Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning.

1 alpha,25-Dihydroxyvitamin D3[1 alpha,25(OH)2D3], an active form of vitamin D, has roles in many biological phenomena such as calcium homeostasis and bone formation, which are thought to be mediated by the 1 alpha,25(OH)2D3 receptor (VDR), a member of the nuclear hormone receptor superfamily. However, the molecular basis for the actions of 1 alpha,25(OH)2D3 in bone formation, its role during development and VDR genetic polymorphisms for predicting bone mineral density are uncertain. To investigate the functional role of VDR, we generated mice deficient in VDR by gene targeting. We report here that in VDR null mutant mice, no defects in development and growth were observed before weaning, irrespective of reduced expression of vitamin D target genes. After weaning, however, mutants failed to thrive, with appearance of alopoecia, hypocalcaemia and infertility, and bone formation was severely impaired as a typical feature of vitamin D-dependent rickets type II (refs 8, 9). Unlike humans with this disease, most of the null mutant mice died within 15 weeks after birth, and uterine hypoplasia with impaired folliculogenesis was found in female reproductive organs. These defects, such as alopoecia and uterine hypoplasia, were not observed in vitamin D-deficient animals. The findings establish a critical role for VDR in growth, bone formation and female reproduction in the post-weaning stage.

Developmentally and spatially regulated expression of HNK-1 carbohydrate antigen on a novel phosphatidylinositol-anchored glycoprotein in rat brain.

HNK-1 carbohydrate antigen in an epitope expressed commonly in many cell surface adhesion and recognition molecules in the nervous system. We purified and characterized from rat brain a novel phosphatidylinositol (PI)-anchored 150-kD glycoprotein belonging to the HNK-1 family. The molecule (PI-GP150) was detected by combination of PI-specific phospholipase C treatment of brain membranes and Western blot analysis with mAb HNK-1. HNK-1-positive PI-GP150 was purified from the PI-PLC-released materials with three successive chromatographies (Sephacryl S-300, mAb HNK-1-Sepharose 4B, and Mono Q) and proven to be a novel molecule by immunoblot and structural analyses. Polyclonal antibody was raised against PI-GP150 and used to show that (a) PI-GP150 is expressed on the surface of neuronal cell bodies and their processes in culture, and (b) PI-GP150 appears during embryonic development and is present throughout all postnatal life in all brain regions. However, the expression of the HNK-1 epitope on PI-GP150 is regulated in both developmental stage-specific and region-specific manners. In newborn rats, the HNK-1 epitope is expressed on PI-GP150 throughout the brain. The level of HNK-1 epitope on PI-GP150 decreases after postnatal day 7 in hindbrain and becomes completely absent in adult myelencephalon and metencephalon. In contrast, HNK-1 epitope on PI-GP150 was constitutively expressed in telencephalon. Thus, while the HNK-1 carbohydrate epitope is strongly coupled to PI-GP150, its expression can be regulated independently of that of PI-GP150. The differential expression of the HNK-1 epitope at different rostro-caudal axial levels was observed also in other HNK-1 family molecules in brain membranes. These results suggest that the HNK-1 epitope plays an important role in adding region-specific and developmental stage-specific modifications on the function of the cell surface molecules.

R2D5 antigen: a calcium-binding phosphoprotein predominantly expressed in olfactory receptor neurons.

