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Biomedical subjects

M G Erlander

Publications and source records attributed to M G Erlander.

At least 19 recordsLinked to original sources
PubMed2000

TOGA: an automated parsing technology for analyzing expression of nearly all genes.

We have developed an automated, high-throughput, systematic cDNA display method called TOGA, an acronym for total gene expression analysis. TOGA utilizes 8-nt sequences, comprised of a 4-nt restriction endonuclease cleavage site and adjacent 4-nt parsing sequences, and their distances from the 3' ends of mRNA molecules to give each mRNA species in an organism a single identity. The parsing sequences are used as parts of primer-binding sites in 256 PCR-based assays performed robotically on tissue extracts to determine simultaneously the presence and relative concentration of nearly every mRNA in the extracts, regardless of whether the mRNA has been discovered previously. Visualization of the electrophoretically separated fluorescent assay products from different extracts displayed via a Netscape browser-based graphical user interface allows the status of each mRNA to be compared among samples and its identity to be matched with sequences of known mRNAs compiled in databases.

Automation↗
PubMed2000

Cloning of rat histamine H(3) receptor reveals distinct species pharmacological profiles.

The recent cloning and characterization of the human histamine H(3) receptor cDNA marked a significant step toward a greater understanding of the role of this receptor in the central nervous system. We now report the cloning of the rat histamine H(3) receptor cDNA and demonstrate distinct pharmacological species differences. The rat cDNA clone encodes a putative 445-amino acid protein with 93% identity to the human H(3) receptor. Northern blot analysis revealed a major single entity of 2.7-kb in length expressed only in brain. Transfection of the rat receptor cDNA into SK-N-MC cells conferred an ability to inhibit forskolin-stimulated cAMP formation in response to histamine and other H(3) agonists. N-[(3)H]methylhistamine saturably bound to transfected cells with high affinity (K(d) = 0.8 nM). All agonists tested had low or subnanomolar K(i) values similar to that for the human recombinant receptor. The antagonists thioperamide and clobenpropit also bound with high affinity (K(i) = 4 and 0.4 nM, respectively). This is in contrast to the antagonist profile obtained for the human recombinant receptor that showed K(i) values of 58 and 0.6 nM for thioperamide and clobenpropit, respectively. These data suggest that the low affinity of thioperamide for the human H(3) receptor represents a species difference in pharmacology and not a unique pharmacological subtype. It also was found that chloroproxyfan behaved as a full agonist at the rat recombinant receptor. These findings highlight the significance of validating the pharmacology of experimental compounds at both the rat and human H(3) receptors.

Adenylyl Cyclase Inhibitors↗
PubMed1999

Immune-associated nucleotide-1 (IAN-1) is a thymic selection marker and defines a novel gene family conserved in plants.

Positive selection of thymocytes is a complex and crucial event in T cell development that is characterized by cell death rescue, commitment toward the helper or cytotoxic lineage, and functional maturation of thymocytes bearing an appropriate TCR. To search for novel genes involved in this process, we compared gene expression patterns in positively selected thymocytes and their immediate progenitors in mice using the differential display technique. This approach lead to the identification of a novel gene, mIAN-1 (murine immune-associated nucleotide-1), that is switched on upon positive selection and predominantly expressed in the lymphoid system. We show that mIAN-1 encodes a 42-kDa protein sharing sequence homology with the pathogen-induced plant protein aig1 and that it defines a novel family of at least three putative GTP-binding proteins. Analysis of protein expression at various stages of thymocyte development links mIAN-1 to CD3-mediated selection events, suggesting that it represents a key player of thymocyte development and that it participates to peripheral specific immune responses. The evolutionary conservation of the IAN family provides a unique example of a plant pathogen response gene conserved in animals.

Amino Acid Sequence↗
PubMed1999

The pharmacological and functional characteristics of the serotonin 5-HT(3A) receptor are specifically modified by a 5-HT(3B) receptor subunit.

