Simultaneous reduction of the sarcolemmal and SR calcium ATPase activities and gene expression in cardiomyopathic hamster.

Altered calcium regulation is a prominent feature in the hereditary cardiomyopathy of the Syrian hamster. However, the activity of the two systems necessary for intracellular calcium homeostasis in the heart, the sarcolemmal and sarcoplasmic reticulum calcium ATPase pumps, have not been correlated. Using age- and pair-matched myopathic and control hamsters, a simultaneous reduction in gene expression and enzyme activity for these two pumps has been demonstrated. The concomitant alteration in gene expression as early as 1 month of age, preceding noticeable myocytolysis suggests that the depressed activity in these two calcium ATPase systems is not due to cell necrosis but at least in part due to reduction in their mRNA levels. Reduced capacity of the calcium pumps would result in calcium overload as well as impaired contractility that leads to the eventual heart failure in this animal model.

The myelin proteolipid protein gene modulates apoptosis in neural and non-neural tissues.

Mutations of the myelin proteolipid protein gene (Plp) are associated with excessive programmed cell death (PCD) of oligodendrocytes. We show for the first time that PLP is a molecule ubiquitously expressed in non-neural tissues during normal development, and that the level of native PLP modulates the level of PCD. We analyze three non-neural tissues, and show that native PLP is expressed in trophoblasts, spermatogonia, and cells of interdigital webbing. The non-neural cells that express high levels of native PLP also undergo PCD. The level of PLP expression modulates the level of PCD because mice that overexpress native PLP have increased PCD and mice deficient in PLP have decreased PCD. We show that overexpression of native PLP causes a dramatic acidification of extracellular fluid that, in turn, causes increased PCD. These studies show that the level of native PLP modulates the amount of PCD during normal development via a pH-dependent mechanism.

Identification and cloning of a brain autoantigen in neuro-behavioral SLE.

In murine models of SLE, particular patterns of abnormalities of social interaction and memory collectively known as neurobehavioral dysfunction (NBD) correlate with the occurrence of brain reactive autoantibodies. Study of the immunopathogenic effects of these antibodies has been limited by the absence of isolated autoantibodies and antigens. In order to identify the molecular targets, we isolated autoantibodies highly specific for brain plasma membranes from MRL/lpr mice. After immunoscreening a brain expression library with these brain specific autoantibodies, we identified a single cDNA clone of unique sequence and relevant anatomic distribution. Transcript for this cDNA is wide spread among mammalian species but appears to be present only in the brain. Addition features, suggesting this cDNA is pertinent for further study include (1) the expressed protein, called lupus brain antigen 1, reacts with the screening immunoglobulins as well as immunoglobulins from other strains of murine neuro-SLE not used to screen the library, but not with immunoglobulins from normal mice, (2) the transcript distribution within the brain is similar to immunochemical localization of binding of the spontaneous autoantibodies and (3) the localization of transcript within the brain, in the hippocampus, hypothalamus an cingulate gyrus, corresponds to anticipated anatomical regions of clinical dysfunction. Further, the transcript is a large, potentially structural molecule of unique sequence. Antibodies to this molecule may mediate changes in behavior either by direct interactions with the cognate antigen or by indirect influences through neuro-endocrine axes.

Variable subcellular localization of a neuron-specific protein during NTera 2 differentiation into post-mitotic human neurons.

