprey larvae, it may be regarded as a conserved evolutionary feature associated with the formation of the massa caudalis.

Elevated expression of glucose transporter-1 in hypothalamic ependymal cells not involved in the formation of the brain-cerebrospinal fluid barrier.

Glucose transporters play an essential role in the acquisition of glucose by the brain. Elevated expression of glucose transporter-1 has been detected in endothelial cells of the blood-brain barrier and in choroid plexus cells of the blood-cerebrospinal fluid barrier. On the other hand, there is a paucity of information on the expression of glucose transporters in the ependymal cells that line the walls of the cerebral ventricles. The tanycytes are specialized ependymal cells localized in circumventricular organs such as the median eminence that can be segregated into at least three types, alpha, beta1 and beta2. The beta2 tanycytes form tight junctions and participate in the formation of the cerebrospinal fluid-median eminence barrier. Using immunocytochemistry and in situ hybridization, we analyzed the expression of hexose transporters in rat and mouse hypothalamic tanycytes. In both species, immunocytochemical analysis revealed elevated expression of glucose transporter-1 in alpha and beta1 tanycytes. Intense anti-glucose transporter-1 staining was observed in cell processes located throughout the arcuate nucleus, in the end-feet reaching the lateral sulcus of the infundibular region, and in cell processes contacting the hypothalamic capillaries. On the other hand, there was very low expression of glucose transporter-1 in beta2 tanycytes involved in barrier function. In contrast with the results of the cytochemical analysis, in situ hybridization revealed that tanycytes alpha, beta1, and beta2 express similar levels of glucose transporter-1 mRNA. Further analysis using anti-glial fibrillary acidic protein antibodies to identify areas rich in astrocytes revealed that astrocytes were absent from areas containing alpha and beta1 tanycytes, but were abundant in regions containing the barrier-forming beta2 tanycytes. Overall, our data reveal a lack of correlation between participation in barrier function and expression of glucose transporter-1 in hypothalamic tanycytes. Given the virtual absence of astrocytes in areas rich in alpha and beta1 tanycytes, we speculate whether the tanycytes might have astrocyte-like functions and participate in the metabolic coupling between glia and neurons in the hypothalamic area.

Neurite outgrowth in developing mouse spinal cord neurons is modulated by glycine receptors.

The effect of glycine receptor activation on neurite outgrowth and survival was studied in 5 DIV (days in vitro) spinal neurons. These neurons were depolarized by spontaneous synaptic activity and by glycine, but not by glutamate. These responses were accompanied by increases in intracellular calcium concentration measured with Indo-1 and Fluo-3. Glycine (100 microM, 48 h) increased (46 +/- 6%) the number of primary neurites and total neuritic length. This effect was mediated by synaptic activity and calcium influx because TTX (1 microM) and nimodipine (4 microM) blocked the stimulatory effect of glycine. Neuronal survival, on the other hand, was not affected. This study shows for the first time the modulatory effect of glycine receptors on spinal neuron development.

Hydrolysis of kininogens by degranulated human neutrophils and analysis of bradykinin as chemotactic factor for cells isolated from peripheral blood.

Human neutrophils play a pivotal role in acute inflammation including the regulation of vascular permeability. We have examined the capacity of neutrophil enzymes to hydrolyse human kininogens in vitro and have also explored the potentiality of bradykinin to induce chemotactic migration on neutrophils isolated from peripheral blood. Isolated neutrophils were stimulated with either f-Met-Leu-Phe, thrombin or silica particles coated with human IgG. Neutrophil enzymes obtained by degranulation produced, after 45 min of incubation with high and low molecular weight kininogens, the complete transformation of both proteins in polypeptides ranging from 20 to less than 10 kDa in molecular mass. Supernatants obtained from nonstimulated neutrophils did not modify the molecular size of kininogens. The assay used to test the chemoattractant capacity of synthetic bradykinin on human neutrophils showed that this peptide has no chemotactic activity on cells isolated from healthy subjects. Our results show that stimulation of human neutrophils with opsonized silica, thrombin and the chemotactic peptide f-Met-Leu-Phe induces release of kininogen-hydrolyzing enzymes from these cells.

