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M Holst

Publications and source records attributed to M Holst.

52 records · Page 3Linked to original sources
PubMed1989

Effects of a DNA helix-destabilizing protein on transcription in living cells.

The effects of microinjected rat DNA helix-destabilizing protein (HDP) and anti-HDP sera on the transcription of various RNAs in nuclei of Chironomus tentans salivary gland cells were investigated. The results showed that injected antisera have the greatest inhibitory effect on the RNA polymerase II-based transcription of Balbiani ring puffs, about 80%. The inhibition of RNA polymerase I-based transcription of nucleolar preribosomal RNA was about 70%, while the effect on the heterogenous nuclear RNA (hnRNA) from chromosome I to III was about 40%. In all cases, the antiserum against the denatured subunit HDP was more inhibitory than that against the native HDP. In correlative experiments, microinjection of the HDP itself showed stimulated transcription of all RNAs. Indirect localization by immunofluorescence showed immunoreactive HDP to be preferentially concentrated on transcriptionally active Balbiani rings 1 and 2. Western blot analysis of the protein extract from isolated Chironomus tentans salivary gland nuclei with anti-HDP (rat) sera showed cross-reactive protein bands with molecular masses of about 33,000, 42,000 and 65,000 daltons. These results suggest that a homologue of rat HDP and other C. tentans proteins immunologically related to it play an important role in transcription in vivo.

Animals↗
PubMed1988

Phosphorylation of nuclear proteins of peripheral blood T lymphocytes activated by nickel sulfate and mercuric chloride.

The phosphorylation of nuclear proteins of peripheral blood T lymphocytes activated by nickel sulfate or mercuric chloride, and from nickel-allergic subjects, was studied in polyacrylamide gel separations of 32P-labeled proteins. With a preincubation period of the metal salts for 48 h, a marked increase of 32P label into nonhistone proteins, especially the 30- to 40-kilodalton region, was found compared to the control cultures. This increase was most pronounced in mercuric-chloride-treated cultures, which also showed an increase in labeling of histone H4. The increase in nuclear protein phosphorylation probably reflects an activation of the lymphocytes. Moreover, the difference in phosphorylation pattern between mercuric-chloride- and nickel-sulfate-activated lymphocytes might be due to different mechanisms of action for polyclonal and monoclonal activators, respectively.

Cell Nucleus↗
PubMed1988

Binding of mercuric chloride to nuclear proteins in human peripheral blood T lymphocytes.

The binding of labelled (203Hg) mercuric chloride to nuclear proteins of human peripheral blood T lymphocytes was studied by using polyacrylamide gel electrophoresis and autoradiography. Most of the nuclear proteins were found to be able to bind mercuric chloride; however, 4 of the proteins were heavily labelled. Of these 4 proteins, 2 migrated in the histone region, 1 comigrating exactly with histone H3. The other 2 proteins were non-histone proteins with molecular weight of approximately 25 and 43 kD.

Adult↗
PubMed1987

Differential kinase systems are involved in the rapidly turning over phosphorylation of prominent nuclear proteins.

The activity of endogenous nuclear protein kinases has been probed in an vitro assay system of isolated nuclei from Chironomus salivary gland cells. The phosphorylation of a set of seven prominent rapidly phosphorylated non-histone proteins and of histones H3, H2A and H4 was analyzed using ATP or GTP as phosphoryl donor and heparin as protein kinase effector. The core histones H2A and H3 both incorporate 32P from [gamma-32P]ATP as well as from [gamma-32P]GTP but their phosphorylation is differentially affected by heparin. The phosphorylation of H2A is blocked by heparin while that of H3 is even stimulated in the presence of heparin when ATP is used as phosphate donor. H4 is unable to incorporate phosphate groups from GTP but its ATP-based phosphorylation is heparin sensitive. Of the non-histone protein kinase substrates, we could only detect two, the 44-kDa and 115-kDa proteins, which are heparin sensitive with either ATP or GTP and, thus, strictly meet the criteria for casein kinase type II-specific phosphorylation. The investigated histones and non-histone proteins can be grouped into three broad categories on the basis of their phosphorylation properties. (A) Proteins very likely affected by casein kinase NII. (B) Proteins phosphorylated by strictly ATP-specific protein kinases. (C) Proteins phosphorylated by ATP as well as GTP utilizing protein kinase(s) other than casein NII. Category B proteins can be subdivided into proteins phosphorylated in a heparin-resistant (B1) and heparin-sensitive (B2) manner. The phosphorylation of category C proteins may be heparin sensitive with ATP only (C1), heparin sensitive with GTP only (C2), heparin insensitive with both ATP and GTP (C3) or stimulated by heparin (C4).