R2D5 is a mouse monoclonal antibody that labels rabbit olfactory receptor neurons. Immunoblot analysis showed that mAb R2D5 recognizes a 22-kD protein with apparent pI of 4.8, which is abundantly contained in the olfactory epithelium and the olfactory bulb. We isolated cDNA for R2D5 antigen and confirmed by Northern analysis and neuronal depletion technique that R2D5 antigen is expressed predominantly, but not exclusively, in olfactory receptor neurons. Analysis of the deduced primary structure revealed that R2D5 antigen consists of 189 amino acids with calculated M(r) of 20,864 and pI of 4.74, has three calcium-binding EF hands, and has possible phosphorylation sites for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and cAMP-dependent protein kinase (A kinase). Using the bacterially expressed protein, we directly examined the biochemical properties of R2D5 antigen. R2D5 antigen binds Ca2+ and undergoes a conformational change in a manner similar to calmodulin. R2D5 antigen is phosphorylated in vitro by CaM kinase II and A kinase at different sites, and 1.81 and 0.80 mol of Pi were maximally incorporated per mol of R2D5 antigen by CaM kinase II and A kinase, respectively. Detailed immunohistochemical study showed that R2D5 antigen is also expressed in a variety of ependymal cells in the rabbit central nervous system. Aside from ubiquitous calmodulin, R2D5 antigen is the first identified calcium-binding protein in olfactory receptor neurons that may modulate olfactory signal transduction. Furthermore our results indicate that olfactory receptor neurons and ependymal cells have certain signal transduction components in common, suggesting a novel physiological process in ependymal cells.

Intercellular adhesion molecule-5 induces dendritic outgrowth by homophilic adhesion.

Intercellular adhesion molecule-5 (ICAM-5) is a dendritically polarized membrane glycoprotein in telencephalic neurons, which shows heterophilic binding to leukocyte beta(2)-integrins. Here, we show that the human ICAM-5 protein interacts in a homophilic manner through the binding of the immunoglobulin domain 1 to domains 4-5. Surface coated ICAM-5-Fc promoted dendritic outgrowth and arborization of ICAM- 5-expressing hippocampal neurons. During dendritogenesis in developing rat brain, ICAM-5 was in monomer form, whereas in mature neurons it migrated as a high molecular weight complex. The findings indicate that its homophilic binding activity was regulated by nonmonomer/monomer transition. Thus, ICAM-5 displays two types of adhesion activity, homophilic binding between neurons and heterophilic binding between neurons and leukocytes.

The olfactory bulb: coding and processing of odor molecule information.

Olfactory sensory neurons detect a large variety of odor molecules and send information through their axons to the olfactory bulb, the first site for the processing of olfactory information in the brain. The axonal connection is precisely organized so that signals from 1000 different types of odorant receptors are sorted out in 1800 glomeruli in the mouse olfactory bulb. Individual glomerular modules presumably represent a single type of receptor and are thus tuned to specific molecular features of odorants. Local neuronal circuits in the bulb mediate lateral inhibition among glomerular modules to sharpen the tuning specificity of output neurons. They also mediate synchronized oscillatory discharges among specific combinations of output neurons and may contribute to the integration of signals from distinct odorant receptors in the olfactory cortex.

An ICAM-related neuronal glycoprotein, telencephalin, with brain segment-specific expression.

Telencephalin (TLN) is a 130 kd glycoprotein expressed exclusively in neurons of the telencephalon, the most rostral brain segment. In the neurons, TLN is localized to soma-dendritic membrane but not to axonal membrane. In this study, we have cloned cDNA encoding rabbit and mouse TLN. The cDNA-derived primary structure of TLN predicts an integral membrane protein with nine tandem immunoglobulin-like domains in an extra-cellular region, a transmembrane domain, and a short cytoplasmic tail. The distal eight immunoglobulin-like domains of TLN show highest homology with the immunoglobulin-like domains of intracellular adhesion molecules (ICAMs) 1, 2, and 3/R. The structural similarity of TLN with ICAMs provides a new and strong link between immunoglobulin superfamily molecules in the nervous and immune systems. TLN is an example of a dendrite-associated cell adhesion molecule involved in the brain's segmental organization, cell-cell interactions during dendritic development, and maintenance of functional neuronal networks.

BIG-1: a new TAG-1/F3-related member of the immunoglobulin superfamily with neurite outgrowth-promoting activity.

