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N Aronin

Publications and source records attributed to N Aronin.

At least 37 records · Page 2Linked to original sources
PubMed1997

Dde-I restriction endonuclease fragmentation: a novel method of generating cDNA probes for in situ hybridization in brain.

We present a novel procedure for detection of low- and high-abundance messenger RNAs in the brain by in situ hybridization histochemistry, by using fragmented double-stranded cDNA as molecular probes. The procedure involves digesting the cDNA of interest with the restriction endonuclease from Desulfocibrio desulfuricans (Dde I digestion), followed by random primed labeling, which generates a family of high specific activity cDNA fragments. This procedure is a rapid, straightforward, and reproducible method of obtaining sensitive probes for in situ hybridization and is generally applicable to the analysis of the expression of a large number of genes. Here we report the use of this procedure to prepare probes for the detection of synapsin I, p150Glued, neurotensin, c-fos, and c-jun mRNAs in brain, using both isotopic and non-isotopic labeling methods. Because this procedure does not require complex recombinant DNA manipulations or oligonucleotide design, it should prove useful to the non-molecular biologist examining the expression of genes in the central nervous system.

Animals↗
PubMed1997

A kindred with a variant of multiple endocrine neoplasia type 1 demonstrating frequent expression of pituitary tumors but not linked to the multiple endocrine neoplasia type 1 locus at chromosome region 11q13.

Acromegaly is uncommon in kindreds with multiple endocrine neoplasia type 1 (MEN1), whereas primary hyperparathyroidism (PHP) has the highest penetrance of any endocrinopathy. We report an unusual MEN1 kindred with frequent expression of pituitary tumors and a low penetrance of PHP. Four members were found to have disease: PHP in generation I, acromegaly (2 cases) in generation II, and hyperprolactinemia associated with a pituitary tumor in generation III. There was no evidence for PHP in 1 patient with acromegaly (age 60 yr), the patient with hyperprolactinemia and the pituitary tumor (age 22 yr), and 1 asymptomatic obligate carrier (age 50 yr). Screening of 26 members revealed the possible diagnosis of PHP in 1 family member in generation II and possible early acromegaly in 2 members of generation III with elevated serum concentrations of insulin-like growth factor I and insulin-like growth factor-binding protein-3 but normal patterns of pulsatile GH release. Although the predisposing genetic defect in typical MEN1 families has previously been mapped to chromosome location 11q13 without evidence of heterogeneity among the 87 families analyzed, linkage of disease in this family to the MEN1 region is unlikely based on haplotype analysis. Localization of the gene(s) responsible for disease in such atypical families may aid in the understanding of the pathogenesis of MEN1. In addition, further study of the earliest changes in patterns of pulsatile GH release in familial acromegaly may allow more insight into the pathogenesis and natural history of this disease.

Acromegaly↗
PubMed1996

Expression of normal and mutant huntingtin in the developing brain.

Huntington's disease (HD) is caused by a genetic mutation that results in a polyglutamine expansion in huntingtin. The time course of neuronal loss in the HD striatum and other affected brain regions before the onset of symptoms is unknown. To determine the potential influence of huntingtin on brain development, we examined its expression in the developing mouse and in human control and HD brain. By Western blot, huntingtin was detected throughout the adult mouse brain and at all stages of embryonic and postnatal brain development. The protein increased significantly between postnatal day 7 (P7) and P15, which marks a period of active neuronal differentiation and enhanced sensitivity to excitotoxic injury in the rodent striatum. Immunoreactivity was found in neurons throughout the brain and localized mostly to the somatodendritic cytoplasm and to axons in fiber bundles. Staining was variable in different groups of neurons and within the same cell population. In developing brain, huntingtin was limited primarily to neuronal perikarya. Increased immunoreactivity in large neurons followed the gradient of neurogenesis and appeared in the basal forebrain and brainstem by embryonic days 15-17, in regions of cortex by P0-P1, and in the striatum by P7. In human brain at midgestation (19-21 weeks), huntingtin was detected in all regions. The brain of a 10-week-old infant with the expanded HD allele expressed a higher molecular weight mutant form of huntingtin at levels comparable to those of the wild-type protein. Thus, mutant huntingtin is expressed before neuronal maturation is complete. Results suggest that huntingtin has an important constitutive role in neurons during brain development, that heterogeneity in neuronal expression of the protein is developmentally regulated, and that the intraneuronal distribution of huntingtin increases in parallel with neuronal maturation. The presence of mutant huntingtin in the immature HD brain raises the possibility that neurons may be affected during brain development and possibly in the postnatal period when vulnerability to excitotoxic injury is at its peak.

