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H A van Heugten

Publications and source records attributed to H A van Heugten.

At least 19 recordsLinked to original sources
PubMed2000

Endothelin-1 responsiveness of a 1.4 kb phospholamban promoter fragment in rat cardiomyocytes transfected by the gene gun.

The transcriptional regulation of an isolated rat phospholamban (PL) promoter fragment in rat cardiomyocytes was analyzed by applying a new method to reach substantially higher transfection efficiencies: gene gun biolistics. The gene gun transfection method was optimized for application to primary cultures of rat neonatal cardiomyocytes. Cells, cultured at different densities (0.75-1.50x10(5)cells/cm(2)) in serum-free medium, were transfected with DNA coated gold particles. A transfection efficiency of up to 10% could be achieved (compared to <1% with other methods) by the gene gun as checked using a RSV- beta-Gal construct. Cardiomyocytes were stimulated by endothelin-1 (ET-1) (10(-8)M) to induce hypertrophy, thereby yielding the characteristic changes in gene expression (upregulation of Atrial Natriuretic Factor (ANF) and downregulation of PL). The basal activity of an ANF promoter fragment (increasing from the lowest to highest density 2.6-fold) and its ET-1 inducibility (only significant upregulation of 2.6-fold, at lowest density) appeared to be dependent on the plating density of the cardiomyocytes. A PL promoter fragment was isolated, sequenced and 1.4 kb was subcloned in a luciferase reporter vector. The basal activity of the PL promoter fragment was not dependent on the plating density. ET-1 did not downregulate the PL promoter, rather a significant upregulation (1.4-fold) was found at the highest plating density. In conclusion, plating density of the cardiomyocytes can influence promoter activity as shown with an ANF promoter fragment. A newly isolated and sequenced rat PL promoter fragment did not direct gene expression as expected on basis of downregulation of the PL gene by ET-1 observed in this model.

Animals↗
PubMed1998

Sarcoplasmic reticulum Ca2+ ATPase promoter activity during endothelin-1 induced hypertrophy of cultured rat cardiomyocytes.

OBJECTIVES: Characterization of an in vitro model of endothelin-1 induced hypertrophy of cultured neonatal rat ventricular myocytes and subsequent analysis of transcription regulation of the rat promoter of the sarcoplasmic reticulum Ca2+ ATPase gene. METHODS: Neonatal rat ventricular myocytes were cultured in serum free medium and hypertrophy was induced by addition of endothelin-1 to 10(-8) M up to 48 h. Hypertrophy was characterized biochemically, and gene expression regulation was evaluated by Northern blotting. A sarcoplasmic reticulum Ca2+ ATPase promoter fragment, isolated from a rat library was cloned in a reporter vector. Promoter activity during hypertrophy was assessed after transfection of the reporter plasmid to cultured cardiomyocytes. RESULTS: Stimulation with endothelin-1 resulted in increased cell size, as indicated by protein/DNA ratio as well as by augmented protein synthesis. When compared to angiotensin II or alpha 1-adrenergic agonist, endothelin-1 was the strongest inducer of hypertrophy (protein/DNA ratio) after 48 h of stimulation. Endothelin-1 induced hypertrophy was accompanied by a twofold increase in total RNA content per cell as well as to increased glyceraldehydephosphate dehydrogenase mRNA levels. The level of atrial natriuretic factor mRNA was increased more than twofold, relative to glyceraldehydephosphate dehydrogenase, while the expression of the sarcoplasmic reticulum Ca2+ pump and phospholamban genes was decreased (by 26 and 49%, respectively) after induction of hypertrophy by stimulation with endothelin-1. In the same model, a 1.9 kb sarcoplasmic reticulum Ca2+ pump gene promoter fragment (including 0.4 kb of the 5' UTR of the mRNA) directed down-regulation of the expression of the reporter gene to the same magnitude as endogenous Ca2+ pump mRNA relative to glyceraldehydephosphate dehydrogenase mRNA. However, absolute mRNA level per cell did not change for either the reporter gene or the endogenous Ca2+ pump. CONCLUSIONS: Endothelin-1 can induce phenotypic changes in cultured rat ventricular myocytes that are reminiscent of hypertrophy in vivo. In this model, a 1.9 kb sarcoplasmic reticulum Ca2+ pump promoter fragment directed gene expression of a reporter gene identical to the endogenous regulation of the Ca2+ pump. Furthermore, expression of the Ca2+ pump during hypertrophy was only downregulated when compared to (increased levels of) glyceraldehydephosphate dehydrogenase mRNA, but absolute Ca2+ ATPase mRNA amounts remained unchanged. This suggests that the Ca2+ pump promoter is not responding to the increase in transcriptional activity that accompanies hypertrophy.