The current report describes the molecular characterization of the human (the D4S234 locus) and mouse (the m234) homologs of a gene that was isolated during our genomic analysis of the Huntington disease gene region. Sequence comparisons of full-length cDNA clones revealed that the mouse and human homologs encoded evolutionarily conserved 21-kDa proteins with greater than 90% amino acid sequence identity. Extensive sequence identity between the D4S234 gene and the rat p1A75 gene (a previously identified rat neuron-specific gene) showed that these genes are interspecies homologs. Furthermore, the D4S234 protein exhibited significant amino acid similarity to a 19-kDa mouse protein that localizes to the Golgi apparatus of embryonic neurons. However, nonconservative sequence differences suggested that these genes are independent members of a multigene family. Northern analyses revealed that rodent D4S234 expression occurred predominantly in the brain and included all brain regions. Neuron-specific expression was demonstrated using Northern analysis of cultured glial cells and quinolinic acid-treated rat brain samples. Minimal amounts of the rodent D4S234 mRNA were detected prenatally; however, elevated adult levels were detected within 1 month of birth. Sequence analyses of the human and mouse D4S234 proteins identified an evolutionarily conserved hydrophobic sequence and a consensus nuclear localization signal in both genes. Immunofluorescence microscopy, using an antipeptide antibody, established that the human D4S234 protein preferentially localized to the nucleus of mitotic cultured cells. Since the rat p1A75 protein was previously mapped to the neuronal cytoplasm by in situ hybridization, the subcellular localization of the D4S234 protein was subsequently examined during differentiation of the NTera 2 (NT2) cell line. Following differentiation into postmitotic NT2-N neurons, the D4S234 protein demonstrated cytoplasmic staining and reduced or undetectable nuclear staining in many cells. The variation in the intracellular localization of the D4S234 protein in mitotic and nonmitotic cells suggests that the subcellular localization of this protein is developmentally regulated and provides clues about the biochemical function of this protein.

The identification of a functional nuclear localization signal in the Huntington disease protein.

Positional cloning has shown that the Huntington disease (HD) mutation is an expanded trinucleotide repeat in the IT15 gene. Although this mutation clearly produces the HD phenotype, the function of the Huntington disease protein remains undefined. One recent immunocytochemical study suggested that the IT15 protein preferentially localizes to the nucleus of affected neuronal cells. If this result is accurate, it could link the biochemical function of this protein to nuclear activities such as gene regulation. To examine the nuclear transport of the Huntington disease protein, we searched for basic peptide motifs that could produce nuclear localization. One peptide (RRKGKEK) was identified that is highly homologous to a consensus nuclear localization signal. When fused to the cytoplasmic reporter protein, beta-galactosidase, nuclear localization was observed in stably transformed human cell lines. In a complementary study, an anti-peptide polyclonal antibody, raised against a sequence adjacent to the putative nuclear localization sequence, detected the IT15 protein in the nucleus of human cells. These results extend and confirm the previous localization studies and identify an IT15 peptide motif that can function for nuclear localization.

Timing the excitotoxic induction of heat shock protein 70 transcription.

The time course of heat-shock protein 70 (HSP70) transcriptional induction was compared with neuronal- and non-neuronal-specific mRNAs following intrastriatal quinolinate (QA) injection. Within one hour of QA exposure, immediate-early gene (IEG; c-fos) activation preceded slight increases in glutamic acid decaroxylase (GAD) mRNA levels. However, glial fibrillary acid protein (GFAP) and HSP70 mRNA levels remained constant. After one hour, HSP70 mRNA levels surged 6 fold during a delayed transcriptional phase that was induced between 4-12 hours. This phase was characterized by massive increases in c-fos and GFAP mRNAs while GAD transcripts fell drastically suggestive of neuronal death. Therefore HSP70 genes may play an important role in glial/immune activation following rapid excitotoxic damage by direct injections of quinolinate.

Transcription of the Huntington disease gene during the quinolinic acid excitotoxic cascade.

Although Huntington disease (HD) is characterized by the selective neurodegeneration of the basal ganglia and cerebral cortex, efforts to define the disease pathology have been complicated by the widespread expression of the disease gene (IT15) throughout the body. In this study, we examined IT15 mRNA levels during the quinolinic acid (QA) excitotoxic cascade to determine whether neuronal and/or glial expression is regulated by neurodegeneration. Following an initial increase between 1 h and 6 h, IT15 mRNA levels declined in a pattern homologous to a group of neuron-specific genes. Decreased mRNA levels after 24 h demonstrated that glial transcription is not activated by neurodegeneration or gliosis. The 1 h and 24 h mRNA levels strongly suggest that IT15 transcription preferentially localizes to degenerating neurons.