Expression of the hexose transporters GLUT1 and GLUT2 during the early development of the human brain.

We used immunohistochemistry with anti-glucose transporter antibodies to document the presence of facilitative hexose transporters in the fetal human brain. GLUT1 is expressed in all regions of the fetal brain from ages 10 to 21 weeks. GLUT1 was present in the endothelial cells of the brain capillaries, the epithelial cells of the choroid plexus and neurons. High expression of GLUT2 was observed in the granular layer of the cerebellum in brains 21 weeks old, but GLUT2 immunoreactivity was absent at earlier stages. GLUT3 and GLUT4 immunoreactivities were absent at all stages studied. GLUT5 immunoreactivity was evident only in the cerebellar region of 21-week old fetal brains. We conclude that GLUT1 plays a fundamental role in early human brain development. The data also suggest that the cerebellum of the developing brain has the capacity to transport fructose, a substrate that has not been previously identified as a source of metabolic energy in the adult human brain.

Partial sequencing of Reissner's fiber glycoprotein I (RF-Gly I).

The bulk of the secretion of the subcommissural organ is formed by glycoproteins that appear to be derived from two precursor forms of 540 and 320 kDa. Upon release into the ventricle, these glycoproteins aggregate to form Reissner's fiber. We report the isolation of three cDNA clones from a cDNA library prepared from bovine subcommissural organ RNA, by using an anti-Reissner's fiber serum for immunoscreening. Inserts of 0.7, 1.2, and 2.5 kb were amplified by the polymerase chain reaction, subcloned into pUC18 vector, and sequenced. Although restriction mapping of the three inserts initially suggested that all of them were derived from the same mRNA, sequence analysis showed that a short non-homologous region was present in the 0.7-kb insert when compared with the 1. 2-kb and 2.5-kb inserts, suggesting that they corresponded to two different, although highly homologous, mRNAs. Northern analyses showed a single mRNA species of approximately 9.5 kb present in the subcommissural organ and missing in the choroid plexus, brain cortex, and liver. In situ hybridization confirmed that the expression of the RNA was restricted to cells of the bovine subcommissural organ. Polyclonal antibodies raised against a synthetic peptide, whose amino-acid sequence was deduced from the 2.5-kb cDNA, reacted specifically with the bovine and rat subcommissural organ-Reissner's fiber complex. In immunoblots of bovine subcommissural organ, this antibody revealed the precursor 540-kDa form and its putative processed form of 450 kDa. It is concluded that the cloned cDNA encodes for the major constitutive glycoprotein of Reissner's fiber, here designated as RF-Gly I. The sequenced region of RF-Gly I displays a high degree of homology with some regions of the von Willebrand factor and certain mucins; it also displays two motifs homologous with repeats present in proteins of the spondin family and other proteins. A core sequence of the RF-Gly I repeats suggests that this molecule displays protein-binding properties.

Expression of granulocyte-macrophage colony-stimulating factor receptors in human prostate cancer.

We studied the expression and function of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor in the human prostate carcinoma cell line LNCaP and looked for its presence in normal and neoplastic human prostatic tissue. The GM-CSF receptor is composed of two subunits, alpha and beta. While the isolated alpha subunit binds GM-CSF at low-affinity, the isolated beta subunit does not bind GM-CSF by itself; but complexes with the alpha subunit to form a high-affinity receptor. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed expression of mRNAs encoding the alpha and beta subunits of the GM-CSF receptor in LNCaP cells, and the presence of the alpha and beta proteins was confirmed by immunolocalization with anti-alpha and anti-beta antibodies. Receptor binding studies using radiolabeled GM-CSF showed that LNCaP cells have about 150 high-affinity sites with a kd of 40 pmol/L and approximately 750 low-affinity sites with a kd of 2 nmol/L. GM-CSF signaled, in a time- and dose-dependent manner, for protein tyrosine phosphorylation and induced the proliferation of the LNCaP cells. Immunolocalization studies showed low level expression of GM-CSF alpha and beta subunits in normal prostate tissue, with substantial expression in benign prostatic hyperplasia and prominent expression in neoplastic prostate tissue. Maximal expression of both subunits was observed in prostatic carcinomas metastatic to lymph node and bone. Tumor cells that stained positively with anti-alpha subunit antibodies were also reactive with anti-beta subunit antibodies, indicating that they express high-affinity GM-CSF receptors. Our data show that the LNCaP cells express functional GM-CSF receptors and that prostatic carcinomas have prominent GM-CSF receptor expression. These findings imply that both hyperplastic and neoplastic prostatic tissues may be responsive to GM-CSF.

