Increase In Steady-state Levels Research Articles

Monokine-induced Neutrophil Chemotactic Factor Gene Expression in Human Fibroblasts

Although fibroblasts are important in providing a structural framework for most tissues, they also appear to be active participants in the inflammatory process via the production of specific mediators. The production of inflammatory mediators by fibroblasts is especially important in relation to their strategic location within connective tissue as they may act as a cellular communication bridge between the interstitium and vasculature. In this paper, we demonstrate that fibroblasts may participate in these inflammatory reactions by the production of a neutrophil chemotactic factor (NCF) with characteristics similar to a recently isolated and cloned monocyte-derived NCF. Either tumor necrosis factor-alpha-, interleukin-1 alpha-, or interleukin-1 beta-stimulated fibroblasts showed both a time- and dose-dependent increase in steady-state levels of NCF mRNA and secretion of chemotactic activity. In contrast, lipopolysaccharide and interleukin-6 failed to induce fibroblast-derived NCF. The expression of fibroblast-derived NCF mRNA was first detectable by 30 min poststimulation, whereas chemotactic activity was significantly observed 3-4 h postchallenge. Heat-inactivated monokine (100 degrees C) failed to induce NCF mRNA expression, suggesting that only the active proteins are capable of inducing NCF. Gel filtration analysis using high pressure liquid chromatography indicated peak chemotactic activity with an approximate molecular mass of 8000 daltons. This peak of NCF activity was found to be relatively stable to both heat and trypsin inactivation. Specificity of the fibroblast-derived neutrophil chemotactic activity was demonstrated with inhibition of chemotaxis by the addition of neutralizing antibody directed against recombinant human neutrophil chemotactic factor. These data provide evidence that monokine-treated fibroblasts can synthesize a potent chemotactic agent with molecular and physicochemical characteristics similar to monocyte-derived NCF and that this factor may contribute to neutrophil-mediated disease processes.

Escherichia coli Endotoxin is a Major Regulator of Procollagenase, Gelatinase, and Stromelysin Secretion by Human Alveolar Macrophages

Metalloproteinases secreted by macrophages may have an important role in tissue injury during the inflammatory response resulting from gram-negative infectious processes. To begin to evaluate the potential direct roles of human alveolar macrophages in extracellular matrix injury, we have examined the regulation of secretion of several metalloproteinases (interstitial collagenase, 72 kD gelatinase/type IV collagenase, 90 kD gelatinase, and stromelysin) and also secretion of a counter-regulatory protein, the tissue inhibitor of metalloproteinases (TIMP). The influence of E coli endotoxin (LPS) on secretory rates of these proteins was assessed by specific ELISA assays, examination of metabolically labeled proteins by SDS-PAGE, and by Northern blot analysis of messenger RNA. We have found that LPS strikingly and selectively (compared with total new protein synthesis) upregulates the secretion of all metalloproteinases, including interstitial collagenase, gelatinases, and stromelysin, while having only a minor stimulatory effect on TIμP secretion. Northern analysis revealed very substantial increases in steady-state levels of messenger RNA for collagenase, suggesting transcriptional modulation of the LPS stimulation of metalloproteinase production.