Animals↗
PubMed1997

Angiotensin II-mediated growth and antigrowth effects in cultured neonatal rat cardiac myocytes and fibroblasts.

Angiotensin II (Ang II) stimulates cardiovascular growth and remodeling via AT1 receptors. Recent experiments have shown that Ang II may also exert antiproliferative effects via AT2 receptors. We studied the effects of Ang II on protein and DNA content and synthesis rate in unstimulated and endothelin-1 (ET-1)-stimulated neonatal rat cardiomyocytes and fibroblasts, isolated from 1-3-day-old Wistar strain pups. Total protein and total DNA, as well as [3H]leucine and [3H]thymidine incorporation were measured following incubation with either vehicle, Ang II, ET-1 or Ang II+ET-1, both in the presence or absence of the AT1 receptor blocker losartan or the AT2 receptor blocker PD123319. In myocytes, ET-1 increased total protein (+38% relative to control) as well as [3H]leucine (+66%) and [3H]thymidine (+77%) incorporation. Ang II did not affect any of these parameters, nor did it influence the ET-1-induced responses. However, in the presence of PD123319 Ang II stimulated [3H]leucine (+24%) and [3H]thymidine (+30%) incorporation. In fibroblasts, ET-1 and Ang II did not significantly affect total DNA and [3H]thymidine incorporation. Ang II tended to increase total protein in these cells, an effect which was significant only in the presence of PD123319 (+17%). Ang II stimulated [3H]leucine incorporation (+24%) in fibroblasts. This effect was absent with losartan and enhanced in the presence of PD123319. These data demonstrate that AT1 receptor-mediated proliferative effects of Ang II in neonatal cardiac cells may become apparent only when its AT2 receptor-mediated antigrowth effects are blocked. The net growth effect of Ang II therefore depends on the cellular AT1/AT2 receptor ratio. Ang II does not appear to interfere with ET-1-induced effects.

Angiotensin II↗
PubMed1997

Cross-talk between receptor-mediated phospholipase C-beta and D via protein kinase C as intracellular signal possibly leading to hypertrophy in serum-free cultured cardiomyocytes.

