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D I Gottlieb

Publications and source records attributed to D I Gottlieb.

At least 19 recordsLinked to original sources
PubMed1990

Characterization of a cDNA coding for rat glutamic acid decarboxylase.

cDNA clones have been isolated for rat glutamic acid decarboxylase (glutamate decarboxylase; EC 4.1.1.15) (GAD) and 3216 bp of the sequence have been determined. This sequence extends the previously reported feline GAD cDNA sequence both in the 5' (67 bp) and 3' (887 bp) directions and contains the polyadenylation signal and tail. The cDNA codes for a 67 kDa mol. wt. protein beginning from the putative initiator methionine found in the feline sequence. Extensive homology to feline GAD was identified at the amino acid level (97% identity) within the coding region. This interspecies homology is high compared to other neurotransmitter synthesizing enzymes and suggests selective pressure to maintain the primary sequence throughout the full length of the protein. Homology is found 5' to the putative initiator methionine. Extensive stretches of homology are also found in the 3' non-coding region. These conserved non-coding regions may play a role in GAD mRNA regulation. The rat cDNA sequence will facilitate investigations into the structure and regulation of the GAD gene.

Amino Acid Sequence↗
PubMed1990

Localization of the NGFI-A protein in the rat brain.

Antibodies are used to localize the NGFI-A protein in the rat brain. The protein is found in a wide variety of neurons. However, not all neurons are stained. The protein is either absent or present at undetectable levels in glial cells. Neuronal nuclei stain intensely, cytoplasmic staining is lighter. Seizures cause a detectable increase in the intensity of staining.

Amino Acid Sequence↗
PubMed1990

Developmentally regulated expression of an exon containing a stop codon in the gene for glutamic acid decarboxylase.

In the adult rat brain, the gene for glutamic acid decarboxylase (GAD; L-glutamate 1-carboxy-lyase, EC 4.1.1.15) is expressed predominantly as a 3.7-kilobase transcript. Earlier data showed that embryonic brain expresses an RNA transcript distinct from the adult form; however, the exact structure of this form was not elucidated. Here, transcripts expressed in the embryonic but not the adult brain were cloned and analyzed. These transcripts include an exon not expressed in the adult inserted into coding sequence. The embryonic exon contains a stop codon that is in-frame with the coding sequence. The exon is found in genomic DNA within the GAD gene where it is flanked by introns with conventional splice sites. On the basis of these structural data, we propose the hypothesis that, early in brain development, transcripts encoding a truncated form of GAD are expressed. The deduced protein cannot function as a decarboxylase because the stop codon in the embryonic exon occurs upstream of the binding site for pyridoxal phosphate, an essential cofactor. Thus, alternative splicing plays a crucial role in the pathway leading to the development of functional GABAergic neurons. The central nervous system-derived cell lines B65 and C6 express a mixture of the adult and embryonic forms of GAD mRNA. They therefore are useful clonal models of central nervous system cells in the early phases of differentiation.

Age Factors↗
PubMed1988

GABAergic neurons.

One tends to think of the nervous system in terms of chains of excitatory signals that tell neurons to fire. The signals that say "Don't fire" also have a major role. Inhibitory signals damp overall neural activity and also fine-tune the responses of particular circuits. The primary carrier of these signals is the inhibitory transmitter known as GABA.

Animals↗
PubMed1988

Pattern of expression of glutamic acid decarboxylase mRNA in the developing rat brain.

The time and pattern of appearance of glutamic acid decarboxylase (glutamate decarboxylase; EC 4.1.1.15) (GAD) mRNA during the development of the rat brain were analyzed. RNA transfer blot analysis of poly(A)+ RNA from whole brain shows that a 3.7-kilobase transcript is the most abundant form of the message from embryonic day 15 (E15) through adulthood. By E15 this form is present at about 50% of its adult abundance relative to other poly(A)+ mRNA species. At birth the abundance is approximately the same as in the adult. In contrast, the enzyme activity level is only 8% of the adult level at birth and takes 3 weeks to reach adult levels. There are qualitative changes in GAD mRNA during development. Several large (7-9 kilobases) transcripts with strong homology to GAD are enriched in early developmental stages but are barely detectable in the adult. A nuclease protection assay shows a developmentally regulated heterogeneity in a coding portion of the mRNA.

Animals↗
PubMed1988

Purification and characterization of an antigen that is spatially segregated in the primary olfactory projection.