Increased facilitated transport of dehydroascorbic acid without changes in sodium-dependent ascorbate transport in human melanoma cells.

Many cell types transport vitamin C solely in its oxidized form, dehydroascorbic acid, through facilitative glucose transporters. These cells accumulate large intracellular concentrations of vitamin C by reducing dehydroascorbic acid to ascorbate, a form that is trapped intracellularly. Certain specialized cells can transport vitamin C in its reduced form, ascorbate, through a sodium-dependent cotransporter. We found that normal human melanocytes and human malignant melanoma cells are able to transport vitamin C using both mechanisms. Melanoma cell lines transported dehydroascorbic acid at a rate that was at least 10 times greater than the rate of transport by melanocytes, whereas both melanoma cells and melanocytes transported ascorbate with similar efficiency. Dehydroascorbic acid transport was inhibited by deoxyglucose and cytochalasin B, indicating the direct participation of facilitative glucose transporters in the transport of oxidized vitamin C. Melanoma cells accumulated intracellular vitamin C concentrations that were up to 100 times greater than the corresponding extracellular dehydroascorbic acid concentrations, whereas intracellular accumulation of vitamin C by melanocytes never exceeded the extracellular level of dehydroascorbic acid. Melanoma cells transported dehydroascorbic acid through at least two different transporters, each with a distinct K(m), a finding that agreed well with the presence of several glucose transporter isoforms in these cells. Only one kinetic component of ascorbate uptake was identified in both melanocytes and melanoma cells, and ascorbate transport was sodium dependent and inhibited by ouabain. Both cell types were able to accumulate intracellular concentrations of vitamin C that were greater than the extracellular ascorbate concentrations. The data indicate that melanoma cells and normal melanocytes transport vitamin C using two different transport systems. The transport of dehydroascorbic acid is mediated by a facilitated mechanism via glucose transporters, whereas transport of ascorbic acid involves a sodium-ascorbate cotransporter. The differential capacity of melanoma cells to transport the oxidized form of vitamin C reflects the increased expression of facilitative transporters associated with the malignant phenotype.

Immunochemical analysis of the subcommissural organ-Reissner's fiber complex using antibodies against alkylated and deglycosylated glycoproteins of the bovine Reissner's fiber.

Reissner's fiber (RF) has been isolated, solubilized, and used to raise polyclonal antibodies. The present investigation has been designed: (1) to obtain antibodies against RF-glycoproteins in their native form (anti-RF-BI), after irreversible denaturation by alkylation (anti-RF-A), and after alkylation and deglycosylation by using endoglycosidase F (anti-RF-DE); (2) to use these antisera for a comparative immunocytochemical study of the subcommissural organ (SCO)-RF complex; (3) to establish the molecular mass of the deglycosylated RF-glycoproteins. Anti-RF-BI reacts with the SCO of all the species investigated. Anti-RF-A and anti-RF-DE only react with bovine SCO and RF. The three antisera stain the same bands in immunoblots of extracts of bovine SCO and RF, but anti-RF-A and anti-RF-DE reveal additional bands. The epitope common to all species reacting with anti-RF-BI is thus probably conformational in nature and associated with the integrity of the disulfide bonds. The lack of antibodies against conserved sequential epitopes in anti-RF-A does not support previous assumptions on the conserved nature of the SCO secretion. After deglycosylation by using endoglycosidase F, the RF-glycoproteins present a reduction in their molecular mass ranging between 10% and 25%. The three larger compounds (450, 300, and 230 kDa) lose their affinity for Limax flavus agglutinin (affinity = sialic acid), whereas the 190-kDa compound (170 kDa after deglycosylation) keeps its affinity for this lectin suggesting that it has N-linked and O-linked carbohydrate moieties, the three larger proteins probably having only N-linked carbohydrates.