We have cloned a rat cDNA for a novel brain-derived immunoglobulin (Ig) superfamily molecule, BIG-1, by using PCR based on the amino acid sequences of the two closely related and well-known Ig superfamily members, rat TAG-1 and mouse F3. BIG-1 is a glycosylphosphatidylinositol-anchored membrane protein with six Ig-like domains and four fibronectin type III repeats, belonging to the TAG-1/F3 subgroup. The expression of BIG-1 mRNA is developmentally regulated with the highest level in the adult brain. It is restricted to subsets of neurons such as Purkinje cells of the cerebellum, granule cells of the dentate gyrus, and neurons in the superficial layers of the cerebral cortex. Recombinant BIG-1 protein has a neurite outgrowth-promoting activity when used as a substrate for neurons in vitro. These results suggest that BIG-1 may be involved in the formation and maintenance of neuron type-specific networks in the brain.

A genetic approach to visualization of multisynaptic neural pathways using plant lectin transgene.

The wiring patterns among various types of neurons via specific synaptic connections are the basis of functional logic employed by the brain for information processing. This study introduces a powerful method of analyzing the neuronal connectivity patterns by delivering a tracer selectively to specific types of neurons while simultaneously transsynaptically labeling their target neurons. We developed a novel genetic approach introducing cDNA for a plant lectin, wheat germ agglutinin (WGA), as a transgene under the control of specific promoter elements. Using this method, we demonstrate three examples of visualization of specific transsynaptic neural pathways: the mouse cerebellar efferent pathways, the mouse olfactory pathways, and the Drosophila visual pathways. This strategy should greatly facilitate studies on the anatomical and functional organization of the developing and mature nervous system.

Superficial zone chondrocytes in normal and osteoarthritic human articular cartilages synthesize novel truncated forms of inter-alpha-trypsin inhibitor heavy chains which are attached to a chondroitin sulfate proteoglycan other than bikunin.

OBJECTIVE: We have examined the occurrence of the inflammation-associated inter-alpha-trypsin inhibitor (IalphaI) components, bikunin, heavy chain (HC)1 and HC2 in normal cartilage and osteoarthritis (OA) cartilage and synovial fluids. DESIGN/METHODS: Cartilage extracts from normal donors and late-stage OA patients, and synovial fluids from OA patients were studied by Western blot with multiple antibodies to bikunin, HC1 and HC2. Cell and matrix localization was determined by immunohistochemistry and mRNA by RT-PCR. RESULTS: Bikunin.chondroitin sulfate (CS) and IalphaI were abundant in OA cartilages, but virtually undetectable in normal. In both OA and normal cartilages, HCs were largely present in a novel C-terminally truncated 50-kDa form, with most, if not all of these being attached to CS on a proteoglycan other than bikunin. Synovial fluids from OA patients contained bikunin.CS and full-length (approximately 90 kDa) HCs linked to hyaluronan (HA) as HC.HA (SHAP.HA). Immunohistochemistry showed intracellular and cell-associated staining for bikunin and HCs, consistent with their synthesis by superficial zone chondrocytes. PCR on multiple human normal and OA cartilage samples detected transcripts for HC1 and HC2 but not for bikunin. In OA cartilages, immunostaining was predominantly matrix-associated, being most intense in regions with a pannus-like fibrotic overgrowth. CONCLUSION: The truncated structure of HCs, their attachment to a proteoglycan other than bikunin, PCR data and intracellular staining are all consistent with synthesis of HC1 and HC2 by human articular chondrocytes. The presence of bikunin.CS and IalphaI in OA cartilage, but not in normal, appears to be due to diffusional uptake and retention through fibrillated (but not deeply fissured) cartilage surfaces.

cDNA cloning and characterization of mouse nifS-like protein, m-Nfs1: mitochondrial localization of eukaryotic NifS-like proteins.