The monoclonal antibody RB-8 heavily labels axons from the ventrolateral olfactory epithelium and their terminals in the glomeruli of the ventrolateral olfactory bulb, but leaves the axons from the dorsomedial epithelium unstained or lightly stained. RB-8 reacts with a 125 kDa membrane protein in both olfactory nerve and other parts of the CNS (Schwob and Gottlieb, 1986). Here we report further characterization of the molecular nature and cellular localization of the RB-8 antigen. The RB-8 antigen is exposed on the surface of olfactory axons. Individual axons and axon bundles stain when explant cultures of the fetal olfactory epithelium are incubated with monoclonal RB-8 antibody while living. The cell membrane is demonstrably intact, and access to the cell interior is blocked under these conditions, since the living axons do not stain if exposed to an antibody against a known intracellular constituent. The RB-8 antigen is an integral membrane protein. When assayed by direct radioimmunoassay (RIA), the antigen remains associated with brain membranes after extraction at pH 11, which solubilizes numerous other protein bands. The 125 kDa RB-8 antigen was purified to homogeneity from whole rat brains by extracting membranes with sodium deoxycholate, immunoaffinity chromatography over an RB-8 antibody column, and preparative one-dimensional SDS-PAGE. The NH2-terminal amino acid sequence is apparently unique among neuron-specific proteins that have been sequenced and has only an insignificant degree of homology with other known proteins. Two polyclonal rabbit antisera raised against the purified antigen recognize only the 125 kDa protein on immunoblots. Immunohistochemical staining of the primary olfactory projection with the antisera exactly matches that seen with monoclonal RB-8 antibody. Thus, the RB-8 antigens in brain and in olfactory nerve are highly homologous, if not identical. Furthermore, the results with the antisera suggest that the expression of the entire 125 kDa protein is regulated differentially between ventral and dorsal zones of the olfactory epithelium. The additional characterization of the RB-8 antigen reported here places constraints on the potential functions of this protein. The availability of polyclonal antisera may prove useful in assessing the role of this spatially segregated antigen in the primary olfactory projection.

Amino Acid Sequence↗
PubMed1988

Characterization of the proteins purified with monoclonal antibodies to glutamic acid decarboxylase.

Immunoaffinity columns are prepared from the monoclonal antibody (MAb) GAD-1. These columns are used to enrich glutamic acid decarboxylase (GAD) from the cytosolic fraction of rat brain homogenates and from Triton X-100 extracts of the brain membrane fraction. In each case enzyme activity is enriched over 400-fold. The immunopurified fractions were analyzed by SDS-PAGE. Fractions purified from the cytosol consisted of a quantitatively major band of 59 kDa, and one band of 63 kDa, as well as a group centered around 55 kDa. Fractions purified from membranes consisted primarily of the 59 and 63 kDa components; only traces of the lower-molecular-weight components were present. The entire set of proteins purified on GAD-1 immunoaffinity columns is strongly recognized by 2 widely used antisera to GAD, those described in Saito et al. (1974) and Oertel et al. (1981). The 59 kDa protein from the cytosolic fraction was purified to homogeneity by preparative SDS-PAGE; a partial amino acid sequence of this protein was obtained. The 59 kDa protein has a high degree of sequence homology with the deduced amino acid sequence of the protein that was coded for by a cDNA for feline GAD (Kaufman et al., 1986; Kobayashi et al., 1987). Thus, these proteins are either products of a single gene that diverged during the evolution of rat and cat from a common ancestor, or are members of a closely related set of genes found in both species. The MAb GAD-6 recognizes the 59 kDa band and the group of bands centered around 55 kDa on Western blots. Therefore, these proteins are immunochemically related. GAD-6 does not recognize the 63 kDa band. In Western blots of unfractionated homogenates of the whole brain, the only band recognized by GAD-6 is a 59 kDa band.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Monoclonal antibodies to glutamic acid decarboxylase.

Five monoclonal antibodies that recognize chicken brain glutamic acid decarboxylase (GAD) have been selected and designated GAD-1 to -5. GAD-1 to -5 were selected on the basis of their ability to immunoprecipitate active GAD from crude brain extracts. GAD-1 recognizes an epitope that is conserved in many vertebrates; the epitope recognized by GAD-5 is restricted to the chicken. Radioimmunoassays with GAD-1 indicate that GAD is highly enriched in brain relative to other tissues. GAD was localized immunocytochemically with GAD-1 and GAD-2 in rat cerebellum, spinal cord, and retina. The staining pattern is in agreement with that obtained previously with polyclonal antisera to GAD. GAD from the chicken brain was purified by chromatography on an immunoaffinity column made of GAD-1. NaDodSO4/PAGE analysis of the immunoaffinity-purified GAD fractions shows a major band of 59 kDa and minor bands at 63 and 54 kDa.

