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Biomedical subjects

L T Jensen

Publications and source records attributed to L T Jensen.

At least 19 recordsLinked to original sources
PubMed2002

Hypertension and renovascular disease: follow-up on 100 renal vein renin samplings.

The clinical value of renal vein renin sampling (RVRS) as a prognostic tool in the treatment of renovascular hypertension was evaluated. One hundred consecutive patients were included over a 4-year period of time. About half of the patients (49%) were treated interventionally by PTRA (21%), nephrectomy (20%), or vascular surgery (8%). Seven patients (15%) were cured and 15 (32%) had improved (reduction in antihypertensive medicine) after 6 months follow-up, whereas three patients (6%) were cured and 12 (26%) improved after 3-4 years follow-up. Thus, the number of patients cured or improved is comparable with the results from our department reported 20 years ago. However, in the present report, more than twice as many patients were enrolled, leading to double costs. Different indices of lateralisation of the renin generation were calculated for the use in cases of a shrunken kidney (functional share < or =15%). None of the indices clearly discriminated between the patients who did benefit from intervention, and those who did not. The only positive finding was that a peripheral renin concentration lower than 8 mlU/l predicted no effect of intervention, which might lead to the exclusion of 11% of the patients before entering the diagnostic programme. We conclude that the RVRS demands a very restrictive referral pattern if it should be of prognostic value for the blood pressure outcome after intervention. No indices of lateralised renin concentrations proved high predictive value. However, a peripheral renin concentration low in the normal range seems useful as an indicator of no benefit from intervention.

Adult↗
PubMed2001

A fraction of yeast Cu,Zn-superoxide dismutase and its metallochaperone, CCS, localize to the intermembrane space of mitochondria. A physiological role for SOD1 in guarding against mitochondrial oxidative damage.

Cu,Zn-superoxide dismutase (SOD1) is an abundant, largely cytosolic enzyme that scavenges superoxide anions. The biological role of SOD1 is somewhat controversial because superoxide is thought to arise largely from the mitochondria where a second SOD (manganese SOD) already resides. Using bakers' yeast as a model, we demonstrate that Cu,Zn-SOD1 helps protect mitochondria from oxidative damage, as sod1Delta mutants show elevated protein carbonyls in this organelle. In accordance with this connection to mitochondria, a fraction of active SOD1 localizes within the intermembrane space (IMS) of mitochondria together with its copper chaperone, CCS. Neither CCS nor SOD1 contains typical N-terminal presequences for mitochondrial uptake; however, the mitochondrial accumulation of SOD1 is strongly influenced by CCS. When CCS synthesis is repressed, mitochondrial SOD1 is of low abundance, and conversely IMS SOD1 is very high when CCS is largely mitochondrial. The mitochondrial form of SOD1 is indeed protective against oxidative damage because yeast cells enriched for IMS SOD1 exhibit prolonged survival in the stationary phase, an established marker of mitochondrial oxidative stress. Cu,Zn-SOD1 in the mitochondria appears important for reactive oxygen physiology and may have critical implications for SOD1 mutations linked to the fatal neurodegenerative disorder, amyotrophic lateral sclerosis.

Cell Compartmentation↗
PubMed2000

Effect of the two conserved prolines of human growth inhibitory factor (metallothionein-3) on its biological activity and structure fluctuation: comparison with a mutant protein.

