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Biomedical subjects

M Holst

Publications and source records attributed to M Holst.

At least 19 recordsLinked to original sources
PubMed1995

Steroid 21-hydroxylase in the kidney: demonstration of levels of messenger RNA which correlate with the level of activity.

Steroid 21-hydroxylase activity was assayed in low-speed supernatants prepared from whole cell homogenates of mouse and rat tissues. Kidney supernatants had an activity which was approximately 2-5% that of adrenal preparations while heart muscle was found to be without 21-hydroxylase activity. When the enzyme kinetics were characterized, both adrenal and kidney low-speed supernatants demonstrated saturation kinetics, but with very different Vmax and Km values. Using polymerase chain reaction amplification after reverse transcriptase synthesis of cDNA from isolated RNA (RT-PCR), we found low levels of mRNA for steroid 21-hydroxylase in mouse kidney, but none in heart muscle. Thus, extra-adrenal steroid 21-hydroxylase activity in the kidney may be mediated by the same enzyme as found in adrenals.

Adrenal Glands↗
PubMed1994

The salivary gland 42-kDa phosphoprotein is a single-stranded DNA-binding protein with characteristics of the epithelial casein kinase N42 in Chironomus tentans.

The DNA-binding and phosphorylation properties of a rapidly phosphorylated nuclear 42-kDa phosphoprotein and of its two structurally related proteins, pp43 and pp44 in Chironomus tentans salivary glands were investigated. pp42, pp43 and pp44 bind promoter probes of the ecdysterone controlled I-18C gene and of the joint histone H2A/H2B genes in a sequence-selective and single-stranded DNA (ssDNA) specific manner. Rapid phosphorylation appears to give pp42 and pp43 uniquely hydrophilic characters making them soluble in the aqueous phase during phenol treatment. Dephosphorylation of the nuclear proteins markedly stimulates the ssDNA-binding activity of pp42 but not of pp43 and pp44. All three phosphoproteins are sensitive to heparin and the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) in vitro, but their sensitivity to heparin is more than one order of magnitude lower than that of casein kinase II. The heparin sensitivity of pp42 and pp43 is, however, similar to that described for a previously identified nuclear 42-kDa phosphoprotein in a Chironomus tentans epithelial cell line, casein kinase N42 (CKN42). pp42 and pp43 bind with high affinity to a Phosvitin-Sepharose matrix, like casein kinase I, II and N42, and can be eluted with high salt buffers from the affinity column. In intact salivary gland cells, microinjected (gamma-32P)GTP labels pp42 in a heparin sensitive manner, and this GTP-phosphorylation of pp42 could be competed out by a large excess of phosvitin. (gamma-32P)ATP-based phosphorylation of pp42 was uninfluenced by phosvitin in intact cells. The experimental data suggest that the salivary gland 42-kDa phosphoprotein, pp42, is a ssDNA-binding protein with characteristics of the epithelial CKN42.

Adenosine Triphosphate↗
PubMed1994

Protein electrostatics: rapid multigrid-based Newton algorithm for solution of the full nonlinear Poisson-Boltzmann equation.

A new method for solving the full nonlinear Poisson-Boltzmann equation is outlined. This method is robust and efficient, and uses a combination of the multigrid and inexact Newton algorithms. The novelty of this approach lies in the appropriate combination of the two methods, neither of which by themselves are capable of solving the nonlinear problem accurately. Features of the Poisson-Boltzmann equation are fully exploited by each component of the hybrid algorithm to provide robustness and speed. The advantages inherent in this method increase with the size of the problem. The efficacy of the method is illustrated by calculations of the electrostatic potential around the enzyme Superoxide Dismutase. The CPU time required to solve the full nonlinear equation is less than half that needed for a conjugate gradient solution of the corresponding linearized Poisson-Boltzmann equation. The solutions reveal that the field around the active sites is significantly reduced as compared to that obtained by solving the corresponding linearized Poisson-Boltzmann equation. This new method for the nonlinear Poisson-Boltzmann equation will enable fast and accurate solutions of large protein electrostatics problems.

Algorithms↗
PubMed1991

Analysis of the structural relationships between the DNA-binding phosphoproteins pp42, pp43 and pp44 by in situ peptide mapping.