We have isolated a mouse cDNA which shows significant sequence similarity to the yeast nifS-like gene (y-NFS1), and termed it m-Nfs1. The deduced protein sequence (459 amino acids long) has several characteristic features common to those of bacterial NifS proteins, but distinct from them by its amino-terminal extension which contains a typical mitochondrial targeting presequence. m-Nfs1 was found to be a soluble 47-kDa protein in the matrix fraction of mouse liver mitochondria. The m-Nfs1 gene was ubiquitously expressed in most tissues, suggesting its housekeeping function in vivo. We also found that the gamma-NFS1 protein was localized in the mitochondrial matrix in yeast cells. These results suggest that both eukaryotic NifS-like proteins may play some roles in mitochondrial functions.

Odor stimulation causes disappearance of R4B12 epitope on axonal surface molecule of olfactory sensory neurons.

Monoclonal antibodies R4B12 and R1D1 label the same subsets of rabbit primary olfactory axons. In the present study, we characterized the R4B12 antigens using immunohistochemical, immunoelectron-microscopic, and biochemical techniques. The R4B12 antigens are expressed on the surface membrane of a subset of primary olfactory axons. Western blot analysis revealed the existence of two forms (115,000 and 90,000 mol.wt) of the R4B12 antigens with different membrane-anchoring structures. Of the two forms, the smaller antigen (90,000 mol. wt) is anchored to the plasma membrane via a phosphatidylinositol linkage and expressed exclusively by the olfactory system. When the rabbit olfactory epithelium was stimulated by odors for 2-8 h in situ, the R4B12 immunoreactivity disappeared from the primary olfactory axons in the glomeruli of the olfactory bulb. These results suggest that the cell surface antigens R4B12 expressed by subsets of primary olfactory axons undergo stimulus-dependent changes by odor stimulation and may be involved in plasticity of olfactory sensory neurons.

Increase in cytoplasmic free Ca2+ elicited by noradrenalin and serotonin in cultured local interneurons of mouse olfactory bulb.

Effects of noradrenalin and serotonin on cytoplasmic free Ca2+ concentrations ([Ca2+]i) were studied by using the fluorescent indicator fura-2 in cultured local interneurons of mouse olfactory bulb. Application of noradrenalin (0.1-100 microM) caused a rapid and concentration-dependent rise in [Ca2+]i, while isoproterenol was ineffective at concentrations up to 100 microM. The noradrenalin (1 microM)-induced increase in [Ca2+]i was completely inhibited by pretreatment with alpha 1-antagonist, prazosin (100 nM), whereas the inhibitory effect of alpha 2-antagonist, yohimbine, was about 100-times less potent. Serotonin (0.1-100 microM) also caused the dose-dependent rise in [Ca2+]i, which was inhibited by serotonin2 antagonist, ketanserin. Even in the absence of the extracellular calcium, the noradrenalin- or serotonin-induced increase in [Ca2+]i was observed. These results indicate that both noradrenalin and serotonin elicit the rise in [Ca2+]i in local interneurons of the olfactory bulb. They also suggest that the rise in [Ca2+]i is mediated by alpha 1-adrenergic and serotonin2 receptors, and that the increased calcium is mainly derived from intracellular calcium storage sites. The above results provide evidence to suggest that in the olfactory bulb, noradrenergic and serotonergic centrifugal fibers exert modulatory influences on synaptic interactions between mitral/tufted cells and local interneurons by increasing cytoplasmic Ca2+ in local interneurons.

Abnormal kainic acid receptor density and reduced seizure susceptibility in dystrophin-deficient mdx mice.

Duchenne muscular dystrophy is characterized by a defect in dystrophin, which often causes mental retardation in addition to progressive muscular weakness. As dystrophin is localized in synaptic regions of the CNS, cognitive abnormalities associated with Duchenne muscular dystrophy are attributable to synaptic dysfunction. We report that dystrophin-deficient mdx mice were more resistant to kainic acid-induced seizures but not to GABA antagonist-induced seizures compared with the control mice. The kainic-acid receptor density in the brain was significantly lower in the mdx than in the control, although the density of muscarinic cholinergic receptors, another important neurotransmitter receptor for cognitive function, was normal. Moreover, mdx had significantly lower Timm staining intensity in the mossy fibers, which originate from the dentate granule cells and terminate on the pyramidal cells in the CA3 of the hippocampus. These results suggest that an instability of neurotransmitter receptors, such as kainate-type glutamate receptors, on synaptic membranes due to the disruption of dystrophin complex induces inefficient neurotransmission in Duchenne muscular dystrophy patients.