Human neuronal growth inhibitory factor, a metalloprotein classified as metallothionein-3 (MT-3), impairs the survival and the neurite formation of cultured neurons. In these studies the double P7S/P9A mutant (mutMT-3) and single mutants P7S and P9A of human Zn(7)-MT-3 were generated, and their effects on the biological activity and the structure of the protein were examined. The biological results clearly established the necessity of both proline residues for the inhibitory activity, as even single mutants were found to be inactive. Using electronic absorption, circular dichroism (CD), magnetic CD (MCD), and (113)Cd NMR spectroscopy, the structural features of the metal-thiolate clusters in the double mutant Cd(7)-mutMT-3 were investigated and compared with those of wild-type Cd(7)-MT-3 [Faller, P., Hasler, D. W., Zerbe, O., Klauser, S., Winge, D. R., and Vasák, M. (1999) Biochemistry 38, 10158] and the well characterized Cd(7)-MT-2a from rabbit liver. Similarly to (113)Cd(7)-MT-3 the (113)Cd NMR spectrum of (113)Cd(7)-mutMT-3 at 298 K revealed four major and three minor resonances (approximately 20% of the major ones) between 590 and 680 ppm, originating from a Cd(4)S(11) cluster in the alpha-domain and a Cd(3)S(9) cluster in the beta-domain, respectively. Due to the presence of dynamic processes in the structure of MT-3 and mutMT-3, all resonances showed the absence of resolved homonuclear [(113)Cd-(113)Cd] couplings and large apparent line widths (between 140 and 350 Hz). However, whereas in (113)Cd(7)-mutMT-3 the temperature rise to 323 K resulted in a major recovery of the originally NMR nondetectable population of the Cd(3)S(9) cluster resonances, no such temperature effect was observed in (113)Cd(7)-MT-3. To account for the observed NMR features, a dynamic structural model for the beta-domain is proposed, which involves a folded and a partially unfolded state. It is suggested that in the partially unfolded state a slow cis/trans isomerization of Cys-Pro(7) or Cys-Pro(9) amide bonds in (113)Cd(7)-MT-3 takes place and that this process represents a rate-limiting step in a correct domain refolding. In addition, closely similar apparent stability constants of human MT-3, mutMT-3, and rabbit MT-2a with Cd(II) and Zn(II) ions were found. These results suggest that specific structural features dictated by the repetitive (Cys-Pro)(2) sequence in the beta-domain of MT-3 and not its altered metal binding affinity compared to MT-1/MT-2 isoforms are responsible for the biological activity of this protein.

Amino Acid Sequence↗
PubMed2000

A dual role for zinc fingers in both DNA binding and zinc sensing by the Zap1 transcriptional activator.

The Zap1 transcriptional activator of Saccharomyces cerevisiae controls zinc homeostasis. Zap1 induces target gene expression in zinc-limited cells and is repressed by high zinc. One such target gene is ZAP1 itself. In this report, we examine how zinc regulates Zap1 function. First, we show that transcriptional autoregulation of Zap1 is a minor component of zinc responsiveness; most regulation of Zap1 activity occurs post-translationally. Secondly, nuclear localization of Zap1 does not change in response to zinc, suggesting that zinc regulates DNA binding and/or activation domain function. To understand how Zap1 responds to zinc, we performed a functional dissection of the protein. Zap1 contains two activation domains. DNA-binding activity is conferred by five C-terminal C(2)H(2) zinc fingers and each finger is required for high-affinity DNA binding. The zinc-responsive domain of Zap1 also maps to the C-terminal zinc fingers. Furthermore, mutations that disrupt some of these fingers cause constitutive activity of a bifunctional Gal4 DNA-binding domain-Zap1 fusion protein. These results demonstrate a novel function of Zap1 zinc fingers in zinc sensing as well as DNA binding.

Cell Nucleus↗
PubMed2000

Role of Saccharomyces cerevisiae ISA1 and ISA2 in iron homeostasis.

The budding yeast Saccharomyces cerevisiae contains two homologues of bacterial IscA proteins, designated Isa1p and Isa2p. Bacterial IscA is a product of the isc (iron-sulfur cluster) operon and has been suggested to participate in Fe-S cluster formation or repair. To test the function of yeast Isa1p and Isa2p, single or combinatorial disruptions were introduced in ISA1 and ISA2. The resultant isaDelta mutants were viable but exhibited a dependency on lysine and glutamate for growth and a respiratory deficiency due to an accumulation of mutations in mitochondrial DNA. As with other yeast genes proposed to function in Fe-S cluster assembly, mitochondrial iron concentration was significantly elevated in the isa mutants, and the activities of the Fe-S cluster-containing enzymes aconitase and succinate dehydrogenase were dramatically reduced. An inspection of Isa-like proteins from bacteria to mammals revealed three invariant cysteine residues, which in the case of Isa1p and Isa2p are essential for function and may be involved in iron binding. As predicted, Isa1p is targeted to the mitochondrial matrix. However, Isa2p is present within the intermembrane space of the mitochondria. Our deletion analyses revealed that Isa2p harbors a bipartite N-terminal leader sequence containing a mitochondrial import signal linked to a second sequence that targets Isa2p to the intermembrane space. Both signals are needed for Isa2p function. A model for the nonredundant roles of Isa1p and Isa2p in delivering iron to sites of the Fe-S cluster assembly is discussed.