Animals↗
PubMed1985

Transport and metabolism of adenosine in relation to the transcriptional activity of hnRNA genes in Chironomus salivary gland cells.

The transport and metabolism of adenosine in explanted salivary gland cells of Chironomus tentans have been investigated. The adenosine transport is rapid and reaches a maximum velocity within seconds after administration. Nevertheless, a transmembrane equilibrium in adenosine concentrations could never be attained because of the efficiency of the intracellular trapping reaction. Only about 10% or less of the extracellular adenosine concentration could be maintained intracellularly. The rapidity of adenosine phosphorylation did not allow us the assessment of transport kinetics with any degree of accuracy. At lower external [3H]adenosine doses, [3H]ATP was the predominating metabolite, yielding a [3H]ATP/[3H]AMP ratio of 2.5-3.5, while at higher concentrations the [3H]ATP/[3H]AMP ratio was lowered to below 0.9. The [3H]AMP fraction derived from [3H]adenosine-treated cells was not uniform, but rather it consisted of 3H-labeled 5'AMP, 3'AMP and 2'AMP isomers. Whereas the accumulation of 3H-labeled 5'AMP and ATP attained steady-state levels after 30-60 min of incubation at higher exogenous adenosine concentrations, the content of 3H-labeled 3'AMP and 2'AMP continuously and linearly increased. The data indicate that the metabolism of adenosine to 2'AMP and 3'AMP represents a salvage pathway operating at unphysiological adenosine levels and that the well-known inhibitory effect of adenosine on polymerase-II-promoted RNA transcription is not exerted by its phosphorylated metabolites.

Adenosine↗
PubMed1985

Posttranslational phosphorylation of specific chromosomal proteins and transcription of hnRNA genes in isolated nuclei: retention of in vivo sensitivity to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB).

The rapidly turning over phosphorylation of specific nuclear nonhistone proteins, especially 42-, 33-, and 30-kDa polypeptides, and its relation to the transcriptional activity of hnRNA genes was investigated in isolated nuclei from salivary gland cells of Chironomus tentans. Incubation conditions promoting the phosphorylation of nonhistone proteins as well as the transcriptional activity of RNA polymerase II were established. The pattern of 32P incorporation into the nonhistone proteins found in isolated nuclei resembled that obtained in experiments with intact cells, and the endogenous RNA polymerase II retained its ability to reinitiate the transcription under in vitro assay conditions. In addition, the in vivo sensitivity of the phosphorylation of 42-, 33-, and 30-kDa polypeptides, like the sensitivity of the initiation of hnRNA transcription to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), were preserved in the nuclear preparation. The experimental data taken together provide further support for the idea that the activation of hnRNA genes is causally related to the phosphorylation of specific nonhistone proteins.

Animals↗
PubMed1985

Five years experience of coniotomy.

The view held over the last six decades that coniotomy often leads to subglottic stenosis has recently been called into question. In this study 203 electively performed coniotomies were analysed. The operation was found to be much simpler than tracheostomy. No severe intra- or postoperative complications occurred. Six months after decannulation 61 patients operated on were alive and accessible to follow-up examination. No evidence of subglottic stenosis was found. The main disadvantage was a relatively high frequency of voice disorder.