Correlations between heparan sulfate metabolism and hepatoma growth

A rat hepatoma cell line (Gershenson et al., Science, 170:859-861, 1970) contains a dynamic steady-state pool of free heparan sulfate (HS) chains in the nucleus that increases in amount when growing cells reach confluence (Fedarko and Conrad, J. Cell Biol., 102:587-599, 1986). In logarithmically growing cells labeled with 35SO4(2-) steady-state levels of [35SO4]HS in the nucleus are altered by a variety of culture conditions. Rapidly dividing cells (doubling time = 18-22 h) growing under optimized conditions had steady-state levels of nuclear HS within the range of 40-50 pmol 35SO4 in nuclear HS/10(6) cells. The steady-state levels of nuclear HS were lowered by several changes in culture conditions, including 1) additions of 1 mM p-nitrophenyl-beta-D-xyloside, 0.25-0.5 mM (+)-catechin, 0.5 ng/ml transforming growth factor beta, 20 ng/ml phorbol-12-myristate-13-acetate, 1 mM dibutyryl cAMP, or 1 mM inositol-2-PO4; 2) decreased levels of D-glucose; or 3) deletions of serum, insulin, or inositol. In all cases lowering of the nuclear HS level was accompanied by an increase in the cell doubling times, suggesting a correlation in which nuclear HS levels must be optimized for maximal growth rates. When cells cultured under optimal growth conditions reached confluence, the level of nuclear HS increased threefold and the cells stopped dividing. The same culture conditions that lowered the steady-state levels of HS in the logarithmically growing cells prevented this rise in the nuclear HS as the cells reached confluence and resulted in loss of contact inhibition and overgrowth of the confluent cultures. These observations suggest a second correlation in which elevated nuclear HS levels are found when cell growth is inhibited at confluence; prevention of this rise results in continued growth. Consistent with this correlation between elevated nuclear HS and reduced growth rates, it was observed that addition of either 0.5 microgram/ml hydrocortisone or 0.05 microgram/ml retinoic acid to the culture medium of logarithmically growing cultures resulted in increases in steady-state levels of nuclear HS that were accompanied by increased cell doubling times. The two agents that increased the levels of nuclear HS in logarithmically growing cultures had little effect on levels of nuclear HS in confluent cells or on contact inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of ornithine decarboxylase gene expression in normal and transformed hamster embryo fibroblasts following stimulation by 12-O-tetradecanoylphorbol-13-acetate.

We have compared the regulation of ornithine decarboxylase (ODC) gene expression in primary cultures of hamster embryo fibroblasts and in two independently transformed hamster embryo cell lines. Previous studies have demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) can greatly potentiate the serum growth factor induction of ODC enzyme activity in transformed cells, but not in normal hamster embryo fibroblasts. Treatment of either normal or transformed cells with both TPA and serum yielded greater accumulations of ODC mRNA than with either treatment alone, which is consistent with changes at the protein level. However, treatment of the transformed cells with TPA and serum resulted in a greater increase in steady state levels of ODC mRNA than that observed using normal fibroblasts. The time course for the induction of ODC mRNA was similar for both normal and transformed cells with maximal accumulations 4-8 h after treatment. Studies with actinomycin D further suggests that ODC mRNA is comparatively long-lived in both normal and transformed cells. The accumulation of ODC mRNA after stimulation with TPA and serum is blocked by cycloheximide in normal hamster fibroblasts suggesting that this induction is dependent upon protein synthesis. In contrast, cycloheximide did not affect the accumulation of ODC mRNA under similar treatment conditions in transformed cells. This altered regulation of ODC gene expression in transformed hamster embryo fibroblasts cannot be explained by either gene rearrangement or the amplification of an ODC gene. These data suggest that transformation of hamster embryo cells results in a loss of cellular control over ODC gene regulation which includes an alteration in the requirement for protein synthesis for ODC mRNA accumulation.

Changes in gene expression associated with IFN-beta and IL-2-induced augmentation of human natural killer cell function.

NK function can be augmented by a variety of agents, including the cytokines IL-2 and IFN. The mechanisms associated with IL-2- and IFN-mediated augmentation of NK function are largely unknown. In order to learn more about the regulation of NK activity, we have studied changes in gene expression that occur upon treatment of a cloned line of NK cells (NK 3.3) with rIL-2 and rIFN-beta. Both IL-2 and IFN-beta induced rapid augmentation of lysis mediated by NK 3.3, which was significant within 1 h, peaked at 6 h of treatment, and declined by 12 h. This enhancement of lytic function was independent of proliferation and associated with a corresponding increase in steady state levels of RNA coding for both the nuclear proto-oncogene c-myb and for the IL-2R. These changes were specific in that RNA levels of another nuclear proto-oncogene, c-myc, were increased by IL-2 but not by IFN-beta, whereas HLA class I RNA levels were relatively unchanged by either IL-2 or IFN-beta treatment. Treatment of NK 3.3 with the combination of IL-2 and IFN enhanced both lysis and c-myb expression in an additive fashion. These findings suggest that c-myb may play a regulatory role in the cytolytic activity of NK cells.