Amino Acid Sequence↗
PubMed1999

The yeast transcription factor Mac1 binds to DNA in a modular fashion.

Mac1 is a metalloregulatory protein that regulates expression of the high affinity copper transport system in the yeast Saccharomyces cerevisiae. Under conditions of high copper concentration, Mac1 represses transcription of genes coding for copper transport proteins. Mac1 binds to DNA sequences called copper response elements (CuREs), which have the consensus sequence 5'-TTTGC(T/G)C(A/G)-3'. Mac1 contains two zinc binding sites, a copper binding site, and the sequence motif RGRP, which has been found in other proteins to mediate binding to the minor groove of A/T-rich sequences in DNA. We have used hydroxyl radical footprinting, missing nucleoside, and methylation interference experiments to investigate the structure of the complex of the DNA binding domain of Mac1 (called here Mac1(t)) with the two CuRE sites found in the yeast CTR1 promoter. We conclude from these experiments that Mac1(t) binds in a modular fashion to DNA, with its RGRP AT-hook motif interacting with the TTT sequence at the 5' end of the CTR1 CuRE site, and with another DNA-binding module(s) binding in the adjacent major groove in the GCTCA sequence.

Amino Acid Sequence↗
PubMed1998

Identification of a copper-induced intramolecular interaction in the transcription factor Mac1 from Saccharomyces cerevisiae.

Mac1 mediates copper (Cu)-dependent expression of genes involved in high-affinity uptake of copper ions in Saccharomyces cerevisiae. Mac1 is a transcriptional activator in Cu-deficient cells, but is inhibited in Cu-replete cells. Mac1 resides within the nucleus in both Cu-deficient and Cu-loaded cells. Cu inhibition of Mac1 appears to result from binding of eight copper ions within a C-terminal segment consisting of two Cys-rich motifs. In addition, two zinc ions are bound within the N-terminal DNA-binding domain. Only 4-5 mol. eq. Cu are bound to a mutant Mac1 (His279Gln substitution) that is impervious to Cu inhibition. The CuMac1 complex is luminescent, indicative of copper bound in the Cu(I) state. Cu binding induces a molecular switch resulting in an intramolecular interaction in Mac1 between the N-terminal DNA-binding domain and the C-terminal activation domain. This allosteric interaction is Cu dependent and is not observed when Mac1 contained the mutant His279Gln substitution. Fusion of the minimal DNA-binding domain of Mac1 (residues 1-159) to the minimal Cu-binding activation domain (residues 252-341) yields a functional Cu-regulated transcriptional activator. These results suggest that Cu repression of Mac1 arises from a Cu-induced intramolecular interaction that inhibits both DNA binding and transactivation activities.

Amino Acid Substitution↗
PubMed1998

Metalloregulation of FRE1 and FRE2 homologs in Saccharomyces cerevisiae.

The high affinity uptake systems for iron and copper ions in Saccharomyces cerevisiae involve metal-specific permeases and two known cell surface Cu(II) and Fe(III) metalloreductases, Fre1 and Fre2. Five novel genes found in the S. cerevisiae genome exhibit marked sequence similarity to Fre1 and Fre2, suggesting that the homologs are part of a family of proteins related to Fre1 and Fre2. The homologs are expressed genes in S. cerevisiae, and their expression is metalloregulated as is true with FRE1 and FRE2. Four of the homologs (FRE3-FRE6) are specifically iron-regulated through the Aft1 transcription factor. These genes are expressed either in cells limited for iron ion uptake by treatment with a chelator or in cells lacking the high affinity iron uptake system. Expression of FRE3-FRE6 is elevated in AFT1-1 cells and attenuated in aft1 null cells, showing that iron modulation occurs through the Aft1 transcriptional activator. The fifth homolog FRE7 is specifically copper-metalloregulated. FRE7 is expressed in cells limited in copper ion uptake by a Cu(I)-specific chelator or in cells lacking the high affinity Cu(I) permeases. The constitutive expression of FRE7 in MAC1 cells and the lack of expression in mac1-1 cells are consistent with Mac1 being the critical transcriptional activator of FRE7 expression. The 5' promoter sequence of FRE7 contains three copper-responsive promoter elements. Two elements are critical for Mac1-dependent FRE7 expression. Combinations of either the distal and central elements or the central and proximal elements result in copper-regulated FRE7 expression. Spacing between Mac1-responsive sites is important as shown by the attenuated expression of FRE7 and CTR1 when two elements are separated by over 100 base pairs. From the three Mac1-responsive elements in FRE7, a new consensus sequence for Mac1 binding can be established as TTTGC(T/G)C(A/G).