Phospholipase C-beta (PLC-beta) signalling via protein kinase C (PKC) has been recognized as a major route by which stimuli such as alpha1-adrenergic agonists, endothelin-1 (ET-1) and angiotensin II (Ang II) induce hypertrophy of myocytes. The goal of this study was to evaluate the role of phospholipase D (PLD) in contributing to the formation of the PKC activator 1,2-diacylglycerol (1,2-DAG) and to study the mechanism(s) of PLD activation by agonists. Stimulation of serum-free cultured neonatal rat cardiomyocytes with ET-1 (10(-8)M), phenylephrine (PHE, 10(-5)M) or Ang II (10(-7)M) resulted in a rapid (0-10 min) activation of PLC-beta to an extent (ET-1>PHE>Ang II) that correlated with the magnitude of stimulation of protein synthesis ([3H]leucine incorporation into protein) measured after 24 h. Phorbol 12-myristate 13-acetate (PMA, 10(-6)M) and ET-1 were equipotent in stimulating protein synthesis. ET-1 and PMA, but not PHE and Ang II stimulated [3H]choline formation from labelled PtdCho after a lag-phase of about 10 min. That this [3H]choline formation was due to the action of PLD was confirmed by measurement of phosphatidylgroup-transfer from cellular [14C]palmitoyl-phosphatidylcholine to exogenous ethanol. ET-1 and PHE, to much lesser extent, produced a rapid (0-5 min) translocation of PKC- immunoreactivity from the cytosol to the membrane fraction, whereas no intracellular redistribution of PKC-alpha, -delta and -xi immunoreactivities was observed. PMA caused translocation of PKC-alpha, PKC-epsilon as well as PKC-delta. Cellular redistribution of PKC activity measured by [32P]-incorporation into histone III-S was not observed with ET-1 and PHE, but only with PMA stimulation. Down-regulation of PKC isozymes by 24 h pretreatment of cells with PMA or blockade of PKC by chelerythrine (10(-4)M) inhibited ET-1 and PMA stimulated [3H]choline production. Staurosporine (10(-6)M) had, however, no effect. In conclusion, the results indicate that in serum-free cultured cardiomyocytes, ET-1 initially activates PLC-beta and after a lag-phase PLD, whereas PHE and Ang II activate only PLC-beta. PLC-beta stimulated by ET-1, may cross-talk with PLD via translocation of PKC-epsilon. These signals are possibly linked to the hypertrophic response.

Angiotensin II↗
PubMed1995

Alpha-adrenergic agonist and endothelin-1 induced intracellular Ca2+ response in the presence of a Ca2+ entry blocker in cultured rat ventricular myocytes.

Previously we demonstrated that stimulation of cultured neonatal rat ventricular myocytes by either alpha 1-adrenergic agonist or endothelin-1 resulted in a rapid formation of total inositolphosphates, although the levels of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate did not rise significantly. The aim of this study was to examine whether stimulation by alpha 1-adrenergic agonist and endothelin-1 could still elicit phosphatidylinositol cycle mediated intracellular Ca2+ mobilization in these cells. The intracellular free Ca2+ concentration ([Ca2+]i) was measured by single cell imaging dual wavelength fluorescence microscopy in Fura-2-loaded cardiomyocytes. The interference of agonist induced [Ca2+]i responses by the beat to beat variation of [Ca2+]i was prevented by arresting the cells with the Ca2+ entry blocker diltiazem (10 microM). The [Ca2+]i response (expressed as % of baseline ratio of fluorescence intensities of Fura-2 at 340 nm and 380 nm excitation wavelength), induced by phenylephrine (10(-4) M) and endothelin-1 (10(-8) M) was small, up to 20% of baseline after 9-20 min. In contrast, Ca(2+)-influx induced by incubation in Na(+)-free buffer caused a steep increase of [Ca2+]i up to 150% of baseline after 30 s. Analysis of single cells following stimulation with phenylephrine or endothelin-1 showed heterogeneity with respect to a rise in [Ca2+]i. However, if rapid Ca(2+)-influx was induced by incubation in Na(+)-free buffer, [Ca2+]i responses in individual myocytes occurred homogeneously. It is concluded that the alpha 1-adrenergic agonist and endothelin-1 induced [Ca2+]i responses are delayed in time, small and quite heterogeneous among cells. The findings are in agreement with earlier observations which revealed no detectable overall increase of the Ca2+ releasing inositolphosphates under these conditions and suggest that other second messengers, such as 1,2-diacylglycerol, are involved in the agonist mediated Ca2+ signals.

Adrenergic alpha-Agonists↗
PubMed1995

Endothelin-1-induced phospholipase C-beta and D and protein kinase C isoenzyme signaling leading to hypertrophy in rat cardiomyocytes.