Sequence changes preceding a Shine-Dalgarno region influence trpE mRNA translation and decay

In studies with a trpE promoter-strength measuring system we observed that constructs containing the Escherichia coli trp promoter and its adjacent transcribed region yielded lower levels of trpE protein than were expected. To analyze this observation we introduced mutational changes in the nucleotide sequence preceding the trpE Shine-Dalgarno region and examined their effects on trpE mRNA synthesis, translation and decay. We found that certain deletion, insertion and substitution mutations in the pre-Shine-Dalgarno region caused a two- to fivefold increase in trpE enzyme activity. These increases were accompanied by increases in steady-state levels of trpE mRNA. Pulse-chase analyses of trpE mRNA degradation revealed that the observed steady-state trpE mRNA levels correlated with changes in trpE mRNA stability. These findings are interpreted in terms of alternative models in which the primary effect of mutational changes that elevate trpE expression is to increase trpE mRNA translation, versus increasing trpE mRNA stability.

Regulation of CAD gene expression in mouse fibroblasts during the transition from the resting to the growing state

We have analyzed the steady-state levels of CAD mRNA and ATCase activity in BALB/c 3T3 mouse fibroblasts at quiescence and at various time points following the initiation of serum stimulation. Steady-state levels of CAD mRNA in 3T3 cells following 12 h of serum stimulation increased 10-fold over levels measured at quiescence. In contrast to the observed increase in steady-state levels of CAD mRNA, its rate of transcription increased only 3-fold, suggesting that the expression of CAD gene in these cells is regulated at both the transcriptional and post-transcriptional levels, to a major extent by the latter. These increases in CAD mRNA in serum-stimulated cells were followed by parallel increases in ATCase activity as well. When comparing DNA synthesis ([ 3H]thymidine uptake) to the accumulation of CAD mRNA and ATCase activity, it was observed that this accumulation occurred during the mid- to late-G 1 phase of the cell cycle. These results suggest that the expression of CAD gene is cell growth dependent and may be a prerequisite to DNA synthesis.

Regulation of 4F2 heavy-chain gene expression during normal human T-cell activation can be mediated by multiple distinct molecular mechanisms.

The 4F2 molecule belongs to the set of cell surface antigens which is induced following lectin- or antigen-mediated T-cell activation. The increase in 4F2 cell surface expression following lectin-mediated stimulation has been shown to be accompanied by a parallel increase in the steady-state levels of 4F2 heavy-chain (4F2HC) mRNA. The studies described in this report were designed to further elucidate the molecular mechanisms responsible for induction of 4F2HC gene expression following activation of normal resting human peripheral blood T cells. The low levels of mature 4F2HC mRNA in resting T cells were shown to be the result of a block to transcription elongation within the exon 1-intron 1 region of the 4F2HC gene rather than promoter inactivity. Phorbol myristate acetate stimulation of resting T cells resulted in a 20-fold increase in steady-state 4F2HC mRNA levels which was mediated by removal of this block to transcription elongation. The phorbol myristate acetate-induced increase in 4F2HC gene expression is distinct from previously described AP-1-mediated, phorbol ester-induced gene expression in that it requires new protein synthesis. Treatment of resting T cells with ionomycin plus PMA resulted in a 60-fold increase in 4F2HC mRNA levels. This induction was mediated by both an increase in promoter utilization and removal of the block to transcription elongation. Finally, by increasing the half-life of 4F2HC mRNA, cycloheximide treatment of resting T cells induced an approximately five fold increase in the levels of 4F2HC gene expression, although the physiologic significance of this mechanism remains unclear. These results demonstrate that the level of 4F2HC gene expression in normal peripheral blood T cells can be regulated by at least three distinct molecular pathways: (i) changes in promoter utilization, (ii) modulation of a block to transcription elongation, and (iii) alteration in mRNA stability.

