Biomedical subjects

J Nunez

Publications and source records attributed to J Nunez.

At least 19 recordsLinked to original sources

PubMed1996

New forms of HMW MAP2 are preferentially expressed in the spinal cord.

The high molecular weight forms of microtubule-associated protein 2 (MAP2a and b) play a central role in the specification of dendrites. RT-PCR amplification of a portion of the N-terminal and middle MAP2b domains of rat spinal cord cDNAs allowed identification of new variants containing both exon 8 (246 bp) and a new exon, 7A (237 bp), located at the beginning of the middle MAP2b region. The brain and the spinal cord express transcripts containing exon 8, whereas exon 7A alone or exons 7A+8 were detected, whatever the developmental stage, only in the spinal cord.

Amino Acid Sequence↗

PubMed1996

Four repeat high-mol-wt MAP2 forms in rat dorsal root ganglia.

The high-mol-wt forms of brain microtubule-associated protein 2 (MAP2a/b) segregate within the dendrites during neuronal differentiation, whereas a low-mol-wt variant, MAP2c, is distributed within all the neuronal domains. Both MAP2b and MAP2c contain three tubulin binding repeats, whereas another low-mol-wt form, MAP2d, contains four repeats. Since high-mol-wt MAP2 species with four repeats have been cloned so far only from the sensory ND cell line, we have studied in this work the high-mol-wt forms expressed by dorsal root ganglia (DRG). Different clones obtained from PCR amplification products of portions of the C-terminal and of the 3' end of the middle MAP2b domains contained either three or four tubulin binding repeats at adult stages and only three postnatally. In adulthood, two exons located at the 3' end of the MAP2b middle domain were missing in several clones: exon 10 within clones with three or four repeats, exon 11 only within those containing four repeats. Several other clones obtained from PCR amplification products of portions of the N-terminal and of the 5' end of the middle MAP2b domains revealed exons 7A and 8. In contrast, Northern analysis revealed exon 8 but not exon 7A, which is probably expressed in trace amounts in the DRG. In this article, we have identified for the first time high-mol-wt MAP2 transcripts containing four tubulin binding repeats that seem to be expressed only by the DRG and also differing from brain MAP2b by a number of other exons.

Aging↗

PubMed1995

Tau and microtubule-associated protein 2c transfection and neurite outgrowth in ND 7/23 cells.

Neuronal hybrid ND 7/23 cells, which display sensorylike properties, develop neurites when cultured in the presence of either dibutyryl cyclic AMP plus nerve growth factor (DBcAMP + NGF) or retinoic acid or a phorbol ester derivative, although they express only trace amounts of the microtubule-associated tau proteins and low levels of microtubule-associated protein 2c (MAP2c). Nondifferentiated ND cells transfected with tau cDNAs did not develop neurites, whereas very short cell processes were formed in MAP2c-transfected cells. tau and MAP2 antibodies labeled microtubule bundles displayed in a ring array underneath the surface of the transfected cells and short microtubules starting from the cell center. After differentiation in the presence of DBcAMP + NGF, the same bundle organization was observed in the transfected cells. In addition, tau and MAP2 antibodies stained a short section of the formed neurites. These data demonstrate that the expression of tau protein is not sufficient to induce neurite extension and that other proteins induced by morphogens are more important to initiate morphological differentiation of this cell line.

Animals↗

PubMed1995

Tumor necrosis factor receptors in neuroblastoma SKNBE cells and their regulation by retinoic acid.

The human neuroblastoma cell line SKNBE can be differentiated either by serum removal or by adding to the culture medium different morphogens, for instance, retinoic acid (RA), cyclic AMP derivatives, and phorbol esters. Both the differentiated and undifferentiated cells express the two types of membrane tumor necrosis factor (TNF) receptors (TNFRs) of 55 and 75 kDa (p55 and p75 TNFR, respectively) and also their soluble forms. After RA addition the number of the surface TNFRs per cell is increased approximately twofold, but the kinetics of expression are different, depending on the receptor type. The level of the mRNAs of 2.4 and 4.2 kb, which, respectively, encode the p55 and p75 TNFRs, is also increased during the time course of differentiation, and the kinetics of their expression are biphasic. In contrast, the number of TNFRs and the level of their encoding mRNAs remain unchanged after exposure of the cells to both a phorbol and a cyclic AMP derivative.