A procedure for in situ hybridization combined with retrograde labeling of neurons: application to the study of cell adhesion molecule expression in Dil-labeled rat pyramidal neurons.

We have devised a simple method that combines retrograde labeling of projecting neurons and in situ hybridization histochemistry to examine mRNA expression in the retrogradely labeled neurons. First, projecting neurons were retrogradely labeled in vivo by injection of the lipophilic neuronal tracer Dil. The fluorescence of the labeled neurons in the brain slices was photoconverted into stable DAB precipitate by green light illumination. The slices were cut into thinner sections and processed for detection of specific mRNA by in situ hybridization. Using this highly sensitive method, we demonstrate here that the corticospinal tract neurons in newborn rats express mRNA for the cell adhesion molecule L1. TAG-1 mRNA was not detected in these neurons. Therefore, the present method provides an important tool to study the molecular expression of projection neurons during the development of neuronal circuitry.

Expression of prostaglandin endoperoxide synthase in rat brain.

Developmental and regional expression of prostaglandin endoperoxide synthase (PES) transcript was examined in the rat brain and in primary mixed cultures of neurons and glial cells from neonatal brain. Although the PES mRNA level in the brain was much lower than that in peripheral rat tissues such as lung, liver, spleen and kidney, a significant 3.0 kb band was detected in brain samples by Northern blot analysis. During development, PES mRNA was first detectable at postnatal day 7, and increased thereafter toward adulthood. The highest level of 3.0 kb PES mRNA was observed in the olfactory bulb, midbrain, and hypothalamus; and the lowest level in the hippocampus. In primary cultures of neonatal brain cells, the level of 3:0 kb transcript of PES transiently and dramatically increased about 30-fold on the third day after plating. Simultaneously, two cross-hybridizing signals were detected at 4.0 and 7.0 kb. This increase in PES mRNAs was completely inhibited by addition of cytosine-1-beta-D-arabinofuranoside. The induction of PES mRNA was in parallel with the increase in PES protein, as assessed by Western blot analysis. Immunostaining of cultured cells with anti-PES monoclonal antibody revealed that PES protein was induced mainly in neurons but not in glial cells. These results suggest that PES is expressed in the central nervous system at a low concentration under normal conditions, and that the neuronal cells possess an ability to express high levels of PES mRNA and protein.

Use of an SDS-gel-separated protein band as a ligand for affinity chromatography: procedure and application to the purification of domain-specific antibodies against alpha-actinin.

This paper describes a simple and efficient method for preparing affinity columns. We used protein separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis as a ligand. Protein bands detected in a polyacrylamide gel were electrophoretically transferred to CNBr-activated Sepharose using a buffer containing Nonidet P-40. The amount of ligand protein coupled to activated Sepharose by our method was almost comparable to that obtained by conventional coupling procedure with a native ligand protein. Using affinity columns prepared by this method, we have successfully purified anti-alpha-actinin antibody and antibodies highly specific to the rod domain of alpha-actinin from the antiserum. This new method should be useful for separating a specific antibody from an antiserum that has been raised against multiple antigens. In addition, the use of the SDS-gel-fractionated band facilitates the coupling of proteins that have low solubility under the coupling conditions.

The complete amino acid sequence of subunit d of rat liver mitochondrial H(+)-ATP synthase.