A structural homology is established between three DNA-binding phosphoproteins located in the 42 to 44 kDa range, referred to as pp42, pp43 and pp44, from Chironomus tentans salivary gland cells by in situ peptide mapping. The staining patterns of pp42, pp43 and pp44 which resulted from digestion with Staphylococcus aureus V8, trypsin or papain proteases show the presence of 8 to 15 spots majority of which have identical mobility. In the patterns of the digests generated by treatments with trypsin about 10 spots appear in common between any pair of the protein substrates. In addition, each pattern includes two to three peptides of mobility not present in the other. Thus the peptide mapping of pp42, pp43 and pp44 based on the staining patterns of proteolytic digests suggest the existence of structural homology between the three unlabelled substrates. The proteolytic peptides carrying the rapidly turning over phosphate groups form markedly different electrophoretic patterns than the unlabelled peptides visualized by staining. Treatment of 32P-labelled pp42, pp43 and pp44 with V8 generates only one labelled fragment in the 30 kD range. The cleavage patterns of pp44 produced by chymotrypsin or papain contain seven to ten labelled fragments while those of pp42 and pp43 contain only two. The 32P-labelled tryptic peptides of pp42, pp43 and pp44 exhibit a ladder pattern for each substrate which probably arise by a consecutive removal of 25 to 35 amino acid residues from the primary digestion products pp29, pp29.5 and pp30 by cleavage of four to five putative interdomain regions. The possibility that these three structurally related phosphoproteins belong to the category of transcription factors is discussed.

Animals↗
PubMed1991

Effect of the ANF analog A68828 in cisplatin-induced acute renal failure.

Experiments were conducted to determine the effects of the reduced-size atrial natriuretic factor (ANF) analog, A68828, on renal function in rats with cisplatin (CP)-induced acute renal failure. CP was given as a single intraperitoneal injection (7.5 mg/kg) 3 days before experiments. In separate groups of rats, the renal response to intravenous infusion of A68828 at 3, 10 or 30 micrograms/kg/min or ANF[1-28] at 0.03, 0.1 or 0.3 micrograms/kg/min for 2 hr was evaluated. Another group of CP-treated rats were infused with the vehicle (0.1% bovine serum albumin in 0.9% NaCl). CP treatment resulted in a marked decline in glomerular filtration rate (GFR), arterial pressure, heart rate and reabsorption of water and electrolytes compared to untreated control animals. Infusion of A68828 produced a dose-dependent improvement in the glomerular filtration rate. The highest dose of A68828 produced a nearly 3-fold increase in the glomerular filtration rate, whereas arterial pressure was decreased; heart rate was unchanged. Despite producing a significant diuresis and natriuresis, net tubular reabsorption of water and sodium was also increased. Similar dose-dependent effects were observed with the native peptide, ANF[1-28]. These data indicate that infusion of the reduced-sized analog of ANF, A68828, can significantly improve glomerular and tubular function in rats with acute renal failure induced by CP.

Acute Kidney Injury↗
PubMed1990

Errors in tracheal pressure recording in patients with a tracheostomy tube--a model study.

The recording of intratracheal pressure in patients breathing through a tracheostomy tube is marred by methodological problems. In model experiments it has been shown that the introduction of a recording catheter into the tracheostomy tube alters the recorded pressure by as much as 20-40%. To avoid such inaccuracy, a specially designed tracheostomy tube is proposed with an externally placed channel for introduction of the recording catheter. In in vivo measurements, recordings can be made at a distance of up to about 20 mm below the end of tube. Pressure recordings from this part of a model trachea are compared with results obtained from a reference pressure port 100 mm below the tube ending. The results show that it is possible to record expiratory pressure with good accuracy but that inspiratory pressure is still overestimated by 9-16%.

Airway Resistance↗
PubMed1990

Vocal dysfunction following cricothyroidotomy: a prospective study.

Voice dysfunction is a known consequence of cricothyroidotomy, but few detailed analyses have been published. This study reports an investigation of voice function in 19 patients who underwent cricothyroidotomy during a 12-month period (54 were patients operated on, 29 survived the primary disease or injury, and 19 agreed to participate in the investigation). The patients were interviewed regarding signs of dysfunction of the cricothyroid muscles, or a diminished external tensor effect on the vocal folds. Laryngoscopy with a magnifying-angled endoscope, stroboscopy, phonetography, determination of mean speaking fundamental frequency, perceptual analysis of the voice, and electromyogram of the cricothyroid muscle were all performed. Four patients had signs of diminished external tensor effect on the vocal folds (mild or moderate voice dysfunction), 5 patients had other voice disorders, and 10 were classified as having normal voice. Elective cricothyroidotomy should be avoided in patients with professional or other activities that place heavy demands on the voice.

Adult↗
PubMed1989

The rapidly phosphorylated chromosomal 42-kDa protein is a subunit of larger protein complexes.

We have isolated, purified and characterized a 42-kDa phosphoprotein which has been found to be preferentially associated with active gene loci of salivary gland cells of Chironomus tentans. The rapidly phosphorylated form of this protein could be extracted with 0.2 M NaCl. Chromatographic analysis by gel filtration revealed that a significant fraction of labelled 42-kDa polypeptide elutes with an apparent molecular mass of 150 to 200 kDa. The result suggests that a portion of the phosphorylated 42-kDa polypeptide in native state forms a multisubunit protein complex consisting of rapidly phosphorylated 42-kDa polypeptide chains alone.

Animals↗
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↗