Blood Physiological Phenomena↗

PubMed1995

Thyroid hormones and brain development.

Thyroid hormone is a major physiological regulator of mammalian brain development. Cell differentiation, migration and gene expression are altered as a consequence of thyroid hormone deficiency or excess. The physiological role of thyroid hormone can perhaps be defined so as to ensure the timed coordination of different developmental events through specific effects on the rate of cell differentiation and gene expression. All triiodothyronine (T3) receptor isoforms are expressed in the brain and their spatial and temporal patterns of expression suggest unique and complementary functions for the different isoforms. Cell biology studies suggest a role for T3 and its receptors in oligodendroglial and neuronal differentiation and the control of cell death. Some of the effects on neuronal differentiation might be due to an action of thyroid hormone on the production of neurotropins and their receptors. In recent years a number of T3-dependent genes have been identified in the rat brain, such as myelin protein-encoding genes or specific neuronal genes, and thyroid hormone-responsive elements have been demonstrated in some of these genes. The identification of the gene network regulated by thyroid hormone during brain development, the elucidation of the mechanism of regulation and the clarification of the physiological roles of the regulated genes remain major goals for future studies.

Animals↗

PubMed1995

Identification of a new exon of the brain microtubule-associated protein 2.

The 5'-region of the transcripts encoding the HMW-(MAP2b) and LMW-(MAP2c) microtubule associated proteins in the brain was amplified by RT-PCR. The sequencing of cloned PCR fragments allowed to identify a new variant of brain HMW-MAP2 which contained, compared to MAP2b, an insertion of 246 bp located downstream the 5'-junction between MAP2b and MAP2c and that does not alter the open reading frame of MAP2b. The number of amino acid residues encoded by this insertion increases the molecular weight by 8.5 kDa i.e. corresponds to the difference in apparent size between MAP2a and MAP2b. A LMW-MAP2 PCR amplification product containing this insertion has been also identified. Genomic Southern blot analysis confirmed that this region belongs to the MAP2 gene and is located on a single exon.

Amino Acid Sequence↗

PubMed1994

Two novel HMW MAP2 variants with four microtubule-binding repeats and different projection domains.

The brain microtubule-associated protein MAP2 is composed of two high molecular (MAP2a and b) and one low molecular (MAP2c) weight isoforms. All these forms were thought to contain three repeated microtubule-binding domains in their C-terminal region but a MAP2c variant containing four repeats has recently been identified. We report here the existence of two high molecular weight MAP2 isoforms with four microtubule-binding domains in the sensory neuronal cell line ND 7/23. A stretch of 135 bp is missing in one of these forms suggesting that several HMW MAP2 variants can be produced by alternative splicing.

Alternative Splicing↗

PubMed1994

Tear lactoferrin levels and ocular bacterial flora in HIV positive patients.

Keratoconjunctivitis Sicca(4) has recently been reported to occur at a greater rate in HIV-positive symptomatic patients. We looked at HIV positive asymptomatic patients, compared to age matched HIV negative patients to study external ocular resistant factors, namely lactoferrin levels in tears, bacterial flora in lid margins, conjunctiva and tears, and evidence of dry eyes using a Schirmer test and tear osmolarity. Eighteen eyes of nine HIV positive patients and eighteen eyes of HIV negative controls were studied. Results showed markedly decreased lactoferrin levels in HIV positive asymptomatic patients with a mean of 85.8 mgs/dcl compared to HIV negative patients with a mean 156 mgs/dcl (P < 0.01). There were increased numbers of colonies of bacterial flora on the lids of HIV positive asymptomatic patients with an average colony count 4.1 colonies/patient compared to 1.5 colonies/patients in the control group (P < 0.025). Seventy eight percent of the study group had bacterial growth compared to 33% in the control group. The tear osmolarity in both groups had no significant difference; mean in HIV positive being 312 mosml/litre; mean in control 306 mosml/litre. The Schirmer test also showed no significant difference, with the mean in HIV positive patients being 11 mm wetting, and in control patients being 12.7 mm wetting. Therefore, despite no symptomatic or clinical evidence of dry eyes, asymptomatic HIV-positive patients had markedly decreased levels of lactoferrin in tears and increased colony counts of bacterial flora in the lids.