Adult↗
PubMed1984

Phosphorylation of some chromosomal nonhistone proteins in active genes is blocked by the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB).

The distribution of rapidly phosphorylated chromosomal proteins between chromosome I, chromosome II + III, chromosome IV, and nuclear sap including the matrix was investigated in salivary gland cells of Chironomus tentans. Chromosome IV, which carries most active nonribosomal genes in the cell, was found to be enriched in four rapidly phosphorylated nonhistone polypeptides (Mr = 25,000, 30,000, 33,000, and 42,000) in parallel with the transcriptional activity rather than with the DNA content of the chromosome. Also the histones H2A and H4 are rapidly phosphorylated but the phosphorylation is proportional to the DNA content of each chromosome sample. The 32P-labeled Mr = 42,000 polypeptide immunologically cross-reacted with an antibody elicited against the transcription stimulatory factor S-II isolated from Ehrlich ascites tumor cells (Sekimizu, K., D. Mizuno, and S. Natori, 1979, Exp. Cell Res., 124:63-72). In addition, indirect immunofluorescence studies on chromosome IV with antisera against the stimulatory factor II revealed a selective staining of the active gene loci. The incorporation of 32P into three chromosome IV nonhistone polypeptides, especially into the Mr = 42,000 polypeptide, was lowered by 70-85% shortly after administration of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a likely inhibitor of heterogeneous nuclear RNA transcription at initiation level. The possibility of a causal relationship between inhibited phosphorylation of chromosomal proteins and blocked transcription of heterogeneous nuclear RNA genes by DRB is discussed.

Amanitins↗
PubMed1984

[Elective coniotomy. Experiences with 103 personal cases].

The view held for the last 60 years that coniotomy often leads to subglottic stenosis has recently been called into question. In the present study 103 elective coniotomies are analysed. The operation was much easier than tracheotomy, and there were no severe complications during or after operation. Six months after decannulation 31 patients were alive and 28 available for follow-up examination. No evidence of subglottic stenosis was found. The main disadvantage of this operation would seem to be a tendency for the development of voice changes.

Adult↗
PubMed1981

Adenosine selectively inhibits labeling of chromosomal RNA, especially hnRNA, probably by acting at or near the site of chain initiation.

The effects of adenosine on labeling of nucleolar preribosomal RNA, chromosomal plus nuclear sap hnRNA, and 4-5S RNA in explanted salivary gland cells of chironomus tentans has been studied. Of chromosomal transcripts it is the labeling of polymerase II-promoted RNA that is interrupted preferentially, but 4-5S RNA is influenced as well. The labeling of hnRNA and 4-5S RNA is diminished by 70-90 percent and 45-60 percent, repectively, while the incorporation into the nucleolar preribosomal RNA remains essentially unchanged. Labeled adenosine is transported efficiently across the plasma membrane and becomes phosphorylated to AMP, ADP, and ATP, of which ATP predominates at noninhibitory concentrations. The rate of the formation of [(3)H]AMP is, however, enhanced in response to the increase in external adenosine doses, whereas that of [(3)H]ATP increases only slowly or remains essentially unaltered. A rise in exogenous [(3)H] adenosine concentration to 200 muM yields a [(3)H]ATP/[(3)H]AMP ratio that is about one order of magnitude lower than that at 20 muM of the nucleoside. In parallel with this, there is a gradual repression of the labeling of chromosomal RNA. A similar treatment with guanosine produces only minor reduction in GTP/GMP quotient and does not influence significantly the labeling of any sizable RNA fraction. Thus the experimental data strongly indicate that the purine ribonucleoside adenosine, but not guanosine, gives rise to a markedly diminished triphosphate/monophosphate quotient simultaneously with a selective suppression of the labeling of chromosomal RNA, especially hnRNA, when applied in overdoses. The sequence of hnRNA events during inhibition by adenosine resembles the effect of the purine nucleoside analogue 5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole, indicating that the site of inhibitory action is at or close to the initiation of transcription.