Preprotachykinin and preproenkephalin mRNA expression within striatal subregions in response to altered serotonin transmission.

The effects of lowered serotonin (5-hydroxytryptamine; 5-HT) neurotransmission on preprotachykinin (PPT) and preproenkephalin (PPE) mRNA levels were examined in subregions of the striatum. Adult male rats were treated systemically with para-chlorophenylalanine (pCPA; 350 mg/kg single i.p. injection) which reduced forebrain 5-HT amounts to approximately 20% of saline-injected controls at 24 and 48 h. As measured by Northern analysis, PPT and PPE mRNA levels were elevated 50% and 160% respectively in the anterior ventromedial striatum (region included nucleus accumbens). PPT mRNA levels were raised 90% in posterior striatum (at the level of the globus pallidus) by 48 h post-pCPA injection. To determine if increased PPT and PPE mRNA levels represented a transient response to brief 5-HT inhibition, additional experiments were performed to provide continual suppression of 5-HT within the striatum. First, rats received daily intraperitoneal injections of saline or the 5-HT1A receptor agonist, 8-OH-DPAT (1 mg/kg), for 7 days to reduce 5-HT release from raphestriatal terminals. In a parallel experiment, the serotonin neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT, 5 micrograms), was stereotaxically injected into the striatum as a means to permanently remove 5-HT terminals. Although levels of each mRNA species were differentially sensitive to 5,7-DHT or 8-OH-DPAT, PPT and PPE mRNAs were lowered between 30-55% within the anterior dorsolateral and ventromedial striatum. Although these results support previous studies suggesting an overall positive regulatory role of serotonin on striatal tachykinin biosynthesis, PPT and PPE gene regulation in certain striatal subregions may by differentially sensitive to lowered 5-HT neurotransmission. This suggestion is supported by observations that acute systemic stimulation of 5-HT2A/C receptors with DOI (7 mg/kg single i.p. injection) raised PPT and PPE mRNA levels within anterior dorsolateral (30-60%) and posterior (100-200%) striata, but not within the anterior ventromedial striatum.

T-cell-dependent antibody responses in the rat: forms and sources of keyhole limpet hemocyanin matter.

The T-cell-dependent antibody response (TDAR) is a functional assay used in immunopharmacology and immunotoxicology to assess ability to mount an antibody response to immunization. Keyhole limpet hemocyanin (KLH) is extensively used as the immunogen of choice in non-clinical and clinical settings. Native KLH is comprised of high molecular weight (HMW; 4-8 MDa) assemblies of KLH subunit dimers (> 600-800 kDa). It is not known how the different forms (HMW vs subunit) and manufacturing processes (commercial sources) may impact the nature of anti-KLH immune responses (e.g. magnitude and inter-animal variability). Anti-KLH IgM and IgG responses were studied in Sprague-Dawley rats immunized with different forms and commercial sources of KLH: 100 µg of HMW KLH from two different sources or subunit KLH from three different sources. Biophysical and biochemical analyses were conducted to characterize the KLH formulations. Anti-KLH IgM and IgG responses were measured using a proprietary indirect quantitative electrochemiluminescence immunoassay. The HMW KLH preparations showed a greater number of sub-visible particles (2-150 µm size range) than the subunit KLH preparations. All HMW KLH and all subunit KLH were equivalent on SEC (hydrodynamic volume), PAGE (size and charge), and SDS-PAGE (molecular radius). Robust primary and secondary anti-KLH responses were detected for both sources of HMW KLH. The subunit KLH immunizations resulted in lower IgG and IgM responses compared to the HMW KLH, with the exception of Stellar Biotechnologies subunit KLH that produced both robust primary and secondary responses, which approached the HMW KLH responses. Inter-animal variability for IgM and IgG responses was lower with HMW KLH than with subunit KLH. In conclusion, different forms and commercial sources of KLH were associated with different magnitudes and inter-animal variability in IgM and IgG responses, a critical finding to take into consideration when designing TDAR studies for robust immunotoxicology or immunopharmacology testing.