Animals↗
PubMed1986

The primary olfactory projection has two chemically distinct zones.

The sensory neurons of the olfactory epithelium form an anatomically uniform population but are differentially excited by odorants. We have discovered an unexpected biochemical heterogeneity within this population that extends to its axonal projection onto the olfactory bulb. This heterogeneity is recognized by a newly generated monoclonal antibody, designated RB-8, that differentially stains the primary olfactory projection in rats and divides it into 2 nonoverlapping zones. With light-microscopic immunohistochemistry, RB-8 densely labels the fascicles of the olfactory nerve from the ventral and lateral parts of the olfactory epithelium, where there is also some epithelial staining. This area, which we designate RB-8-positive, comprises about two-thirds of the epithelial sheet. RB-8 labeling of the other third of the epithelium, which includes the dorsal recess and medial tips of the dorsal turbinals, is not detectable, and the fascicles from these RB-8-negative areas are only weakly stained. These RB-8-negative areas form a contiguous zone on flattened maps of the epithelial sheet. In the olfactory bulb, RB-8 staining of the glomeruli in the ventrolateral part is correspondingly dense, while that in the dorsomedial glomeruli is undetectable or very light. In the labeled glomeruli, the RB-8 staining is precisely coextensive with anti-olfactory marker protein staining, which serves as a marker for the olfactory axons and terminals. In addition, knife-cut lesions of the olfactory nerve totally eliminate the RB-8 staining in the glomeruli where the destruction of the olfactory terminals is complete. There is also a good correlation between the staining patterns in the bulb and epithelium and what is known from tract-tracing studies of the arrangement of the axonal projection of the epithelium onto the bulb. This evidence strongly suggests that, in the olfactory nerve and glomeruli, RB-8 stains the olfactory axons and their terminals. A survey of the CNS and peripheral tissues demonstrates that staining with RB-8 is nervous system-specific; not all components of the CNS and PNS are stained. The antigen recognized by RB-8 was characterized in immunoblots and by use of a direct radioimmunoassay (RIA) which assessed binding of 125I-RB-8. With this assay, the RB-8 binding sites in whole brain are shown to be membrane-associated, saturable, immunologically specific for RB-8, and trypsin-sensitive. In SDS-PAGE immunoblots of membrane proteins, the antigen in rat forebrain and in the olfactory nerve is a protein of 125 kDa Mr, which comigrates in mixtures of membranes from the 2 sources.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Neurons of the olfactory epithelium in adult rats contain vimentin.

In the developing nervous system, the intermediate filament protein vimentin is found in the proliferating neuroepithelium and neural crest. As development proceeds, postmitotic neurons cease vimentin expression and neurofilament proteins begin to accumulate. We have shown that olfactory receptor neurons deviate from the general pattern of neuronal intermediate filament expression, in that they continue to express vimentin or a highly vimentin-like protein rather than neurofilament proteins in the adult rat. With light-microscopic immunohistochemistry, three independently derived antibodies to vimentin label all portions of the primary olfactory projection, including the sensory neuron cell bodies in the olfactory epithelium, the fascicles of the olfactory nerve, and their axonal arbors in the glomeruli of the olfactory bulb. In contrast, anti-neurofilament antisera stain only rare scattered receptor cells and a small number of axons in the olfactory nerve. Electron-microscopic immunohistochemistry shows dense staining of olfactory axons with anti-vimentin. The vimentin-like immunoreactive material in the olfactory nerve layer was characterized by SDS-PAGE and by immunoblotting. On immunoblots of homogenates of the olfactory nerve, the anti-vimentin monoclonal antibody SBV-21 (Blose et al., 1984) stains only a single protein of Mr = 55 kDa. This band comigrates with vimentin in crude cytoskeletal material from the neonatal rat brain prepared according to the method of Dahl et al. (1981). SBV-21 does not stain neurofilament triplet proteins or glial fibrillary acidic protein, which are also present in these blots. These results demonstrate that the vast majority of olfactory receptor neurons and their axons contain vimentin or a protein of similar immunological character and electrophoretic mobility, while identifiable expression of neurofilament proteins is confined to a very small subpopulation. Hence, the switch in intermediate filament proteins that normally accompanies neuronal maturation is arrested in most olfactory neurons, and a "juvenile" biochemical marker is retained. This population of neurons is also unique among mammalian neurons in several other respects, including that olfactory neurons die during normal adult life or following injury and then are replaced from a proliferating pool of stem cells.