Regulation of 4F2 heavy-chain gene expression during normal human T-cell activation can be mediated by multiple distinct molecular mechanisms.

The 4F2 molecule belongs to the set of cell surface antigens which is induced following lectin- or antigen-mediated T-cell activation. The increase in 4F2 cell surface expression following lectin-mediated stimulation has been shown to be accompanied by a parallel increase in the steady-state levels of 4F2 heavy-chain (4F2HC) mRNA. The studies described in this report were designed to further elucidate the molecular mechanisms responsible for induction of 4F2HC gene expression following activation of normal resting human peripheral blood T cells. The low levels of mature 4F2HC mRNA in resting T cells were shown to be the result of a block to transcription elongation within the exon 1-intron 1 region of the 4F2HC gene rather than promoter inactivity. Phorbol myristate acetate stimulation of resting T cells resulted in a 20-fold increase in steady-state 4F2HC mRNA levels which was mediated by removal of this block to transcription elongation. The phorbol myristate acetate-induced increase in 4F2HC gene expression is distinct from previously described AP-1-mediated, phorbol ester-induced gene expression in that it requires new protein synthesis. Treatment of resting T cells with ionomycin plus PMA resulted in a 60-fold increase in 4F2HC mRNA levels. This induction was mediated by both an increase in promoter utilization and removal of the block to transcription elongation. Finally, by increasing the half-life of 4F2HC mRNA, cycloheximide treatment of resting T cells induced an approximately five fold increase in the levels of 4F2HC gene expression, although the physiologic significance of this mechanism remains unclear. These results demonstrate that the level of 4F2HC gene expression in normal peripheral blood T cells can be regulated by at least three distinct molecular pathways: (i) changes in promoter utilization, (ii) modulation of a block to transcription elongation, and (iii) alteration in mRNA stability.

Isolation and structural characterization of the human 4F2 heavy-chain gene, an inducible gene involved in T-lymphocyte activation.

The human 4F2 cell surface antigen is a 120-kilodalton (kDa) disulfide-linked heterodimer which is composed of an 80- to 90-kDa glycosylated heavy chain (4F2HC) and a 35- to 40-kDa nonglycosylated light chain (4F2LC). 4F2 belongs to a family of inducible cell surface molecules which are involved in T-lymphocyte activation and growth. To better understand the molecular mechanism(s) that controls 4F2HC gene expression in both resting and activated T cells, a 4F2HC human genomic clone was isolated and structurally characterized. The 4F2HC gene spans 8 kilobases of chromosome 11 and is composed of nine exons. The 5' upstream region of the gene displays several properties which are characteristic of housekeeping genes. It is G+C rich and hypomethylated in peripheral blood lymphocyte DNA and contains multiple binding sites for the Sp1 transcription factor while lacking TATA or CCAAT sequences. This region of the gene also displays sequence homologies with several other inducible T-cell genes, including the interleukin-2, interleukin-2 receptor alpha chain, dihydrofolate reductase, thymidine kinase, and transferrin receptor genes. A 255-base-pair fragment of the 4F2HC gene which contains 154 base pairs of the 5' flanking sequence was able to efficiently promote expression of the bacterial chloramphenicol acetyltransferase gene in human Jurkat T cells, indicating that it contains promoter or enhancer (or both) sequences. Analyses of chromatin structure in resting and lectin-activated T cells revealed the presence of stable DNase I-hypersensitive sites within both the 5' flanking and intron 1 regions of the 4F2HC gene. Although the 4F2HC gene displayed many of the structural features characteristic of a constitutively expressed gene, lectin-mediated activation of resting peripheral blood T lymphocytes resulted in a dramatic increase in steady-state levels of 4F2HC mRNA.

Isolation and structural characterization of the human 4F2 heavy-chain gene, an inducible gene involved in T-lymphocyte activation.