Immediate early gene activation during the initial phases of the excitotoxic cascade.

Direct brain injections of the N-methyl-D-aspartate receptor agonist quinolinic acid (QA) trigger an excitotoxic cascade characterized by rapid neuronal death and glial/immune cell activation. The present study compared the timing of immediate early gene (IEG; c-fos, c-jun, jun-B, and zif/268) induction with the response of neuronal transcripts during the first 24 hr of a QA lesion within the rodent striatum. Following QA exposure, IEG mRNA induction periods extended from 30 min to 24 hr. Several characteristics of this prolonged transcriptional response suggest that separate cell populations (neuronal vs. glial) originate individual IEG phases during the first day of the lesion. The first IEG phase was rapid and peaked at 60 min. This initial IEG phase, likely neuronal in origin, was dominated by robust increases in the expression of c-fos, jun-B, and zif/268 mRNAs in contrast to small increases in c-jun expression. A second, delayed IEG phase was initiated after the first hour and extended to 24 hr. This IEG phase was more intense and continued beyond the period of neuronal survival as detected by the loss of neurotransmitter-specific mRNAs (preprotachykinin, preproenkephalin, and glutamic acid decarboxylase). During this phase, c-jun mRNA levels coordinately increased with c-fos. Interestingly, the transcriptional peak of the delayed IEG phase occurred between 4 and 12 hr, the time which corresponded to the rapid decline of neuronal transcripts.(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation-defective mutant of adenovirus type 5 containing a single altered E1a mRNA species.

A mutant of adenovirus type 5 containing an octanucleotide insert in region E1a of the viral genome was constructed. The insert was present in only one (13s) of the three overlapping mRNA's synthesized from this region. The insert was within the sequences removed by RNA splicing during the production of the other two nRNA's. The insertion resulted in a shift in the translational reading frame of the 13s mRNA and the probable premature termination of translation. The mutant was defective for viral DNA replication in HeLa cells and the transformation of rat embryo and baby rat kidney cells, indicating that a product encoded by the 13s nRNA is required for these two processes. Other early regions of the genome were expressed in HeLa cells infected by this mutant although in some cases the expression was decreased as compared with wild-type-infected cells.

Chromosomal localization of human gene for histidyl-tRNA synthetase: clustering of genes encoding aminoacyl-tRNA synthetases on human chromosome 5.

The gene encoding histidyl-tRNA synthetase was localized to human chromosome 5 using human-Chinese hamster ovary cell hybrids. Seven of the functionally related genes encoding the 20 different aminoacyl-tRNA synthetases have not been mapped in humans and four are located on a single chromosome, number 5, which represents less than 7% of the total genome. Thus, there appears to be significant clustering of this group of genes which may reflect their evolutionary relatedness or common factors involved in regulating their expression.

Molecular approach to analyzing the human 5p deletion syndrome, cri du chat.

DNA unique or low-copy fragments were isolated from a genomic DNA library specific for the short (p) arm of human chromosome 5. These chromosome 5p-specific DNA fragments were used to analyze, by Southern blot experiments, somatic cell hybrids that retained either a normal chromosome 5 homolog or a homolog with a partial deletion of 5p, which was derived from either of two persons with the common human deletion syndrome, cri du chat or 5p- syndrome. In these studies, two classes of DNA fragments were identified, those located outside the region deleted in the persons with cri du chat and those located within the deleted region. This latter class of DNA probes will help to define, at the molecular level, a region of 5p that is critical in producing the phenotype associated with the cri du chat syndrome.

A Sau3A polymorphism in the 5' end of the IT15 gene that nonrandomly segregates with the Huntington disease trinucleotide expansion.

Genomic clones encompassing the Huntington disease (HD) mutation were used to isolate a probe that detects size changes in the restriction fragments that contain the HD trinucleotide repeat (TNR). This probe also detects a frequent Sau3A polymorphism (allele sizes 1.8-kb and 2.7kb), which maps approximately 950bp from the TNR. Examination of a number of HD families established that the frequency of the Sau3A alleles did not differ significantly between control and HD populations; however, the HD expansion was always present on a chromosome that contained the 1.8-kb Sau3A allele. This association between a specific allele and the HD TNR expansion was significant and could provide a clue to the chromosomal elements that produce the trinucleotide expansion on the Huntington disease chromosome.