Acquired Immunodeficiency Syndrome↗

PubMed1994

High molecular weight tau distribution and microtubule stability in neuroblastoma N115 cells.

The localization of high molecular weight (HMW) tau proteins in neuroblastoma N115 cells and of their transcripts was compared to that of non-tyrosinated and tyrosinated tubulin before and after treatment with depolymerizing drugs. Microtubules stained by tau antibodies were present both in a limited region of the cell center and in the cell processes, whereas tau transcripts were detected only in the cell body. The microtubules localized in the cell center and labeled by tau antibodies resisted colcemid treatment, whereas those in the neurites were completely depolymerized by the drug. Microtubules containing stable and unstable microtubule tracts were identified in the neurites after colcemid treatment. These composite microtubules were not labeled by tau antibodies. It is concluded that stable and unstable polymers--localized in the cell center and in the neurites, respectively--contain HMW tau proteins, whereas composite microtubules displayed in the cell processes do not. Microtubule stability in this cell line does not therefore seem to be related to the association of tau proteins to the polymers but, rather, to posttranslational modifications of the tubulin subunits.

Animals↗

PubMed1994

Diversity of high-molecular-weight tau proteins in different regions of the nervous system.

We show in this work that high-molecular-weight (HMW) tau transcripts are present in all the regions of the CNS and PNS studied, i.e., the dorsal root ganglia (DRG), spinal cord, cerebellum, and forebrain. However, the relative amount of HMW and low-molecular-weight (LMW) tau variants and their sequence vary depending on the region. Two HMW tau variants that contain either both exons 4A and 6 or only exon 6 have been identified in the adult spinal cord by PCR amplification and sequenced. In contrast, a single HMW tau variant that contains exon 4A but not exon 6 was detected in the adult rat DRG. This means that at least part of the HMW tau expressed in the spinal cord is produced locally and not transported within this structure by fibers arising in the DRG. The expression of the HMW tau isoforms is developmentally regulated both quantitatively and qualitatively in the spinal cord. At immature stages very low levels of the HMW tau transcript containing both exons 4A and 6 are expressed, whereas the tau species containing only exon 6 is absent. Rat forebrain, rat cerebellum, and human forebrain express much lower levels of HMW tau transcripts compared with the spinal cord and the DRG. Their sequence contains both exons 4A and 6, i.e., is identical to that of the major HMW tau transcript detected in the adult rat spinal cord. The minor spinal cord species that contains only exon 6 was not identified in the rat forebrain and rat cerebellum but was present among the human brain PCR fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Heterogeneity of the high molecular weight tau proteins in N115 neuroblastoma cells.

The sequence of a high molecular weight (HMW) tau cDNA cloned from a neuroblastoma N115 library contains, in addition to the C- and N-terminal and middle regions present in the low molecular weight mouse brain tau proteins, a 711-bp nonhomologous domain (exon 4a) and a region of 198 bp corresponding to exon 6 of the tau gene. Protein immunoblot analysis, performed with antibodies specific either for a sequence present in the N-terminal region of all the tau variants or for exon 4a revealed several bands suggesting that more than one tau form is expressed in this cell line. Northern blot experiments performed with a number of cDNA probes spanning domains common and uncommon to low molecular weight and HMW tau allowed the identification of four tau transcripts differing in the size of their coding and noncoding regions. All these transcripts contain the sequence encoded by exon 6, but two of them lack exon 4a. As shown by RNase protection assays, the N-terminal region of these transcripts is also variable and contains either exon 1, or exons 1 and 2, or exons 1-3. Yet all these HMW tau forms contain four homologous repeats in their C-terminal domain both in the differentiated and nondifferentiated cells, i.e., have adult characteristics. In conclusion, the data reported in this article demonstrate that several HMW tau variants are expressed in neuroblastoma N115 cells and that the transition between immature to mature tau forms occurring during brain development is not required for neurite outgrowth during morphological differentiation of this cell line.