The human 4F2 cell surface antigen is a 120-kilodalton (kDa) disulfide-linked heterodimer which is composed of an 80- to 90-kDa glycosylated heavy chain (4F2HC) and a 35- to 40-kDa nonglycosylated light chain (4F2LC). 4F2 belongs to a family of inducible cell surface molecules which are involved in T-lymphocyte activation and growth. To better understand the molecular mechanism(s) that controls 4F2HC gene expression in both resting and activated T cells, a 4F2HC human genomic clone was isolated and structurally characterized. The 4F2HC gene spans 8 kilobases of chromosome 11 and is composed of nine exons. The 5' upstream region of the gene displays several properties which are characteristic of housekeeping genes. It is G+C rich and hypomethylated in peripheral blood lymphocyte DNA and contains multiple binding sites for the Sp1 transcription factor while lacking TATA or CCAAT sequences. This region of the gene also displays sequence homologies with several other inducible T-cell genes, including the interleukin-2, interleukin-2 receptor alpha chain, dihydrofolate reductase, thymidine kinase, and transferrin receptor genes. A 255-base-pair fragment of the 4F2HC gene which contains 154 base pairs of the 5' flanking sequence was able to efficiently promote expression of the bacterial chloramphenicol acetyltransferase gene in human Jurkat T cells, indicating that it contains promoter or enhancer (or both) sequences. Analyses of chromatin structure in resting and lectin-activated T cells revealed the presence of stable DNase I-hypersensitive sites within both the 5' flanking and intron 1 regions of the 4F2HC gene. Although the 4F2HC gene displayed many of the structural features characteristic of a constitutively expressed gene, lectin-mediated activation of resting peripheral blood T lymphocytes resulted in a dramatic increase in steady-state levels of 4F2HC mRNA.

Elastase regulates the synthesis of its inhibitor, alpha 1-proteinase inhibitor, and exaggerates the defect in homozygous PiZZ alpha 1 PI deficiency.

The net balance of neutrophil elastase, an enzyme that degrades many components of the extracellular matrix, and its inhibitor, alpha-1-proteinase inhibitor (alpha 1 PI), is thought to be a critical determinant in the development of destructive lung disease, especially in individuals with homozygous alpha 1 PI deficiency. Synthesis and secretion of alpha 1 PI has been recently demonstrated in cells of mononuclear phagocyte lineage, including peripheral blood monocytes and tissue macrophages. In this study we show that alpha 1 PI gene expression in human monocytes and bronchoalveolar macrophages is affected by a novel mechanism, whereby elastase directly regulates the synthesis of its inhibitor. In nanomolar concentrations, neutrophil or pancreatic elastase mediates a dose- and time-dependent increase in steady state levels of alpha 1 PI mRNA and in the rate of synthesis of alpha 1 PI in human monocytes and bronchoalveolar macrophages. Antisera to neutrophil elastase or pretreatment of elastase with the serine proteinase inhibitor diisopropylfluorophosphate abrogates the effect of elastase on alpha 1 PI expression. Elastase also stimulates the synthesis of alpha 1 PI in monocytes from homozygous PiZZ alpha 1 PI-deficient individuals, but has no effect on the rate of secretion; hence, the enzyme mediates an effect on alpha 1 PI that increases the intracellular accumulation of inhibitor and exaggerates the intrinsic defect in secretion of alpha 1 PI that characterizes the homozygous PiZZ alpha 1 PI deficiency.

Alpha 1-adrenergic stimulation of cardiac gene transcription in neonatal rat myocardial cells. Effects on myosin light chain-2 gene expression.