Bovine Reissner's fiber (RF) and the central canal of the spinal cord: an immunocytochemical study using a set of monoclonal antibodies against the RF-glycoproteins.

The subcommissural organ secretes N-linked complex-type glycoproteins into the cerebrospinal fluid. These glycoproteins condense to form Reissner's fiber (RF), which extends along the fourth ventricle and central canal of the spinal cord. A set of three monoclonal antibodies (Mabs 3E6, 3B1, and 2A5) has been obtained using these glycoproteins as immunogens. Competitive and sandwich enzyme-linked immunoassay methods have demonstrated that the three monoclonal antibodies are directed against different epitopes, and that there is no competition among them for their binding to glycoproteins of RF. Mab 3E6 displays immunoblotting properties that are similar to those of a polyclonal antibody against the pool of glycoproteins from RF, but that are different from those of Mabs 3B1 and 2A5. All three antibodies immunostain the bovine subcommissural organ and RF. A population of ependymal cells is stained by the polyclonal antibody, and Mabs 2A5 and 3E6, but not by Mab 3B1. The material present in a population of ependymal cells of the central canal, and the glycoproteins secreted by the subcommissural organ thus probably have partial chemical identity. Some evidence suggests that the immunoreactive ependymal cells are secretory cells. The luminal surface of the central canal is coated by a thin layer of material with immunocytochemical characteristics different from those of the ependymal cells; such a coat may correspond to material released from RF.

Alpha 1-antitrypsin expression in human thyroid papillary carcinoma.

Alpha 1-antitrypsin is a plasma serine protease inhibitor originally used as a marker for tumors of histiocytic origin. Our casual finding of immunoreactive alpha 1-antitrypsin in one case of thyroid papillary carcinoma led us to investigate its presence in 10 thyroid papillary carcinomas by applying immunocytochemical and immunochemical techniques to tissue sections and Western blots of tissue homogenates prepared from neoplastic tissue and from uninvolved normal areas in the vicinity of each tumor. The immunocytochemical study was performed in both thyroid tissue and metastatic regional lymph nodes. This analysis revealed immunoreactivity for alpha 1-antitrypsin in nine of the 10 cases studied. Immunoreactivity was intense in some of the cells forming the papillar and follicular structures. These cells were intermingled with completely unstained tumoral cells. In contrast to neoplastic tissue, the normal thyroid tissue present in the vicinity of each tumor showed no staining for alpha 1-antitrypsin. The electrophoretic analysis performed on homogenates prepared from both tumoral and normal thyroid tissue revealed a drastic reduction in the band corresponding to thyroglobulin in the tumoral tissue compared with normal thyroid extracts, where it represented the major protein. Western blotting and immunoprinting with a polyclonal alpha 1-antitrypsin antibody confirmed the results obtained with immunocytochemistry about the presence of this protease inhibitor in neoplastic thyroid tissue. Immunoprinting with the anti-alpha 1-antitrypsin antibody revealed an intense immunoreactive band of 53 kDa in the extracts prepared from tumoral tissue. This band had exactly the same apparent molecular mass previously described by others for alpha 1-antitrypsin purified from plasma and was identical to the molecular mass of the purified commercial standard employed.

Expression of the fructose transporter GLUT5 in human breast cancer.

The primary metabolic characteristic of malignant cells is an increased uptake of glucose and its anaerobic metabolism. We studied the expression and function of the glucose transporters in human breast cancer cell lines and analyzed their expression in normal and neoplastic primary human breast tissue. Hexose uptake assays and immunoblotting experiments revealed that the breast carcinoma cell lines MCF-7 and MDA-468 express the glucose transporters GLUT1 and GLUT2, isoforms expressed in both normal and neoplastic breast tissue. We also found that the breast cancer cell lines transport fructose and express the fructose transporter GLUT5. Immunolocalization studies revealed that GLUT5 is highly expressed in vivo in human breast cancer but is absent in normal human breast tissue. These findings indicate that human breast cancer cells have a specialized capacity to transport fructose, a metabolic substrate believed to be used by few human tissues. Identification of a high-affinity fructose transporter on human breast cancer cells opens opportunities to develop novel strategies for early diagnosis and treatment of breast cancer.