Animals↗
PubMed1996

c-Fos expression in the rat intergeniculate leaflet: photic regulation, co-localization with Fos-B, and cellular identification.

Ambient light alters the level of the transcriptional regulatory protein c-Fos in the suprachiasmatic nucleus, the site of an endogenous circadian clock in mammals, and in one other retino-recipient area, the intergeniculate leaflet of the lateral geniculate complex. Complementing previous work by ourselves and others on the photic and temporal regulation of c-Fos expression in the suprachiasmatic nucleus, the present studies investigated c-Fos regulation in the rat intergeniculate leaflet, revealing some important differences between the two brain regions. In the intergeniculate leaflet, the levels of c-fos mRNA (by in situ hybridization) and immunoreactive c-Fos protein (by immunohistochemistry) were elevated by light pulses administered either during the subjective day or subjective night. The regulation of immunoreactive Fos-B protein was similar to c-Fos, and 98% of Fos-B-expressing cells were also c-Fos-positive (by double-label immunofluorescence). By combining c-Fos immunofluorescence with stereotaxic injections of the retrograde tract tracer FluoroGold, we found photically-induced c-Fos in 15% of intergeniculate leaflet neurons projecting to the suprachiasmatic nucleus and in 34% of those projecting to the contralateral intergeniculate leaflet. Intergeniculate leaflet cells that express c-Fos after photic stimulation appear to represent a functionally-defined population that does not correspond to anatomically-defined categories based on connectivity or peptidergic phenotype.

Animals↗
PubMed1996

In vitro stimulation of c-Fos protein expression in the suprachiasmatic nucleus of hypothalamic slices.

Light regulates the c-fos protooncogene in the suprachiasmatic nucleus (SCN), with increased expression after animals are exposed to light during the dark phase of a light-dark cycle or during the subjective night in constant darkness. To determine whether this phase-dependent activation of c-Fos persists in an acute in vitro preparation, we prepared horizontal slices of hamster ventral hypothalamus, electrically stimulated the still-attached optic nerves, recorded the resulting evoked potentials in the SCN, and examined c-Fos protein levels in the nucleus by immunohistochemistry. The number of SCN cells labeled for immunoreactive c-Fos was significantly increased in slices stimulated during projected night, but not during projected day, compared to matched, sham-stimulated control slices. These results imply that the phase-dependent mechanism that gates c-Fos photoinduction in vivo is intrinsic to SCN tissue, and they suggest that an in vitro slice preparation will provide a useful model for dissecting the responsible signal transduction elements.

Animals↗
PubMed1996

Unexpected c-fos gene expression in the suprachiasmatic nucleus of mice entrained to a skeleton photoperiod.

Several authors have suggested that the transcriptional regulatory protein c-Fos might be part of the mechanism for photic entrainment of the circadian pacemaker in the suprachiasmatic nucleus (SCN) to environmental light:dark cycles. This hypothesis has been based on evidence gathered using single light pulses administered acutely to animals free-running in constant darkness. In order to begin to analyze SCN c-fos gene expression in animals during steady-state entrainment to photic cycles, we exposed male BALB/c mice to a skeleton photoperiod consisting of two 1-h light pulses separating a long (14 h) and a short (8 h) dark interval. The cycle was designed so that stable entrainment could be achieved in either one of two patterns (with rhythmic locomotor activity occurring during either the long or the short dark interval); SCN c-fos mRNA levels could then be measured during entrainment to light pulses at different phases of the circadian cycle, while controlling for the duration of preceding darkness. We found that c-fos was induced equally well by a light pulse that represented ZT 12 or ZT 3. The ZT 12 pulse functioned as an entraining pulse, because animals free-ran after it was removed from the lighting regimen, whereas removing the ZT 3 pulse caused little or no phase shift of activity onset. The data confirm that the expression of SCN c-Fos is not itself sufficient to reset rhythm phase, and they indicate that the role of this gene in the mechanism of photic entrainment is not yet fully understood.