Previous studies have demonstrated that alpha-adrenergic stimulation of cultured, neonatal rat myocardial cells results in an increase in intracellular volume and protein content of cultured neonatal rat myocardial cells. Utilizing this model of cardiac hypertrophy, we have examined the effects of alpha-adrenergic stimulation on the accumulation of sarcomeres and the expression of a rat cardiac myofibrillar gene, myosin light chain-2 (MLC-2). Following alpha-adrenergic stimulation, cultured myocardial cells displayed a severalfold increase in the number of sarcomeric units, as assessed by electron microscopy, an increase in cellular MLC-2 content, and a 2-3 fold increase in the steady state levels of MLC-2 mRNA. This effect of alpha-adrenergic stimulation was accompanied by a 2-3-fold increase in total transcriptional activity, which was dependent on the concentration and duration of exposure to the agonist, and displayed alpha 1-adrenergic receptor specificity. The transcriptional response was not immediate, with a lag period of at least 1 h, and a maximal effect required continuous occupancy of the receptor. The increase in steady state levels of MLC-2 mRNA is regulated, in part, at the level of transcription of the cardiac MLC-2 gene. These results suggest that alpha 1-adrenergic stimulation may be important in the growth of the neonatal heart through the activation of total transcriptional activity. In addition, increases in the levels of myofibrillar proteins during myocardial cell growth and hypertrophy, may be mediated in part by the stimulation of transcription of myofibrillar genes.

Functional mapping of Autographa california nuclear polyhedrosis virus genes required for late gene expression.

A plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of an Autographa california nuclear polyhedrosis virus (AcNPV) late gene promoter was constructed. This plasmid (pL2cat) also contained the AcNPV hr5 enhancer element. Transient-expression assay experiments indicated that the late promoter was active in Spodoptera frugiperda cells cotransfected with pL2cat and AcNPV DNA but not when pL2cat was transfected alone. Low levels of CAT activity were observed in cells cotransfected with pL2cat and pIE-1 DNAs. However, CAT activity was not induced in a similar plasmid which lacked the cis-linked enhancer element, indicating that the enhancer was required for expression of the late gene. Cotransfection mapping of pPstI clones of AcNPV DNA indicated that the pPstI-G clone of viral DNA contained a factor which further stimulated late gene expression 3- to 10-fold. Transient-expression assay analysis of subclones of pPstI-G localized the trans-active factor to a 3.0-kilobase XbaI fragment. The nucleotide sequence of this fragment was determined and found to contain three potential open reading frames. A computer-assisted search of a protein database revealed no closely related proteins. One of the predicted amino acid sequences contained potential metal-binding domains similar to those found in nucleic acid-binding proteins. Subcloning and subsequent CAT assay indicated that two of the open reading frames were required for the activation of pL2cat. Nuclease S1 mapping of infected and transfected RNAs indicated that the two open reading frames were transcribed as delayed-early genes. Quantitative nuclease S1 analysis and differential DNA digestion of recovered plasmids indicated that the activation of pL2cat was not due to an increase in steady-state levels of mRNA replication of the viral DNA.

Induction of macrophage Ia antigen expression by rIFN-gamma and down-regulation by IFN-alpha/beta and dexamethasone are mediated by changes in steady-state levels of Ia mRNA.

In previous studies, the induction of Ia antigens on murine peritoneal exudate macrophages by recombinant IFN-gamma (rIFN-gamma) and the antagonism of rIFN-gamma-induced Ia expression by the inhibitors IFN-alpha/beta and glucocorticoids have been examined. In this report, these findings have been extended to an analysis of total or cytoplasmic mRNA from macrophage cultures treated with rIFN-gamma in the absence or presence of these two inhibitors. Recombinant IFN-gamma induced a 5.7- to 6.5-fold increase in steady-state levels of Ia (A alpha-specific) mRNA. Coordinate increases in steady-state mRNA for A beta, and E alpha were observed in response to rIFN-gamma. Maximum induction occurred 24 hr post-treatment and required the continued presence of rIFN-gamma. Induction of A alpha-specific mRNA was sensitive to the protein synthesis inhibitor cycloheximide. Simultaneous treatment of macrophage cultures with rIFN-gamma and IFN-alpha/beta or the glucocorticoid dexamethasone (DEX) resulted in a significant decrease in steady-state, A alpha-specific mRNA levels compared with treatment with rIFN-gamma alone. This analysis suggests that both the induction of Ia expression by rIFN-gamma, and the antagonism of rIFN-gamma-induced Ia gene expression by IFN-alpha/beta and DEX, are regulated by cognate changes in Ia mRNA.