While homomers containing 5-HT(3A) subunits form functional ligand-gated serotonin (5-HT) receptors in heterologous expression systems (Jackson, M. B., and Yakel, J. L. (1995) Annu. Rev. Physiol. 57, 447-468; Lambert, J. J., Peters, J. A., and Hope, A. G. (1995) in Ligand-Voltage-Gated Ion Channels (North, R., ed) pp. 177-211, CRC Press, Inc., Boca Raton, FL), it has been proposed that native receptors may exist as heteromers (Fletcher, S., and Barnes, N. M. (1998) Trends Pharmacol. Sci. 19, 212-215). We report the cloning of a subunit 5-HT(3B) with approximately 44% amino acid identity to 5-HT(3A) that specifically modified 5-HT(3A) receptor kinetics, voltage dependence, and pharmacology. Co-expression of 5-HT(3B) with 5-HT(3A) modified the duration of 5-HT(3) receptor agonist-induced responses, linearized the current-voltage relationship, increased agonist and antagonist affinity, and reduced cooperativity between subunits. Reverse transcriptase-polymerase chain reaction in situ hybridization revealed co-localization of both 5-HT(3B) and 5-HT(3A) in a population of neurons in the amygdala, telencephalon, and entorhinal cortex. Furthermore, 5-HT(3A) and 5-HT(3B) mRNAs were expressed in spleen and intestine. Our data suggest that 5-HT(3B) might contribute to tissue-specific functional changes in 5-HT(3)-mediated signaling and/or modulation.

Amygdala↗
PubMed1999

DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression.

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (alpha), early (beta), early-late (gamma1), and late (gamma2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5' noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for beta, gamma1, and gamma2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.

Base Sequence↗
PubMed1999

Anatomical distribution of prolactin-releasing peptide and its receptor suggests additional functions in the central nervous system and periphery.

A recently identified neuropeptide with PRL-releasing capabilities binds to and activates a previously known orphan G protein-coupled receptor, GPR10. We initiated a study to define the pharmacology of the peptide/receptor interaction and to identify the distribution of the peptide and its receptor in the central nervous system to elucidate sites of action of the peptide. The PRL-releasing peptide (PrRP) is a C-terminally amidated, 31-amino acid peptide derived from a 98-amino acid precursor. Radioiodinated PrRP-(1-31) binds to its receptor with high affinity (1 nM) and stimulates calcium mobilization in CHOK1 cells stably transfected with the receptor. A series of N-terminal deletions reveals that the PrRP-(12-31) amino acid is equipotent to PrRP-(1-31). Further N-terminal deletions reduce the affinity of the ligand considerably, although PrRP-(25-31) is still able to compete for binding and behaves as an agonist. The arginine residues at position 26 and 30 are critical for binding, as substitution with either lysine or citrulline reduces the affinity substantially. In situ hybridization reveals a distinct tissue distribution for both the peptide and receptor messenger RNAs. The receptor is expressed abundantly in the reticular thalamic nucleus, periventricular hypothalamus, dorsomedial hypothalamus, nucleus of the solitary tract, area postrema, anterior pituitary, and adrenal medulla. The peptide messenger RNA is expressed in the dorsomedial hypothalamus, nucleus of the solitary tract, ventrolateral reticular nucleus, and intestine. This tissue distribution suggests an alternative function of PrRP than its purported hypophysiotropic function, such as a potential role for PrRP in the central feedback control of neuroendocrine and autonomic homeostasis. Further work using selective agonists and antagonists should help define additional physiological roles of this novel mammalian neuropeptide.

Animals↗
PubMed1999

Cloning and functional expression of the human histamine H3 receptor.

Histamine regulates neurotransmitter release in the central and peripheral nervous systems through H3 presynaptic receptors. The existence of the histamine H3 receptor was demonstrated pharmacologically 15 years ago, yet despite intensive efforts, its molecular identity has remained elusive. As part of a directed effort to discover novel G protein-coupled receptors through homology searching of expressed sequence tag databases, we identified a partial clone (GPCR97) that had significant homology to biogenic amine receptors. The GPCR97 clone was used to probe a human thalamus library, which resulted in the isolation of a full-length clone encoding a putative G protein-coupled receptor. Homology analysis showed the highest similarity to M2 muscarinic acetylcholine receptors and overall low homology to all other biogenic amine receptors. Transfection of GPCR97 into a variety of cell lines conferred an ability to inhibit forskolin-stimulated cAMP formation in response to histamine, but not to acetylcholine or any other biogenic amine. Subsequent analysis revealed a pharmacological profile practically indistinguishable from that for the histamine H3 receptor. In situ hybridization in rat brain revealed high levels of mRNA in all neuronal systems (such as the cerebral cortex, the thalamus, and the caudate nucleus) previously associated with H3 receptor function. Its widespread and abundant neuronal expression in the brain highlights the significance of histamine as a general neurotransmitter modulator. The availability of the human H3 receptor cDNA should greatly aid in the development of chemical and biological reagents, allowing a greater appreciation of the role of histamine in brain function.

Adenylyl Cyclase Inhibitors↗
PubMed1998

Up-regulated expression of decay-accelerating factor (CD55) confers increased complement resistance to sprouting neural cells.