Animals↗
PubMed1996

Metastatic papillary thyroid carcinoma to lung diagnosed by bronchoalveolar lavage.

The diagnosis of papillary carcinoma of the thyroid metastatic to the lung frequently requires a battery of noninvasive tests. Occasionally, invasive procedures such as open lung biopsy, transthoracic needle biopsy, and transbronchial lung biopsy are employed to confirm the diagnosis. A 31-yr-old woman with papillary thyroid carcinoma treated previously by a near-total thyroidectomy and 131I ablation presented to our clinic with shortness of breath and a clear chest roentgenogram. A post-131I treatment whole body scan revealed widespread 131I pulmonary uptake, and the presence of papillary thyroid cancer was confirmed by bronchoalveolar lavage. We conclude that bronchoalveolar lavage should be considered when tissue confirmation of metastatic papillary carcinoma to the lung is needed. During the evaluation and follow-up of this patient, we were able to determine that metastatic papillary carcinoma to the lung may cause a methacholine bronchoprovocation test to be falsely positive for asthma.

Adult↗
PubMed1995

Functional role for Sp1 in the transcriptional amplification of a cell cycle regulated histone H4 gene.

The promoter of the cell cycle regulated histone FO108 H4 gene is mediated by two in vivo protein/DNA interaction domains, sites I and II. We have shown previously that site II mediates the cell cycle controlled enhancement of H4 gene transcription at the G1/S phase boundary. Here we show that site I, an element containing both G-rich and ATF-like consensus sequences, confers maximal levels of transcription in proliferating cells. By the combined application of gel shift assays with site-directed mutagenesis, DNase I footprinting, oligonucleotide competition, in vitro expression of recombinant proteins, and specific antibody supershift studies, we demonstrate that the proximal G-rich sequence within site I interacts with the transcription factor Sp1, while the distal portion of site I interacts with members of the ATF family of proteins, including ATF-1. In vitro transcription studies as well as expression assays of transiently and stably transfected genes in HeLa cells reveal that the deletion of site I causes a dramatic decrease in expression. Mutation of the Sp1 element, which abolishes Sp1 binding, results in a 6-10-fold reduction in reporter activity. In addition, overexpression of Sp1 in Sp1-deficient cells results in the dramatic activation of the histone promoter. In contrast, mutation of the asymmetric ATF binding site, located distally within site I, has a more limited effect upon expression. Interestingly, the contribution of the Sp1 site to maximal transcription was cell type dependent. Thus, we demonstrate that the Sp1 binding site of the site I histone H4 promoter in particular is critical for maximal expression in living cells and postulate that this site may act to amplify the cell cycle response.

Base Sequence↗
PubMed1995

CAG expansion affects the expression of mutant Huntingtin in the Huntington's disease brain.

A trinucleotide repeat (CAG) expansion in the huntingtin gene causes Huntington's disease (HD). In brain tissue from HD heterozygotes with adult onset and more clinically severe juvenile onset, where the largest expansions occur, a mutant protein of equivalent intensity to wild-type huntingtin was detected in cortical synaptosomes, indicating that a mutant species is synthesized and transported with the normal protein to nerve endings. The increased size of mutant huntingtin relative to the wild type was highly correlated with CAG repeat expansion, thereby linking an altered electrophoretic mobility of the mutant protein to its abnormal function. Mutant huntingtin appeared in gray and white matter with no difference in expression in affected regions. The mutant protein was broader than the wild type and in 6 of 11 juvenile cases resolved as a complex of bands, consistent with evidence at the DNA level for somatic mosaicism. Thus, HD pathogenesis results from a gain of function by an aberrant protein that is widely expressed in brain and is harmful only to some neurons.