We have previously demonstrated that stimulation of cultured rat neonatal cardiomyocytes by endothelin-1 (ET-1) induces rapid activation of phospholipase C-beta (PLC-beta), accompanied by transient expression of proto-oncogenes and subsequent development of hypertrophy and characteristic phenotypic changes. In the present study we examined the ET-1-induced hypertrophic response in relation to the initial signaling by phospholipase D (PLD) and protein kinase C (PKC). ET-1 (10(-8) M) induced hypertrophy after 48 h, as judged by protein/DNA ratio. The formation (0.5 h) of 14C-labeled phosphatidylethanol ([14C]PEth) in the presence of exogenous ethanol (0.5%) in [14C]palmitate prelabeled cells, which reflects the PLD activity, was increased 1.9- and 5.6-fold by ET-1 and phorbolester (PMA, 10(-6) M), respectively. The translocation of PKC isoforms from the cytosol to the membrane fraction was examined by immunoblot analysis using specific antibodies for PKC-alpha and -epsilon. ET-1 caused a rapid (within 15 s) and sustained disappearance of PKC-epsilon but not of PKC-alpha, from the cytosol. The translocation of PKC-epsilon to the membrane fraction was just detectable. However, PMA (10(-7) M) showed a rapid, sustained, and clearly detectable translocation of PKC-alpha and PKC-epsilon. The results indicate that the ET-1-induced development of hypertrophy via activation of distinct PKC isoenzymes may be initiated not only by PLC-beta but also by PLD signaling.

Animals↗
PubMed1994

Angiotensin II induced expression of transcription factors precedes increase in transforming growth factor-beta 1 mRNA in neonatal cardiac fibroblasts.

Angiotensin II (ANG II), a potent vasoconstricting peptide, may act as a growth factor for cardiac muscle cells and induce hypertrophy. We examined the molecular phenotype of neonatal rat cardiac fibroblasts in relation to ANG II by studying the expression pattern of three transcription factors (Egr-1, c-fos and c-jun) and the transforming growth factor-beta 1 (TGF-beta 1). ANG II did not affect cell proliferation and growth of serum deprived neonatal cardiac fibroblasts as predicted from their DNA and protein contents. The expression of Egr-1 and c-fos was induced as early as 15 min that reached maximal levels at 45 min and declined thereafter, whereas c-jun was induced at 45 min and remained elevated up to 2 hrs of ANG II addition. ANG II up-regulated the expression of TGF-beta 1, which became apparent after 1 hr of incubation and reached a plateau between 16-48 hrs. Our results indicate that ANG II transiently stimulates the expression of transcription factors, which may up-regulate TGF-beta 1, that in turn could contribute to the process of myocardial extra-cellular matrix remodeling in hypertrophy.

Angiotensin II↗
PubMed1994

Distinct alpha 1-adrenergic agonist- and endothelin-1-evoked phosphoinositide cycle responses in cultured neonatal rat cardiomyocytes.

Previously it was shown by us and others in cultured neonatal rat cardiomyocytes that the desensitization of the phenylephrine (PHE)- and endothelin-1 (ET-1)-mediated response of phospholipase C (PLC) was receptor-specific. The aim of this study was to characterize receptor-dependent specificities downstream of PLC. PHE (10(-4)M) as well as ET-1 (10(-8) M) stimulated the total [3H]inositolphosphate ([3H]InsPn, predominantly [3H]Ins(4)P) formation to about the same extent whereas Ins(1,4,5)P3 and Ins(1,3,4,5)P4 did not increase. Yet, ET-1 but not PHE stimulated Ins(1,3,4)P3 and Ins(3,4)Pn formation. Activation of PLC in saponin-permeabilized cells by GTP gamma S- and Ca2+ gave predominantly the formation of Ins(1,4)P2. The PHE- and ET-1-mediated increase of [3H]1,2-diacylglycerol was significant after respectively 16 and 8 min. PHE but not ET-1 stimulated phosphorylation of a 30 kDa protein which was likely of myofibrillar origin. It is concluded that receptor-dependent specificities exist not only at the level of PLC but also downstream.