NMDA receptor overstimulation triggers a prolonged wave of immediate early gene expression: relationship to excitotoxicity.

Exposure of the rodent striatum to quinolinic acid (QA, N-methyl-D-aspartate receptor agonist) induces immediate early gene (IEG; c-fos, c-jun, jun-B, zif/268) expression that may extend 12-24 h after injection. In order to determine the specificity of the prolonged IEG response to the QA injection, the temporal pattern of c-fos mRNA expression was examined during the first 4 h after administration of saline or QA (40 micrograms). As early as 30 min after intrastriatal injection, both saline and QA increased c-fos mRNA levels. In the saline group, this increase in IEG expression was only transient and returned to baseline by 1 h. In contrast, c-fos mRNA levels within QA-injected animals continued to rise significantly at 1 and 4 h. In a second experiment, rats received 4 ng to 40-micrograms injections of QA followed by sacrifice at 6 h to determine if increasing QA doses caused the appearance of the prolonged IEG response phase. The prolonged IEG response was evident at 6 h only in animal groups that received higher dose ranges (4-40 micrograms) of QA. A final experiment was undertaken to determine if blockage of NMDA receptor stimulation would also inhibit the prolonged IEG response at 6 h in relationship to neuronal sparing evidenced at 24 h post-QA injection. The NMDA receptor antagonist, MK-801, blocked the prolonged IEG response at 6 h following QA (40 micrograms) injection while also preventing striatal neuropeptide mRNA decline by 24 h. Delaying the MK-801 administration for 1-2 h post-QA injection revealed that the intensity of the prolonged IEG mRNA response may be predictive of neuronal demise within the QA lesion site. These results suggest that prolonged IEG expression is associated with QA excitotoxicity of the rodent striatum and subsequent neuronal degeneration.

Assignment of human dihydrofolate reductase gene to band q23 of chromosome 5 and of related pseudogene psi HD1 to chromosome 3.

The chromosomal location of the human dihydrofolate reductase (DHFR; EC 1.5.1.3) gene that is amplified in a methotrexate-resistant human cell line has been investigated by screening a number of human-Chinese hamster ovary cell hybrids containing terminal and interstitial deletions in human chromosome 5. A correlation of genomic blotting data with the chromosome 5 constitution of the individual hybrids has allowed the assignment of the human DHFR gene to 5q23. The present work also establishes the location of the related intronless pseudogene psi HD1 in chromosome 3.

Genetic counterselective procedure to isolate interspecific cell hybrids containing single human chromosomes: construction of cell hybrids and recombinant DNA libraries specific for human chromosomes 3 and 4.

Counterselection against genes on human chromosome 5 was applied to interspecific human-Chinese hamster cell hybrids which retained this and one additional human chromosome in order to generate cell hybrids retaining single, nonselected human chromosomes. Using this procedure, stable cell hybrids which retain human chromosome 3 exclusively or human chromosome 4 exclusively were isolated. Complete recombinant genomic DNA libraries were prepared from each hybrid using the lambda cloning vector EMBL-4. These libraries represent sources of human DNA fragments derived specifically from chromosomes 3 and 4, respectively. Low-copy or unique human DNA fragments isolated from both libraries were analyzed to confirm their chromosomal origin and to determine the complexity of their hybridization patterns to total human DNA. These single human chromosome libraries represent a means to efficiently saturate chromosomes 3 and 4 with informative, polymorphic genetic markers. DNA fragments from the chromosome 4 library will be particularly useful in identifying additional genetic markers close to the Huntington's disease gene. The same genetic counterselective procedure can be utilized to derive several additional cell hybrids with single human chromosomes.

The mouse plasma membrane Ca2+ pump isoform 1 promoter: cloning and characterization.