Adult↗
PubMed1994

Circadian regulation of Fos B is different from c-Fos in the rat suprachiasmatic nucleus.

We measured immunoreactive Fos B protein levels in the ventrolateral subdivision of the rat suprachiasmatic nucleus (SCN) as a function of light and time of day. Immunohistochemistry revealed high levels of Fos B that were uniformly expressed throughout the 12 h:12 h light-dark cycle. Levels remained high in constant darkness and were modestly increased (about 2-fold) after a 2 h light pulse administered during the subjective night, but not after a light pulse during the subjective day. Fos B and c-Fos immunoreactivities could be colocalized within individual SCN cell nuclei using a double-label immunofluorescence method. Thus, despite their structural similarities, these two members of the fos gene family exhibit different patterns of expression in the rat SCN. These and previous data suggest that Fos/Jun DNA-binding complexes in the SCN are composed of constant, as well as variable, protein components; in at least some SCN cells, light-induced changes in the composition of these constituent proteins may lead to altered transcription of target genes.

Animals↗
PubMed1994

Temporal regulation of light-induced Fos and Fos-like protein expression in the ventrolateral subdivision of the rat suprachiasmatic nucleus.

We measured c-fos messenger RNA levels and Fos protein immunoreactivity in the suprachiasmatic nucleus of rats as a function of light and time of day. Immunohistochemistry demonstrated a daily rhythm of immunoreactive Fos in the ventrolateral subdivision of the suprachiasmatic nucleus of animals entrained to a 12 h/12 h light-dark cycle; expression was low during the dark phase, peaked about 2 h after light onset at dawn, and remained elevated at an intermediate level for the remainder of the light phase. Immunoblots of nuclear extracts showed a 54,000 mol. wt band that increased in density from the dark phase to the early light phase and decreased again during the late light phase. In situ hybridization using a radiolabeled cDNA probe revealed a c-fos messenger RNA signal that was detected as early as 15 min after dawn, prominent at 30 min, and absent by 2 h. The expression of c-fos messenger RNA and Fos immunoreactivity in the suprachiasmatic nucleus depended on the presence of ambient light. In rats entrained to two daily 1-h light pulses corresponding to dawn and dusk ("skeleton" photoperiod) instead of the complete light-dark cycle, immunoreactive Fos was elicited by the dawn pulse alone and was less persistent than during the complete photoperiod. In rats free-running in constant darkness, c-fos messenger RNA and Fos immunoreactivity were stimulated by 2-h light pulses administered only during the subjective night and early subjective day, but not by light pulses during the middle or late subjective day or in the absence of light pulses.

Animals↗
PubMed1993

Colocalization of D1 and D2 dopamine receptor mRNAs in striatal neurons.

There is evidence that D1 and D2 dopamine receptor subtypes coexist at the cellular level in the striatum and act synergistically to mediate the effects of dopamine. Other data suggest that these receptor subtypes are largely segregated in different striatal projection pathways. We used in situ hybridization in serial adjacent 4 microns sections to determine the extent of colocalization of D1 and D2 receptor mRNAs in rat striatal neurons. Cellular localization of D1 and D2 receptor mRNA was performed on section pairs that were hybridized with 35S-labeled cDNA or oligonucleotide probes directed against non-homologous regions of D1 and D2 receptor mRNAs. We found that 26-27% of striatal cells containing one receptor subtype also contained the other subtype. Thus, although D1 and D2 receptors are segregated in the majority of striatal neurons, a substantial number of striatal neurons coexpress both dopamine receptor mRNA subtypes. Our findings provide anatomic support for many of the functional interactions that have been described for D1 and D2 receptors.