Animals↗
PubMed1994

Calcium and the endothelin-1 and alpha 1-adrenergic stimulated phosphatidylinositol cycle in cultured rat cardiomyocytes.

Cultured neonatal rat cardiac myocytes have been utilized as a model for the study of the effect of variations in cytoplasmic free Ca2+ on the activity of phospholipase C, a key enzyme in agonist-stimulated signal transduction through the phosphoinositide pathway. Cells prelabelled with [3H]inositol were exposed to various agents in an attempt to modulate the cytoplasmic free Ca2+ concentration and the formation of [3H]inositolphosphates (15-30 min) in the presence of Li+ was taken as a measure of phospholipase C activity. Not the basal but the endothelin-1 (10(-8) M) induced [3H]inositolphosphate production (15 min) was stimulated 1.54- and 1.43-fold by A23187 (10 microM external Ca2+) and 50 mM K+ (1.3 mM external Ca2+) treatment of cells, respectively. The phenylephrine (10(-4) M) induced response was also stimulated (1.35-fold) by A23187, however it was 43% inhibited by high K+. Ouabain (10 microM) treatment of cells did not affect either basal or agonist stimulated phosphoinositide turnover. On the other hand, total removal of external free Ca2+ by addition of 50 microM ethylene glycol bis(beta-aminoethyl ether) (N,N,N',N'-tetraacetic acid strongly inhibited (75%) the endothelin-1 induced but not the basal phospholipase C activity. Endothelin-1 binding to its receptor was shown not to be inhibited by the absence of external Ca2+ while resynthesis of [3H]phosphatidylinositol 4,5-bisphosphate was not rate-limiting under this condition. The lack of external Ca2+ eventually resulted in total standstill of the ET-1 induced PtdIns turnover after 30 min. Although not always as predicted, effects on basal and agonist-activated phospholipase C were observed too when cells were treated with low Ca2+ medium, Ca2+ entry blocker nifedipine (1 microM) or Ca(2+)-channel agonist Bay K8644 (1 microM) but most of these effects were only seen after 90 min incubation. Fluorometric (fura-2) measurements showed that total removal of external free Ca2+ for a short period decreased, while short exposure to high K+ increased cytoplasmic free Ca2+ but neither Ca2+ free buffer or nifedipine nor Bay K8644 had any effect. Furthermore, in saponin-permeabilized cardiomyocytes we could demonstrate that basal as well as GTP gamma S (30 microM) stimulated phospholipase C activity was strongly activated by free Ca2+ in the concentration range of 0.1-10 microM. We conclude that in the intact cardiomyocyte the signalling pathway through phospholipase C/phosphatidylinositol 4,5-bisphosphate, stimulated by agonist-receptor interaction that activates GTP-binding proteins as does GTP gamma S, is likely be a Ca2+ dependent process.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Myocardial phosphoinositides do not share the same fatty acid profile.

It is generally assumed that the fatty acid compositions of the phosphoinositides are identical. To investigate this in myocardium, inositol lipids extracted from rat and pig ventricular homogenates were absorbed to neomycin-coated glass beads, eluted and quantitated by fatty acid analysis after thin-layer chromatography. The percentages of stearic, oleic, linoleic and arachidonic acid (20:4n-6) in the rat were, respectively, 49, 4, 7 and 26 for phosphatidylinositol, 62, 1, 4 and 18 for phosphatidylinositol-4-monophosphate and 63, 2, 4, 18 for phosphatidylinositol-4,5-bisphosphate. Equal distribution patterns of fatty acids were found in homogenate and sarcoplasmic reticulum of pig myocardium. Cultured rat ventricular myocytes were utilized to study the incorporation (25 h) of [14C]20:4n-6 relative to that of myo-[3H]inositol into phosphatidylinositol and phosphatidylinositol-4,5-bisphosphate which were, respectively, 1.61 and 1.22. The data indicate that in myocardium phosphatidylinositol-4,5-bisphosphate represents a relatively modest source of 20:4n-6.