We studied gene expression in relation to induced neural differentiation in a human neural crest-derived cell line, Paju. Messenger RNA isolated before and after treatment with phorbol 12-myristate 13-acetate was analyzed by differential display reverse transcription PCR. A strongly up-regulated expression of decay-accelerating factor (DAF, CD55) was found to parallel the induced neural sprouting while the expression of two other complement regulatory proteins (CD59/protectin, CD46/membrane cofactor protein) remained unaltered during neural differentiation. The increased membrane expression of DAF, which was also seen on neural processes and growth cones, conferred elevated resistance to complement-mediated lysis. Our findings suggest that in sprouting neurons DAF expression is up-regulated to provide additional complement resistance to pathfinding axons/dendrites invading new environment. It is also suggested that membrane expression of DAF may constitute a marker of growing and regenerating neurons.

CD55 Antigens↗
PubMed1998

Postdifferential display: parallel processing of candidates using small amounts of RNA.

The necessity of screening differentially expressed candidate genes has imposed a limit on the application of differential display to large-scale analysis of gene expression patterns. Screening candidates has indeed proven a burden because traditional screening methods require the purification of large amounts of RNA. In this article we describe an assay that allows the screening of 240 candidate genes with only 5 microg of total RNA. This assay consists of using cDNA probes synthesized from amplified RNA in differential screening and can be performed in a 96-well plate format.

Cloning, Molecular↗
PubMed1997

Screening differentially expressed cDNA clones obtained by differential display using amplified RNA.

The major obstacle of differential display is not the technique itself but rather the post-differential display issueof discriminating between false positives and the truly differentially expressed mRNAs. This process is arduous and requires large amounts of RNA. We present and validate a method which allows one to screen putative positives from differential display analysis using only micrograms of total RNA. More importantly, we demonstrate that cDNA probes generated from amplified RNA are representative of the starting mRNA population and can be used for differential screening of mRNA species at a detectable limit of sensitivity of>/=1/40 000.

Cloning, Molecular↗
PubMed1996

Cloning differentially expressed mRNAs.

Differential gene expression occurs in the process of development, maintenance, injury, and death of unicellular as well as complex organisms. Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Electronic subtraction (ES), subtractive hybridization (SH), and differential display (DD) are methods commonly used for this purpose. A rigorous examination has been lacking and therefore quantitative aspects of these methods remain speculative. We compare these methods by identifying a total of 58 unique differentially expressed mRNAs within the same experimental system (HeLa cells treated with interferon-gamma). ES yields digital, reusable data that quantitated steady-state mRNA concentrations but only identified abundant mRNAs (seven were identified), which represent a small fraction of the total number of differentially expressed mRNAs. SH and DD identified abundant and rare mRNAs (33 and 23 unique mRNAs respectively) with redundancy. The redundancy is mRNA abundance-dependent for SH and primer-dependent for DD. We conclude that DD is the method of choice because it identifies mRNAs independent of prevalence, uses small amounts of RNA, identifies increases and decreases of mRNA steady-state levels simultaneously, and has rapid output.

Antiviral Agents↗
PubMed1994

G-protein gamma 7 subunit is selectively expressed in medium-sized neurons and dendrites of the rat neostriatum.

We used subtractive hybridization to isolate clones of gamma 7, a 68 residue G-protein gamma subunit. Northern blotting and in situ hybridization reveal that the gamma 7 subunit mRNA is expressed primarily in medium-sized neurons of the neostriatum and nucleus accumbens and neurons of the olfactory tubercle, and at low levels in the dentate gyrus of the hippocampal formation and laminae II-III, and V of the neocortex. The gamma 7 mRNA is translocated into dendrites of neurons in the neostriatum and the dentate gyrus of the hippocampus. gamma 7 is expressed at relatively very low concentrations in peripheral tissues. The selective pattern of gamma 7 expression within the brain is highly reminiscent of those of the striatum-enriched adenylyl cyclase ACST, dopamine receptors, and the alpha subunit of G(olf), suggesting that, in striatum, gamma 7 may be a subunit of a G(olf) alpha-containing G protein that couples dopamine receptors selectively to ACST.

Adenylyl Cyclases↗
PubMed1994

Chromosomal mapping of mouse genes expressed selectively within the central nervous system.