Age Factors↗
PubMed1985

NMB: a human neuroblastoma cell line with specific opiate binding sites.

The human neuroblastoma cell line designated NMB (Brodeur et al., 1977, Cancer 40: 2256) has been shown to have specific opiate binding sites. These sites are highly stereospecific. Two characteristic delta specific peptides, D-Ala2-D-Leu5 enkephalin and D-Thr2-D-Thr6 enkephalin, have high affinity for the binding sites. Morphine binds specifically but with a much lower affinity. Dextrorphan and the mu specific peptide morphiceptin (Tyr-Pro-Phe-Pro-CO-NH2) do not bind to the site. The binding sites are heat and trypsin sensitive. Sodium ions specifically lower agonist binding to the sites. Approximately 14,000 binding sites per cell are found. The binding characteristics of these sites are very similar to those of the delta sites characterized on mouse neuroblastoma cell lines.

Binding Sites↗
PubMed1985

Opiate binding sites in the chick, rabbit and goldfish retina.

The characteristics of opiate binding sites in the retina of the chick, rabbit and goldfish have been investigated. In the newly hatched chick retina, 131 fmol/mg of binding sites for [D-Ala2-D-Leu5]-[3H]enkephalin are present; competition studies with the delta selective peptide [D-Thr-Leu5]-enkephalin (DTLET) and the mu selective peptide morphiceptin show that all of the [D-Ala2-D-Leu5]-[3H]-enkephalin binding sites are of the delta subtype. Dihydro[3H]morphine binds poorly to the chick retina; 13.2 fmol/mg of this binding is displaceable by morphiceptin and corresponds to mu binding sites. Benzomorphan sites are defined as sites occupied by [3H]diprenorphine which is displaceable by low concentrations of ethylketocyclozacine but not by high concentrations of D-Ala2-D-Leu5-enkephalin and morphiceptin. At least 88 fmol/mg of benzomorphan sites are present in the chick retina. [3H]diprenorphine binding to the rabbit and fish retina was measured. The rabbit retina bound 60 fmol/mg, and the fish retina 42 fmol/mg of [3H]diprenorphine. These findings are discussed in the light of the studies on the localization and physiological effects of enkephalin in the retina.

Animals↗
PubMed1985

Immunocytochemical localization of 140 kD cell adhesion molecules in cultured chicken fibroblasts, and in chicken smooth muscle and intestinal epithelial tissues.

A monoclonal antibody (JG22 MAb) that was previously raised to a chick embryo myogenic cell preparation had been shown to produce rounding and other morphological changes in myogenic cells in culture, and, in some cases, their detachment from the substratum. In other studies it was shown that the epitope recognized by JG22 was associated with a set of 140 kD cell surface glycoproteins. It is shown that this antigen occurs in a wide variety of cell types; in cultured fibroblasts, it is distributed equally between the dorsal and ventral cell surfaces shortly after plating, but appears to become concentrated on the ventral surface as cell spreading proceeds; by immunoelectron microscopic labeling experiments, it is absent from the focal adhesion contact sites formed by fibroblasts with their substrata and with one another, but is present in clusters at the edge of focal adhesions, and within the close contact sites and extracellular matrix contact sites; in smooth muscle cells, it is absent from the membrane-associated dense plaques, but is located in clusters at adjacent membrane sites; in intestinal epithelium, it is present in clusters at the basolateral membranes, but not at the microvilli or within junctional complexes of the brush border of the cell layers. These and other results are consistent with the suggestion that the antigen recognized by JG22 MAb is important cell adhesion molecules, and performs a characteristic function in a variety of cell-cell contacts and cell adhesions.

Animals↗
PubMed1977

Studies on cell recognition in the developing brain.

Several lines of evidence demonstrate cell-cell receptors on the surface of developing brain cells. Plasma membrane vesicles with regional and temporal binding specificities can be prepared. Active factors that block cell aggregation can be extracted from these membranes and partially purified. Quantitative studies of cell-cell adhesion demonstrate a gradient of adhesive specificity along the dorsoventral axis of the developing retina.

Animals↗