Cold-induced changes in Ca2+ transport in duckling skeletal muscle mitochondria.

Intermyofibrillar mitochondria were isolated from skeletal muscle (gastrocnemius) of cold-acclimated (4 degrees C) or control (30 degrees C) 4-wk-old ducklings. Ca2+ transport in the mitochondria was studied with the Ca2+-sensitive dye arsenazo III. The mitochondria actively took up Ca2+ but at a lower rate in mitochondria from cold-acclimated than from control ducklings. After addition of the Ca2+ uptake inhibitor ruthenium red, a low rate of Ca2+ release was revealed; this rate was, however, higher in the cold-acclimated than in the control ducklings. Furthermore, these nonmammalian mitochondria were also found to possess a mechanism for Na+-stimulated Ca2+ efflux. This mechanism was specific for Na+ (no effects with choline+ or K+, a small effect with Li+). The effect was maximal with 20 mM Na+, which led to a fourfold increase in rate of Ca2+ efflux. Cold acclimation led to a doubling of the rate of this Na+-stimulated Ca2+ release. Due to these alterations in Ca2+ fluxes caused by cold acclimation, the resultant free extramitochondrial Ca2+ levels were higher in mitochondria from cold-acclimated than from control ducklings. The basal level was doubled (from 0.26 to 0.50 microM) as was the increase in steady-state level caused by 10 mM Na+ (from +0.4 to +0.8 microM). The significance of these alterations for acclimation to cold is discussed.

Haloperidol increases proenkephalin mRNA levels in the caudate-putamen of the rat: a quantitative study at the cellular level using in situ hybridization

Previous immunocytochemical studies have shown that the opioid peptides, Met-enkephalin and Leu-enkephalin, are present in medium-sized, spiny projection neurons of the caudate-putamen. It has also been demonstrated that chronic treatment of rats with the dopamine receptor blocker, haloperidol, results in an increase in the levels of enkephalin peptides and proenkephalin mRNA in this brain region. To determine whether this increase in proenkephalin mRNA content is exhibited by all enkephalinergic neurons of the caudate-putamen or by only a subpopulation, we have used in situ nucleic acid hybridization to examine the haloperidol-induced increase in proenkephalin mRNA levels at the cellular level. Results of in situ hybridization suggest that all enkephalinergic neurons in the caudate-putamen can respond to haloperidol treatment with an increase in steady state levels of proenkephalin mRNA, and that the mean induction is an approximate 3-fold increase in the message levels. This suggests that dopamine exerts a tonic inhibitory effect on the expression of the proenkephalin gene in all of the enkephalinergic neurons of the caudate-putamen. Dot blot analysis indicated a 2.4-fold increase in the tissue levels of this mRNA. The agreement between the in situ hybridization results and dot blot analysis supports in situ hybridization as a reliable method for quantitative studies of alterations in neuropeptide precursor mRNAs in the brain.

Rapid mobilization of cellular Ca2+ in bovine parathyroid cells evoked by extracellular divalent cations. Evidence for a cell surface calcium receptor.

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in dissociated bovine parathyroid cells using the fluorescent indicator quin-2 or fura-2. Small increases in the concentration of extracellular Ca2+ produced relatively slow, monophasic increases in [Ca2+]i in quin-2-loaded cells, but rapid and transient increases followed by lower, yet sustained (steady-state), [Ca2+]i increases in fura-2-loaded cells. The different patterns of change in [Ca2+]i reported by quin-2 and fura-2 appear to result from the greater intracellular Ca2+-buffering capacity present within quin-2-loaded cells, which tends to damp rapid and transient changes in [Ca2+]i. In fura-2-loaded parathyroid cells, other divalent cations (Mg2+, Sr2+, Ba2+) also evoked transient increases in [Ca2+]i, and their competitive interactions suggest that they all affect Ca2+ transients by acting on a common site. In contrast, divalent cations failed to cause increases in steady-state levels of cytosolic Ca2+. Low concentrations of La3+ (0.5-10 microM) depressed steady-state levels of cytosolic Ca2+ elicited by extracellular Ca2+ but were without effect on transient increases in [Ca2+]i elicited by extracellular Ca2+, Mg2+ or Sr2+, suggesting that increases in the steady-state [Ca2+]i arise from the influx of extracellular Ca2+. Mg2+- and Sr2+-induced cytosolic Ca2+ transients persisted in the absence of extracellular Ca2+ but were abolished by pretreatment with ionomycin. These results show that cytosolic Ca2+ transients arise from the mobilization of cellular Ca2+ from a nonmitochondrial pool. Extracellular divalent cations thus appear to act at some site on the surface of the cell, and this site can be considered a "Ca2+ receptor" which enables the parathyroid cell to detect small changes in the concentration of extracellular Ca2+.