Animals↗

PubMed1993

High and low molecular weight tau proteins are differentially expressed from a single gene.

Both high and low molecular weight (HMW and LMW) tau proteins are expressed in the immature and adult mouse spinal cord. Northern blot analysis, performed with probes complementary to domains common and uncommon to the LMW and HMW entities, suggested that HMW tau proteins found in the immature mouse spinal cord are not translated from the single transcript of 6 kb expressed at these stages, but are transported within this nervous structure by axons arising in the periphery. In contrast, another minor transcript of 8 kb was detected in the adult mouse spinal cord by a HMW tau specific probe, suggesting that a small fraction of the HMW tau forms present in adulthood are translated within mouse spinal cord neurons. LMW spinal cord tau forms are encoded by mRNAs of 6 kb that contain three and four homologous repeats at immature and mature stages, respectively, whereas adult HMW entities contain four repeats. PCR analysis performed with mouse genomic DNA also showed that the nonhomologous region specific for HMW tau is a single exon. Southern blot and gene mapping showed that the same gene, located on the murine chromosome 11, encodes all the LMW and HMW tau variants. All these tau forms, therefore, are produced by an alternative splicing mechanism that is neuron-specific and developmentally regulated.

Animals↗

PubMed1993

Expression of high molecular weight tau in the central and peripheral nervous systems.

Using a novel PCR approach, we have cloned a cDNA encoding the entire high molecular weight tau molecule from rat dorsal root ganglia. The resulting 2080 bp cDNA differs from low molecular weight rat brain tau by the insertion of a novel 762 bp region (exon 4a) between exons 4 and 5. This cDNA clone is identical in sequence with a high molecular weight tau (HMW) cDNA from rat PC12 tumor cells and is closely related to a HMW tau cDNA from mouse N115 tumor cells. In vitro transcription/translation produces a protein that migrates on SDS-PAGE with the same apparent molecular weight as HMW tau purified from rat sciatic nerve. The HMW tau protein is generated from an 8 kb mRNA, which can be detected by northern blots in peripheral ganglia, but not in brain. A more sensitive assay using PCR and Southern blot analysis demonstrates the presence of exon 4a in spinal cord and in retina. In combination with immunohistochemical studies of spinal cord, these data suggest that HMW tau, though primarily in the peripheral nervous system, is also expressed in limited areas of the central nervous system, although its presence cannot be detected in the cerebral cortices.

Amino Acid Sequence↗

PubMed1993

Characterization of a PC12 cell sub-clone (PC12-C41) with enhanced neurite outgrowth capacity: implications for a modulatory role of high molecular weight tau in neuritogenesis.

To address the means by which diversity of neuronal morphology is generated, we have isolated and characterized naturally occurring variants of rat PC12 pheochromocytoma cells that exhibit altered neurite outgrowth properties in response to nerve growth factor (NGF). We describe here a PC12 cell sub-clone, designated PC12-clone 41 (PC12-C41), that displays significant increases in neurite abundance and stability when compared with the parental line. This difference does not appear to be due to an altered sensitivity or responsiveness to NGF or to a more rapid rate of neurite extension. Because of the role of the cytoskeleton in neuritogenesis, we examined a panel of the major cytoskeletal proteins (MAP 1.2/1B, beta-tubulin, chartins, peripherin, and high and low molecular weight (HMW and LMW) taus) whose levels and/or extent of phosphorylation are regulated by NGF in PC12 cultures. Although most cytoskeletal proteins showed little difference between PC12 and PC12-C41 cells (+/- NGF treatment), there was a significant contrast between the two lines with respect to tau expression. In particular, while NGF increases the total specific levels of tau in both cell types to similar extents (by about twofold), the proportion comprising HMW tau is threefold higher in the PC12-C41 clone than in PC12 cells. A comparable difference was observed under substratum conditions that were non-permissive for neurite outgrowth and so this effect was not merely a consequence of the differential neuritogenic capacities of the two lines. The distinction between the expression of HMW and LMW taus in PC12 and PC12-C41 cells (+/- NGF) was also observed at the level of the messages encoding these proteins. Such findings indicate that initiation of neurite outgrowth in PC12 cultures does not require a massive induction of tau expression and raise the possibility that HMW and LMW taus may have differential capacities for modulating neuronal morphology.