Adenosine↗
PubMed1979

The size distribution of poly(A) in newly synthesized and old Balbiani ring RNA.

The size distribution of newly synthesized and old poly(A) sequences on transcripts of the giant tissue specific puffs, Balbiani rings in salivary glands of Chironomus tentans has been determined. After labeling with [3H]adenosine, poly(A) containing Balbiani ring RNA(75S RNA) was selectively collected by means of a recently developed technique. This combines electrophoretic fractionation and affinity chromatography in one run by insertion of poly(U) immobilized in glass fiber filters in an agarose gel slab. The majority of short-term labeled poly(A) chains released from poly(A) containing 75S RNA molecules is distributed within a narrow size range migrating as one peak with a mean value of 103 +/- 2 nucleotides, which is probably the initial length of poly(A). The labeling pattern of ribonuclease resistant poly(A) stretches after chase with unlabeled adenosine displays a relatively broad and heterogeneous size spectrum from at least 20 to more than 100 nucleotides. The main peak of labeled adenylate core in newly formed poly(A) containing RNA of non-Balbiani ring origin is dispersed within a broader size range than that of Balbiani ring RNA and possesses an average value of 94 +/- 2 nucleotides. During chase conditions, the relative frequency of occurrence of poly(A) chains of 75S RNA in the size range of 100 nucleotides exhibits a significant decrease in parallel with a rather uniform gain in the size classes between 20--50 nucleotides. However, the results are inconsistent with the existence of an age-dependent shortening of poly(A) chains in the balbiani ring RNA. A significant portion of 75S RNA molecules remain associated with poly(A) segments which are essentially of original size even after 21 hr in the presence of unlabeled adenosine. This finding provides support for the possibility that the initiation of the poly(A) shortening in 75S RNA is a stochastic process.

Animals↗
PubMedDate not supplied

Elective coniotomy. A prospective study.

The view current over the last 60 years or so that coniotomy often leads to subglottic stenosis has recently been called into question. In the present study 103 electively performed coniotomies have been analysed. The operation was found to be much easier than tracheotomy. There was no case of severe peroperative or postoperative complications. Six months after decannulation 28 patients were alive and accessible to follow-up examination. No evidence of subglottic stenosis was found. The main disadvantage of this operation would seem to be a tendency for the development of voice changes.

Adult↗
PubMedDate not supplied

The cricothyroid muscle after cricothyroidotomy. A porcine experimental study.

Cricothyroidotomy in elective use or emergency situations has been discussed by several authors during recent years. The main complication reported has been subglottic stenosis. We have used this method for several years and among our patients there has been no case of stenosis. The major complication in our material has been voice dysfunction. Some of the patients had a change in voice quality consistent with dysfunction in the cricothyroid muscles (diminished voice range and a lowered pitch). The aim of this study was to investigate whether cricothyroidotomy and insertion of a tracheostomy tube caused physiological or morphological changes in the cricothyroid muscle, by direct injury or secondary to a peripheral nerve lesion. In the six animals that were cricothyroidotomized during 7-9 days and examined after 9-15 weeks, we found neither changes in the EMG nor in the muscle morphology indicating injury to the muscle or its peripheral nerve supply.

Animals↗
PubMedDate not supplied

The healing of cricothyroidostomy in pigs. A morphological study.

The advantages and disadvantages with cricothyroidotomy have been discussed by several authors during the last 12 years. The frequency of complications differs considerably probably depending on indications for surgery and methods of follow-up. In our own human material (253 patients cricothyroidotomized and 79 of 102 alive after 6 months followed up) there has been no case of laryngeal or tracheal stenosis. The aim of this study was to analyse the effect of the inflammatory granulation tissue on the surrounding tissues in 8 cricothyroidotomized pigs, and to analyse the regeneration of the mucosa and the healing with special regard to development of stenosis. Despite pronounced stomal infection and formation of granulation tissue we found no case of stenosis, no injuries of the cartilage and a complete regeneration of the ciliated epithelium.

Animals↗