Carboxyl-terminal sequence of entactin deduced from a cDNA clone.

Entactin is a widely distributed basement membrane sulfated glycoprotein of approximately equal to 150 kDa. The entactin gene is expressed early in mouse embryogenesis. Two cDNA clones complementary to rat entactin mRNA were isolated by antibody screening of an oligo(dT)-primed cDNA library constructed in the lambda gt11 expression vector. One of the clones, lambda 1E, was subcloned into plasmid pBR322 and further characterized. The clone contained sequences complementary to an mRNA species 6 kilobases in length. This mRNA was translated in rabbit reticulocyte lysates to yield a polypeptide of 143 kDa that was precipitated with anti-entactin antiserum. The cDNA insert, 1328 base pairs long, was sequenced and found to contain an open reading frame of 729 base pairs that coded for 243 amino acids at the carboxyl terminus of entactin. Analysis of the peptide revealed no extended alpha-helical or beta-sheet secondary structures. Radiolabeled probes prepared by nicktranslation of p lambda 1E were used to monitor the steady-state levels of entactin mRNA in F9 embryonal carcinoma cells that were induced to differentiate by exposure to retinoic acid and dibutyryl cyclic AMP. The increase in steady-state levels of entactin mRNA lagged behind the increase in mRNA for the B2 chain of laminin, suggesting that laminin and entactin are independently rather than coordinately regulated.

Novel induction by herpes simplex virus of hybrid interferon gene transcripts driven by the strong cytomegalovirus IE94 promoter

We have constructed stable DNA-transfected LTK+ cell lines containing two different coselected hybrid interferon (IFN) genes driven by the usually strong and constitutive promoter from the immediate-early 94K protein (IE94) gene of simian cytomegalovirus. Surprisingly, and unlike hybrid IE94-chloramphenicol acetyltransferase gene constructs, both of the IE94-IFN genes (one with and one without the complex spliced intron region) produced relatively low basal titers of biologically active human IFN in the mouse cell lines. However, IFN expression could be stimulated up to 120-fold by superinfection with herpes simplex virus (HSV), although not with cytomegalovirus. To examine the mechanism of this unexpected HSV induction process, we measured the levels of both IE94-IFN mRNA and IFN protein produced under various infection protocols. Compared with similar previously characterized cell lines containing hybrid IFN genes under the control of HSV IE or delayed-early (DE) promoters, activation of IFN expression first occurred at an intermediate time. Both IE94-IFN cell lines also produced an unusual pattern of response to infection with the HSV IE regulation-deficient mutants tsK and tsB7: stimulation of IFN synthesis occurred in the absence of a functional HSV IE175 (or ICP4) gene product, but did not occur in the absence of uncoating of virus capsids. Cycloheximide treatment (without virus infection) also gave a rapid 30-fold increase in steady-state levels of correctly initiated mRNA from both types of IE94-IFN hybrid genes, but had no effect on cells containing the IE175-IFN construct. Therefore, we conclude that the use of the IE94-IFN constructs identifies a novel HSV regulatory response that requires a previously unrecognized function of HSV and does not involve either IE175 or the pre-IE "virion factor" trans-activators that are known to stimulate transcription of HSV IE and DE genes, respectively.