We have used RFLP analysis on DNA from a panel of interspecific (C57BL/6J x Mus spretus) F1 x M. spretus backcross offspring to assign the genes encoding 10 neuron-specific mRNAs and 2 loci corresponding to cyclophilin 2-related sequences to the mouse chromosomal map. The Pss1 locus encoding the forebrain-enriched protein kinase C substrate RC3, a component of dendritic spines, mapped to proximal Chr 9. The Camkl locus encoding the calmodulin-binding protein kinase-like vesicle protein 1G5 mapped to distal Chr 9. The Gng7 locus encoding the gamma 7 G-protein subunit, highly enriched in the striatum and presumptively coupled to dopamine receptors, mapped to mid-Chr 10. The Htr1f, Htr5a, Htr5b, and Htr7loci, encoding four serotonin receptors, mapped to Chr 16, 5, 1, and 19, respectively. The Peplb locus, encoding a CD26 ectopeptidase-like neuronal membrane protein activated by kainate and long-term potentiation, mapped to Chr 5. The D2Sut1e and Cpu3 loci, encoding neural proteins of unknown functions, mapped to Chrs 2 and 9, respectively. Two cyclophilin 2-related loci, Cphn2-r1 and Cphn2-r2, mapped to different regions of Chr 9. Comparison of these 12 newly mapped loci with the existing mouse map and known regions of syntenic homology with the human map, along with the known features and expression profiles of the products of these genes, suggests a few candidates for mouse mutations and human neurological and immunological deficits, including the Tourette syndrome and Bloom syndrome genes.

Animals↗
PubMed1994

Isolation of clones of rat striatum-specific mRNAs by directional tag PCR subtraction.

We report an improved subtractive cDNA cloning procedure, named "directional tag PCR subtraction," for isolating clones of mRNAs enriched in a target tissue compared to a second tissue, the driver. In this method, the target and driver are prepared from directional cDNA libraries constructed in different vectors, and the target cDNA contains tag sequences at both its 5' and 3' ends for PCR amplification. This method avoids several limitations of previous subtraction procedures, and was demonstrated to be technically easy and efficient. Using the directional tag PCR subtraction and improved screening procedures, cDNA clones corresponding to mRNAs expressed in the striatum but not in the cerebellum of the rat brain were efficiently isolated, including mRNAs encoding calmodulin-dependent phosphodiesterase, a transcriptional regulatory protein, and several previously uncharacterized species. Our data suggest that approximately 1% of the striatal polyA+ RNA mass potentially encoding more than 300 distinct proteins corresponds to RNA species reduced in concentration or absent from the cerebellum, of which about one-third are expressed prominently only in the striatum. This unexpected finding suggests that the striatum has a unique biochemical character within the brain, and that characterization of these mRNAs will be important for understanding the biochemical basis of striatal function.

Animals↗
PubMed1994

1G5: a calmodulin-binding, vesicle-associated, protein kinase-like protein enriched in forebrain neurites.

We have characterized cDNA clones of 1G5, an mRNA highly enriched in the mammalian forebrain that encodes a 504-residue protein found in association with perikaryal membranes and neurites. The protein, which accumulates predominantly postnatally, is associated with vesicles in both axons and dendrites. The sequence of the 1G5 protein highly resembles those of protein kinases with serine/threonine specificity; however, although most residues universally conserved among protein kinases are present, a few signature residues are absent from the 1G5 protein. Furthermore, although recombinant 1G5 protein binds calmodulin in the presence of calcium, it lacks kinase activity with a sample substrate.

Amino Acid Sequence↗
PubMed1993

Different distributions of GAD65 and GAD67 mRNAs suggest that the two glutamate decarboxylases play distinctive functional roles.

Two genes encode two forms of glutamate decarboxylase, GAD65 and GAD67. Because the two GADs differ in subcellular distribution and interactions with the cofactor pyridoxal phosphate, the two enzymes may play different roles in gamma-aminobutyric acid (GABA) production. In this study we have used in situ hybridization to compare the regional and cellular distributions of the two GAD mRNAs in rat brain. Both GAD mRNAs are abundant in olfactory bulb, olfactory tubercle, zona incerta, reticular nucleus of the thalamus, oculomotor nuclei, and pontine tegmental area. GAD65 mRNA is more abundant in several structures of the visual system, including the lateral geniculate nuclei, superior colliculi, and olivary pretectal nucleus, as well as in several hypothalamic and pontine nuclei. In contrast, GAD67 mRNA is more abundant in neocortex, the granular layer of olfactory bulb, lateral and medial septum, globus pallidus, inferior colliculi, and cerebellar cortex. Both GAD mRNAs are present in interneurons as well as in projection neurons, and both are present in neurons with different types of synapses, including dendrodendiritic, axosomatic, and axodendritic synapses. GAD65 mRNA predominates in the visual and the neuroendocrine systems, which are more subject to phasic changes, while GAD67 is present at relatively higher concentrations in many tonically active neurons. GAD65 and GAD67 together may provide more flexibility in the regulation of GABA synthesis than either could alone.

Animals↗