Base Sequence↗
PubMed1998

Mapping of the DNA binding domain of the copper-responsive transcription factor Mac1 from Saccharomyces cerevisiae.

Mac1 from Saccharomyces cerevisiae activates transcription of genes, including CTR1 in copper-deficient cells. N-terminal fusions of Mac1 with the herpes simplex VP16 activation domain were used to show that residues 1-159 in Mac1 constitute the minimal DNA binding domain. Mac1-(1-159) purified from Escherichia coli contains two bound Zn(II) ions. Electrophoretic mobility shift assays showed direct and specific binding by Mac1-(1-159) to a DNA duplex containing the copper-responsive element TTTGCTCA. The DNA binding affinity of Mac1-(1-159) for a duplex containing a single promoter element or an inverted repeat was 5 nM for the 1:1 complex. The N-terminal 40-residue segment of Mac1 is homologous to the DNA binding zinc module found in the copper-activated transcription factors Ace1 and Amt1. A MAC1 mutation yielding a Cys11 --> Tyr substitution at the first candidate zinc ligand position relative to Ace1 resulted in a loss of in vivo function. Two TTTGCTCA promoter elements are necessary for efficient Mac1-mediated transcriptional activation. The elements appear to function synergistically. Increasing the number of elements yields more than additive enhancements in CTR1 expression.

Amino Acid Substitution↗
PubMed1998

Metal-ion regulation of gene expression in yeast.

Metal-responsive transcription factors exist in yeast to modulate expression of genes that encode proteins involved in cellular uptake of copper, iron and zinc ions. These signal transduction pathways function in the cellular regulation of the intracellular concentration of free metal ions. A second component of metal homeostasis is the regulation of metal-ion binding through protein-mediated metallation. Copper-specific chaperones exist in yeast that route copper ions to the site of biosynthesis of copper-metalloenzymes.

Copper↗
PubMed1998

The Mycoplasma hominis vaa gene displays a mosaic gene structure.

Mycoplasma hominis contains a variable adherence-associated (vaa) gene. To classify variants of the vaa genes, we examined 42 M. hominis isolated by PCR, DNA sequencing and immunoblotting. This uncovered the existence of five gene categories. Comparison of the gene types revealed a modular composition of the Vaa proteins. The proteins constituted a conserved N-terminal part followed by a varying number of interchangeable cassettes encoding approximately 110 amino acids with conserved sequences boxes flanking the cassettes. The interchangeable cassettes showed a high mutual homology and a conserved leucine zipper motif. The smallest product contained only one cassette and the largest five. Additionally, two types of stop mutations caused by substitutions resulting in the expression of truncated Vaa proteins were observed. Our results expand the known potential of the Vaa system in generating antigen variation.

Adhesins, Bacterial↗
PubMed1998

Mycoplasma contamination of Chlamydia pneumoniae isolates.

We examined 6 C. pneumonia isolates from The American Type Culture Collection (ATCC) and 2 Finnish isolates for Mycoplasma contamination. Three of the ATCC isolates and both of the Finnish isolates were Mycoplasma-contaminated. The contaminants were characterized by means of growth in BEa and BEg media, immunoblotting, polymerase chain reaction and pulsed field gel electrophoresis. Two of the 6 ATCC isolates [ATCC VR1355 (TWAR strain 2043) and ATCC VR1356 (TWAR strain 2023)] were infected with Mycoplasma hominis and 1 isolate [ATCC VR2282 (TWAR strain TW183)] was contaminated with both Mycoplasma hominis and Mycoplasma orale, whereas 3 of the ATCC isolates [ATCC VR1310, ATCC VR1360 (TWAR strain CM-1) and ATCC 53592 (TWAR strain AR39)] were not contaminated. The Finnish C. pneumoniae isolates Kajaani 6 and Parola were found to be contaminated with M. hominis and M. orale, respectively. The contamination of C. pneumoniae stock cultures, frequently used in the microimmunofluorescence test, with human pathogens, could pose a serious problem in C. pneumoniae serology.