Animals↗
PubMed1993

Compositional changes of AP-1 DNA-binding proteins are regulated by light in a mammalian circadian clock.

Recent reports have shown that the nuclear phosphoprotein Fos is induced by light in a mammalian circadian clock, the suprachiasmatic nucleus. To learn how light and circadian phase affect the binding of Fos to DNA, we analyzed the photic and temporal regulation of immunoreactive Jun protein expression and AP-1 DNA-binding activity in the rat suprachiasmatic nucleus. Immunohistochemistry and gel mobility shift assays suggest that AP-1 activity during the night and after a light pulse consists of constant, as well as variable, protein components; JunD could be identified as a constituent of both dark- and light-activated binding complexes, whereas binding by JunB and Fos could be implicated only after photic stimulation. Since JunD or JunB could be colocalized with Fos in individual suprachiasmatic nucleus cell nuclei, light may be acting in at least some suprachiasmatic nucleus cells by altering AP-1 protein composition rather than binding site occupancy.

Animals↗
PubMed1992

Immunohistochemical localization of the D1 dopamine receptor in rat brain reveals its axonal transport, pre- and postsynaptic localization, and prevalence in the basal ganglia, limbic system, and thalamic reticular nucleus.

D1 dopamine receptor localization was examined by immunohistochemistry using a polyclonal anti-peptide antibody which (i) immunoprecipitated a protein fragment encoded by a D1 receptor cDNA and (ii) on Western blots of solubilized striatal and hippocampal membranes recognized two proteins of approximately 50 kDa and 75 kDa, corresponding to reported sizes of D1 receptor proteins. Immunoreactivity overlapped with dopamine-containing pathways, patterns of D1 receptor binding, and mRNA expression. Staining was concentrated in prefrontal, cingulate, parietal, piriform, entorhinal, and hippocampal cortical areas and subcortically in the basal ganglia, amygdala, septal area, substantia inominata, thalamus, hypothalamus, and neurohypophysis. Prominent labeling was seen in the thalamic reticular nucleus, a region known to integrate ascending basal forebrain inputs with thalamocortical and corticothalamic pathways and in fiber bundles interconnecting limbic areas. In striatal neuropil, staining appeared in spines (heads and necks), at postsynaptic sites in dendrites, and in axon terminals; in the pars reticulata of the substantia nigra, labeling was prevalent in myelinated and unmyelinated axons and dendrites. These data provide direct evidence for the regional and subcellular distribution of D1 receptor protein in the brain and for its pre- and postsynaptic localization in the basal ganglia. The prominent immunoreactivity seen in the limbic system and thalamic reticular nucleus supports an important role for this receptor subtype in mediating integrative processes involved with learning, memory, and cognition.

Animals↗
PubMed1992

Downregulation of histone H4 gene transcription during postnatal development in transgenic mice and at the onset of differentiation in transgenically derived calvarial osteoblast cultures.

In vivo regulation of cell cycle dependent human histone gene expression was examined in transgenic mice using a fusion construct containing 6.5 kB of a human H4 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Transcriptional control of histone gene expression, as a function of proliferative activity, was determined. We established the relationship between DNA replication dependent H4 mRNA levels (Northern blot analysis) and H4 promoter activity (CAT assay) during postnatal development in a broad spectrum of tissues. In most tissues sampled in adult animals, the cellular representation of H4 gene transcripts declined in parallel with promoter activity. This result is consistent with transcriptional control of H4 gene expression at the cessation of proliferation. Interestingly, while H4 mRNA was detectable at very low levels post-proliferatively in brain, promoter activity persisted in adult brain, where most of the cells are terminally differentiated. This dissociation between histone gene promoter activity and histone mRNA accumulation points to the possibility of post-transcriptional regulation of histone gene expression in brain. Cultures of osteoblasts were prepared from calvaria of transgenic mice carrying the H4 promoter/CAT reporter construct. In contrast to the brain, in these bone-derived cells, we established by immunohistochemistry that the transition to the quiescent, differentiated state is associated with a transcriptionally mediated downregulation of histone gene expression at the single cell level.