Animals↗
PubMed1992

Occurrence and functions of the phosphatidylinositol cycle in the myocardium.

In the last decade a great deal of attention was awarded to a signal transduction pathway which is utilized primarily by 'Ca2+ mobilizing' signal molecules and which involves the hydrolysis of a quantitatively minor phospholipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by a PtdIns-specific phospholipase C (PLC). The evidence for the existence of receptor-mediated GTP binding protein-coupled PLC in myocardium and its possible functions are briefly summarized. The minireview is concentrated on the following aspects: 1) cellular localization and synthesis of polyphospho-PtdIns from PtdIns, 2) desensitization of the alpha 1-adrenergic agonist and endothelin-1 mediated PtdIns responses, 3) oscillatory Ca2+ transients initiated by PtdIns(4,5)P2 hydrolysis, 4) polyunsaturated fatty acids as constituents of polyphospho-PtdIns and of the protein kinase C activator 1,2-diacylglycerol (DAG), 5) source other than PtdIns(4,5)P2 contributing to the stimulated DAG, 6) role of the PtdIns pathway in cardiomyocyte growth and gene expression during the hypertrophic response.

Adrenergic alpha-Agonists↗
PubMed1992

Interaction of protein synthesis initiation factors with the mRNA cap structure.

The mechanism of mRNA recognition by proteins interacting with the mRNA cap structure was investigated by photochemical cross-linking of proteins with 32P-labelled reoviral RNAs. Using ribosomal washes as a source of eukaryotic protein synthesis initiation factors, we identified the well-known cap binding proteins eIF-4B and -4E, but eIF-2 and eIF-3 as well. The interplay of purified eIF-4A, -4B, and -4F was studied in relation to ATP dependence and cap analogue sensitivity of cap binding. Next to their well-known roles in the initiation process, eIF-2 and eIF-3 also cross-linked to the 5' cap. eIF-2 stimulated eIF-4B and -4E cross-linking, an observation that has been previously described more extensively. The interaction of eIF-2 with the 5' end of mRNA was extremely sensitive to K(+)-ions and was resistant to a high concentration of Mg(2+)-ions; this influence of mono- and divalent ions was in contrast with the cross-linking of eIF-4B and -4E. Optimal interaction of these factors was obtained at moderate K+ concentration and low Mg(2+)-ion concentrations. eIF-2 cross-linking was sensitive to high protein to mRNA ratios indicating a weak affinity as compared to eIF-4E and -4B. The interaction of eIF-3 with the cap of mRNA is also weak as it was counteracted by all other cap binding proteins, leading to an inability to detect the cross-linking of this protein in crude eIF preparations. Time kinetics of formation of complexes suggested eIF-2 to be one of the first factors to interact with mRNA. Preformed RNA-protein complexes were dissociated after cap analogue addition, suggesting reversible interactions between RNA and proteins.

Adenosine Triphosphate↗
PubMed1991

Evidence that eukaryotic initiation factor (eIF) 2 is a cap-binding protein that stimulates cap recognition by eIF-4B and eIF-4F.

We studied the mRNA-binding properties of eukaryotic initiation factor (eIF) 2. This Met-tRNA-binding factor interacts with the cap structure of reoviral mRNA in an ATP-independent manner. Both the beta- and gamma-subunit of eIF-2 are involved in the UV-induced cross-linking of eIF-2 to the cap. The interaction of eIF-2 with a messenger is sensitive to the cap analogue 7-methyl-guanosine 5'-triphosphate as measured by cross-linking and by mRNA retention on nitrocellulose filters. The cap-binding property of eIF-2 does not conflict with the current mRNA-binding model of initiation factors eIF-4A, -4B, and -4F: cross-linking of eIF-4E and of eIF-4B is stimulated by eIF-2. The eIF-2-mediated increase of eIF-4E interaction results in a decrease of the cross-linking of the beta- and gamma-subunits of eIF-2. The presence of GTP in the cross-linking assay interferes with the interaction of eIF-2 with the cap structure but does not inhibit the eIF-2 stimulated eIF-4E and -4B cross-linking. These observations indicate a role for eIF-2 in the mRNA recognition.