Subunit d of H(+)-ATP synthase from rat liver mitochondria was isolated from the purified enzyme by reverse-phase high performance liquid chromatography. The partial amino acid sequence of the subunit was determined by automated Edman degradation of the peptide fragments. The nucleotide sequence of subunit d of rat liver H(+)-ATP synthase was determined from a recombinant cDNA clone isolated by screening a rat hepatoma cell line H4TG cDNA library with a probe DNA. The sequence was composed of 581 nucleotides including a coding region for the import precursor of subunit d and noncoding regions on the 5'- and 3'- sides. The possible precursor of subunit d and its mature polypeptide deduced from the open reading frame consisted of 161 and 160 amino acid residues with molecular weights of 18,763 and 18,631, respectively. Subunit d is a hydrophilic protein with an isoelectric point of 6.19. The sequence of the rat subunit d is highly homologous with that of subunit d of bovine heart and slightly similar to that of the subunit d of the yeast mitochondria. However, it had no homology with the sequence of any of the subunits of bacterial or chloroplast H(+)-ATP synthase.

Human erythrocyte bisphosphoglycerate mutase: inactivation by glycation in vivo and in vitro.

2,3-Bisphosphoglycerate mutase (BPGM) [EC 5.4.2.4] is a multifunctional enzyme that catalyzes both the synthesis and the degradation of 2,3-diphosphoglycerate (2,3-DPG) and contains three types of activities in that it functions as a 2,3-DPG synthetase, a phosphoglycerate mutase and a 2,3-DPG phosphatase. In humans, BPGM occurs only in erythrocytes and plays a pivotal role in the dissociation of oxygen from hemoglobin via 2,3-DPG. The present study shows that the specific activity of BPGM in erythrocytes of diabetic patients is decreased, compared to normal controls as judged by 2,3-DPG synthetase activity and immunoreactive contents. To understand the mechanism by which the enzyme is inactivated, the enzyme was purified from pooled erythrocytes from diabetic patients and subjected to a boronate affinity column. The flow through fraction was active while the bound fraction was completely inactive. The bound fraction was reactive to an anti-hexitollysine antibody, indicating that the enzyme had undergone glycation and inactivation. The primary glycated site of the enzyme was found to be Lys158 as judged by amino acid sequencing and the reactivity with an anti-hexitollysine IgG, after reverse-phase HPLC of the lysyl-endopeptidase-digested peptides. Extensive glycation of recombinant BPGM in vitro indicated that the glycation sites were Lys2, Lys4, Lys17, Lys42, Lys158, and Lys196. From these results, the loss of enzymatic activity appears to be due to the glycation of Lys158 which may be located in the vicinity of the substrate binding site.

Distribution of putative odour receptor proteins in olfactory epithelium.

To investigate immunohistochemically the spatial localization of putative odour receptor proteins, we synthesized a polypeptide which corresponds to a region of a putative odour receptor protein, I3, and raised an antibody to the peptide. In rat olfactory epithelium, the antibody specifically recognized cilia of a small subset of olfactory receptor neurons, suggesting that the odour receptor protein is localized selectively in the cilia. Olfactory receptor neurons having immunoreactive cilia were distributed sparsely throughout the epithelium. This suggests that receptor neurons expressing a similar odour receptor protein are probably distributed similarly in the epithelium.

OCAM reveals segregated mitral/tufted cell pathways in developing accessory olfactory bulb.

Two functional subsets of vomeronasal sensory neurons project their axons to two segregated zones in the accessory olfactory bulb (AOB). Using immunohistochemical methods with antibodies against the novel cell adhesion molecule OCAM, we provide evidence that the segregation of functional pathways is maintained at the level of mitral/tufted (M/T) cells of the mouse AOB and that this pattern emerges early in ontogeny. During embryonic and postnatal development OCAM was strongly expressed by M/T cells in the caudal zone of the AOB where OCAM-negative vomeronasal axons terminated. In contrast, rostral zone M/T cells innervated by OCAM-positive vomeronasal axons displayed no or faint OCAM immunoreactivity. Differential expression of OCAM in segregated M/T cell pathways suggests that OCAM may be involved in defining compartments of connectivity and setting up functional subdivisions in the developing AOB.