Animals↗

PubMed1993

[High molecular weight tau proteins and acquisition of neuronal polarity in peripheral nervous system].

Several variants of the microtubule-associated tau proteins, are expressed during brain development and in adulthood. These entities are required to define the polarity of the neuron and the architecture of the axon but differ in sequence and in their microtubule polymerizing activity. Here, we describe a new group of high molecular weight tau proteins that contain one or two additional exons of 711 and 198 bp in their middle region and a variable N-terminal domain. These high molecular weight tau variants are preferentially expressed in the peripheral nervous system. Immunohistochemical studies showed that they are also present in the dorsal horn of the spinal cord where they are probably transported by sensory fibers arising in the periphery. However, a minor fraction of these proteins is present in the motor neurons of the ventral horn. Similar studies were performed with the neuroblastoma N115 cell line which can be differentiated in vitro and expresses only high molecular weight tau forms. In the non differentiated cells, tau antibodies label the domain of the cell body localized around the centrosome whereas, after differentiation, the cell process facing this structure is also stained. These data suggest that axonal polarity is predetermined by the localization of tau proteins in the domain of the cell body defined by the centrosome.

Animals↗

PubMed1993

Analgesia for ESWL.

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Administration, Rectal↗

PubMed1992

Primary structure of high molecular weight tau present in the peripheral nervous system.

The tau proteins are a family of brain microtubule binding proteins that are required during axonal outgrowth and are found in neurofibrillary tangles in Alzheimer disease. A protein of higher molecular weight, immunologically related to tau, is expressed in the adult peripheral system and in cultured neuronal cell lines of neural crest origin. The predicted amino acid sequence of the high molecular weight tau from N115 cells has been determined from the sequence of its 2340-base-pair cDNA. High molecular weight tau contains an open reading frame encoding 733 amino acid residues. It contains sequences homologous to those present in the N-, middle, and C-terminal domains of adult brain tau proteins, including four homologous repeats, which are the tubulin binding sites, and an amino acid stretch, which is present only in the N-terminal domain of the mature brain variants. The middle region contains a previously unidentified nonhomologous stretch of 237 amino acid residues as well as a domain of 66 residues homologous to exon 6 of the bovine gene that is absent in all bovine, rat, and mouse tau cDNAs sequenced so far. A cDNA probe specific to the nonhomologous tau insert hybridizes to the 8- to 9-kilobase tau mRNA in N115 cells but not to the 6-kilobase tau mRNA in brain. Probes for the domains common to brain tau isoforms hybridize to both messages. The sequence of high molecular weight tau protein also suggests that it, like low molecular weight tau, is an elongated hydrophilic molecule. This cDNA should allow us to study the role of the domains specific to these tau forms in the specialization of the peripheral nervous system and for study of their expression in normal and pathological states.

Amino Acid Sequence↗

PubMed1992

Regulation of tubulin, Tau and microtubule associated protein 2 expression during mouse brain development.

The level of three microtubule proteins, tubulin, Tau and MAP2 and of their encoding mRNA was studied in the mouse brain at an early developmental stage (3 days postnatal) and in adulthood. The level of the mRNA encoding both tubulin and Tau decreased by 85% between these two stages whereas the encoded proteins decreased only by 50% during the same period. Thus, the level of these proteins seems to be regulated both negatively and positively by transcriptional and post translational mechanisms. In vitro transcription assays, performed with nuclei isolated at different postnatal stages, showed that the tubulin and Tau transcripts are produced with some variations during mouse brain development. However these fluctuations are much less important than the drops of the steady state levels of tubulin and Tau mRNA seen in vivo. Thus, the decrease in transcripts levels does not seem to result from reduced transcriptional activities, and can be ascribed to changes in mRNA stability occurring during brain development, i.e. to a post transcriptional mechanism. The situation is even more complex for MAP2: its encoding mRNA level remains constant during development whereas the in vitro transcription activity decreases markedly during the same period. Finally, MAP2 protein level increases during development although its encoding mRNA level remains constant suggesting that this protein is stabilized by a post translational mechanism.

Aging↗