Animals↗
PubMed1985

Comparison of secretion and subcellular localization of von Willebrand protein with that of thrombospondin and fibronectin in cultured human vascular endothelial cells.

Cultured human vascular endothelial cells synthesize von Willebrand protein, thrombospondin and fibronectin. These proteins are secreted in the culture medium and incorporated into the extracellular matrix. We have compared the subcellular localization and the secretion of these proteins in response to stimulants in cultured human umbilical vein endothelial cells. Density gradient centrifugation using colloidal silica showed that the storage and secretion organelle with von Willebrand protein did not contain thrombospondin or fibronectin. Indirect immunofluorescence microscopy indicated that thrombospondin and fibronectin are not located in the rod-shaped organelles containing von Willebrand protein. Thrombin, ionophore A23187 and phorbol myristate acetate did not affect secretion of thrombospondin and fibronectin, while von Willebrand protein secretion was stimulated upon incubation of cells with these agents for 30 min. Prolonged incubation of cultured endothelial cells after a 1-h treatment with phorbol myristate acetate resulted in an increased secretion of von Willebrand protein into the conditioned medium; in contrast, accumulation of thrombospondin and fibronectin in endothelial cell-conditioned medium was decreased. These findings indicate that, unlike in platelets, these major endothelial proteins are not located in the same subcellular compartments. Von Willebrand protein is distinguished from thrombospondin and fibronectin both by its unique subcellular localization and its secretion rate in response to stimuli.

Blood Coagulation Factors↗
PubMed1985

The effect of serum deprivation on the initiation of protein synthesis in mouse neuroblastoma cells.

Growth of mouse neuroblastoma cells becomes stationary when cultured in serum-free medium. Within 60 h, the protein-synthesizing capacity of the cells declines to 25% as compared to that of exponentially growing cells. The transitional activity of the crude ribosomal salt washes from serum-deprived and control cells was compared in in vitro protein-synthesizing pH 5 systems. It appears that the ribosomal salt wash from serum-deprived cells has significantly (70%) lost its ability to support the translation of neuroblastoma poly(A)+ RNA. This activity of the ribosomal wash from serum-deprived cells can be restored to control level with rabbit reticulocyte initiation factor eIF-4B only. The ability of the ribosomal wash from serum-deprived cells to support the translation of encephalomyocarditis virus (EMC) and Semliki Forest virus (SFV) 42 S mRNA was tested. We found that EMC-mRNA is efficiently translated with the ribosomal salt wash from serum-deprived cells, whereas on the other hand the translation of SFV 42 S mRNA is severely impaired. Therefore, we conclude that in serum-deprived neuroblastoma cells protein synthesis is regulated in both a quantitative and a qualitative way. Modulation of the activity of initiation factor of protein synthesis eIF-4B is at least partly responsible for the observed (selective) blockade of protein synthesis in serum-deprived cells.

Animals↗
PubMed1984

Cloning of nitrate reductase genes from the cyanobacterium Anacystis nidulans.

Anacystis nidulans, a non-nitrogen-fixing cyanobacterium, can fulfill its nitrogen requirement by the assimilation of nitrate. The first step in the pathway, the reduction of nitrate to nitrite, is catalyzed by the molybdo-protein nitrate reductase. In this study, newly developed techniques for gene cloning in A. nidulans R2 were used for the isolation of two genes involved in nitrate reduction. One gene was cloned by complementation of the corresponding mutant; the other gene was picked up from a cosmid gene library by using a restriction fragment containing the transposon-inactivated gene as a probe. Both genes were unlinked single-copy chromosomal genes. Transformation studies provided evidence for the existence of a third locus involved in nitrate reduction.

Cloning, Molecular↗