Aging↗
PubMed1992

The subcellular localization of the G-protein Gi alpha in the basal ganglia reveals its potential role in both signal transduction and vesicle trafficking.

The G-protein Gi is known to mediate signal transduction in cells by coupling its 41 kDa alpha-subunit to plasma membrane-bound receptors and inhibiting adenylyl cyclase or affecting ion channel function. Although this G-protein has been functionally associated with D2/dopamine and mu-opioid receptors in striatal membranes, its localization to neurons of the neostriatum, a brain region rich in adenylyl cyclase activity, has not been established. Light and electron microscopic study of the basal ganglia was conducted using the immunoperoxidase method and an antiserum directed against the alpha-subunit of Gi. In the neostriatum, immunoreactivity was localized to medium-sized spiny and aspiny neurons and axon terminals that formed symmetric synapses. Some astrocytes and glial processes that encapsulated axospinous complexes were also labeled. Immunoreactive axon terminals were numerous in the globus pallidus and substantia nigra, where they exhibited a dense pattern of distribution characteristic of neostriatal spiny projection neurons. Gi alpha immunoreactivity was distributed to multiple subcellular compartments. In neostriatal somata and dendrites, labeling was present intermittently along plasma membranes, and on rough and smooth endoplasmic reticulum and microtubules. In axon terminals, reaction product appeared on plasma membranes and heavily labeled the membranes of synaptic vesicles. The presence of Gi alpha in axon terminals was confirmed in purified synaptosome preparations. G-proteins consistent with the masses of Go alpha and Gi alpha, respectively, were ADP-ribosylated in the presence of pertussis toxin in striatal synaptosomes. Western blot analysis in purified synaptosome preparations of the neostriatum, globus pallidus, and substantia nigra with the same antiserum used in the immunohistochemistry demonstrated a predominant 41 kDa protein corresponding to the molecular mass of Gi alpha. Immunohistochemical localization of Gi alpha with the immunogold method in a crude striatal synaptosome preparation showed gold particles associated with synaptic vesicles and plasma membranes. Results provide the first direct evidence that Gi alpha is localized to medium-sized neostriatal projection neurons and interneurons, where it is likely to function in membrane-bound signal transduction at the postsynaptic and presynaptic level. The presence of Gi alpha in synaptic vesicle membranes points to another potentially important role for this G-protein in vesicle trafficking, such as that recently shown for smaller-molecular-mass G-proteins.

Animals↗
PubMed1992

Monoamines in the brain cerebrospinal fluid of facial pain patients.

The purpose of the study was to assay monoamines in cerebrospinal fluid (CSF) obtained from the trigeminal cistern of 64 patients with intractable facial pain. The CSF was analyzed for homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), and 3-methoxy-4-hydroxyphenylglycol (MHPG), end-product markers of activity for the dopamine, serotonin, and norepinephrine systems, respectively. HVA averaged 121 ng/mL in these facial pain patients, compared to 150 to 550 ng/mL in 10 studies of ventricular brain CSF in assorted psychiatric and pain patients. 5-HIAA averaged 29 to ng/mL in our facial pain patients compared to 60 to 120 ng/mL in nine studies of ventricular brain CSF in assorted psychiatric and neurological patients. Trigeminal cistern CSF MHPG averaged 9 ng/mL, similar to the range of 13 studies of lumbar CSF of assorted psychiatric and pain diagnoses. These results indicate that (1) the electrochemical detection method provides a unique way of accurately measuring nanogram concentrations of multiple monoamines in a little as 0.25 mL of CSF; (2) trigeminal cistern and posterior fossa brain CSF monoamine metabolites reflect a different profile of dopaminergic and serotonergic functioning in these facial pain patients from that previously reported with lumbar CSF measurements of other patients; and (3) trigeminal sensory ganglion or brain dopamine and serotonin systems may be concomitantly dysfunctional in intractable facial pain.

Analysis of Variance↗