The expression of plasma membrane Ca2+ pump (PMCA) is regulated by various hormones or agonists via multiple second messenger pathways. Two different 5' segments of the PMCA1 gene (isoform 1) were cloned from a mouse genomic library. While one segment contained the 3' end of intron 1 and exon 2, the other segment was found to encompass the 5'-flanking region of the gene, exon 1, and the 5' portion of intron 1. Sequence analysis of the 5'-flanking region suggested the presence of the putative promoter. Four sites for initiation of transcription (spanning 64 bp) were identified by RNase protection assay and primer extension analysis. The promoter region was very GC-rich, contained no "TATA box," but had a "CAAT box" at -51. Comparison of sequence with known cis-regulatory motifs disclosed that the 5'-flanking region has a number of potential regulatory elements including an AP-1 site at -354, AP-2 binding sites at -267 and -123, Sp1 binding sites at -127, -111, and +3, and a cyclic AMP response element binding protein site at -67. To demonstrate promoter activity, a segment containing 611 bp of the promoter region (from -442 to +169) was subcloned in front of a promoterless chloramphenicol acetyltransferase (CAT) gene. This segment was able to drive the expression of chloramphenicol acetyltransferase in transient transfections of mouse (or human) neuroblastoma cells as well as rat aortic endothelial cells. Deletion analysis demonstrated that a fragment from -256 to +169 showed strong promoter activity, while a fragment from -117 to +169 had CAT activity that was not different from the vector control. The promoter was stimulated threefold by phorbol ester and twofold by cyclic AMP. These results provide further proof indicating up-regulation of the PMCA1 gene by multiple second messenger pathways.

cDNA cloning and chromosomal localization (4q11-13) of a gene for statherin, a regulator of calcium in saliva.

On the basis of the known amino acid sequence of statherin, a human salivary protein, mixed synthetic oligonucleotides were synthesized and used to screen a cDNA library constructed from human parotid-gland mRNA. A cDNA clone coding for statherin was isolated from this library and has been completely sequenced. The cDNA represents a full-length (or nearly full-length) copy of an approximately 640-bp statherin mRNA. Statherin appears to be coded by a single-copy gene that maps to chromosome 4q11-4q13 when somatic-cell hybrids are used.

Phorbol ester induces both gene expression and phosphorylation of the plasma membrane Ca2+ pump.

Regulation of the plasma membrane Ca2+ pump in the cell is of critical importance in maintaining calcium homeostasis. Since protein kinase C is known to regulate functions of cellular proteins by direct phosphorylation or by inducing their gene expression, we investigated the possible involvement of protein kinase C in the regulation of the plasma membrane Ca2+ pump. The Ca2+ pump was isolated by immunoprecipitation from [32P]orthophosphate-labeled cultured rat aortic endothelial cells grown in the absence or presence of phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. PMA treatment of cells led to a rapid increase in the phosphorylation level (1.3-fold) within 5 min and a further increase to 2.9-fold after 3 h. Prolonged PMA treatment also induced the accumulation of the Ca2+ pump mRNA, followed by increased levels of the pump protein. The peak level of the pump mRNA induction occurred at 4 h and was 8-20-fold higher than the control culture without PMA. The rate of the Ca2+ pump protein accumulation was slower, reaching a maximum of 3.5-fold after 6 h. Induction of the pump mRNA was suppressed by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and by down-regulation of protein kinase C. Inactive phorbol ester 4 alpha-phorbol didecanoate also failed to mimic the PMA effect. These results suggest that the induction of Ca2+ pump expression is mediated by a protein kinase C-dependent mechanism. Furthermore, since the induction of the Ca2+ pump mRNA was blocked when cycloheximide and PMA were added together, this suggests that newly synthesized protein factor is needed to produce the mRNA induction. Our results suggest that protein kinase C is involved in the regulation of the Ca2+ pump in endothelial cells. At the protein level, it modifies the Ca2+ pump by phosphorylation, and at the gene level, it stimulates the expression of its mRNA and thereby increases the amount of the pump protein.