Bacteriological Techniques↗
PubMed1998

Antigenic and genomic homogeneity of successive Mycoplasma hominis isolates.

Sixty Mycoplasma hominis isolates were obtained from the cervices of pregnant women and from the ears or pharynges of their newborn babies. The isolates were examined by SDS-PAGE and pulsed-field gel electrophoresis. Antigenic and genomic profiles were obtained for 16 series with two or more successive isolates. Both analyses led to the conclusion that isolates from the same woman were identical or nearly identical, while isolates from different women exhibited a high degree of variation with respect to both genomic and antigenic profiles.

Antigenic Variation↗
PubMed1997

Molecular biology of Mycoplasma.

Mycoplasmas are the smallest free living microorganisms with the smallest genome. The G+C content is in general low (25-33%) and the coding capacity is about 600 proteins. Mycoplasma species are phylogenetically related, they use the genetic codon UGA for tryptophan, and show rapid evolution, with a high rate of divergence. The genomes of Mycoplasma genitalium and Mycoplasma pneumoniae have been fully sequenced. Striking features of the M. genitalium sequencing project are the presence of a high number of membrane proteins with no resemblance to previously sequenced genes and the presence of repeated fragments of the gene encoding the tip-localized 140 kDa adhesin (MgPa). Many Mycoplasma species display a high frequency of antigenic variation, both as phase and size variation of individual antigens. Mycoplasma hominis isolates are known to be antigenic heterogeneous, as reflected in the reactivity with monoclonal antibodies (MAbs). The genetics of the antigenic variation has been studied for three different surface exposed antigens: P120, Lmp, and P50/Vaa. The gene encoding P120 had a hyper-variable region in the N-terminal region. In addition, a second gene with homology to p120 was identified. The gene encoding Lmp, a 135 kDa protein is repeated and both genes are translated and both contain internal repeated sequences. Deletion mutants in the lmp gene were obtained by cultivation of M. hominis PG21 with MAb 552 specific for the repeated part of Lmp. One of the lmp genes had deletions of from four to eight repeats. The other gene was left unaltered. The genes encoding P50/Vaa show a different form of variability where domains of the genes seem to be exchangeable. The genomic maps of five M. hominis strains showed that even though the size of the genomes varied the position of the different genes were in general conserved.

Animals↗
PubMed1997

The aminoterminal propeptide of type III procollagen. Studies on physiology and pathophysiology.

The aim of the present study was to examine the physiological basis for the clinical use of serum PIIINP as a marker of the deposition rate of type III collagen. The assumption was that the serum concentration of PIIINP would reflect the turnover of type III collagen and thus directly reflect the inflammatory response. For the study we assessed commercially available RIAs and optimised one of them. Porcine PIIINP was purified and compared with human PIIINP. The application of a gentle iodination procedure made it possible to perform tracer studies. An experimental model consisting of a thoracic duct-venous shunt in conscious pigs was developed. Double and triple isotope tracer techniques were used for kinetic studies in the animal model and in cultures of tubule cells. The rat model with the induction of granulation tissue was used to investigate catabolic states. The anabolic state was studied in humans receiving growth hormone therapy. We conclude: 1) That, for our purpose, the best method of determining PIIINP is the PIIINP RIA, owing to the profile of the substances determined. It was possible to improve the quality of the tracer and to increase sensitivity by changing the assay procedure. 2) That porcine PIIINP is similar to human PIIINP, therefore the human assay is suitable for studies in pigs. 3) That PIIINP most likely escapes from the extracellular space by bulk flow, similar to that of albumin. That the major part of the PIIINP synthesised is drained via the lymphatics. That intact PIIINP is not, or only to a minor extent, degraded through the lymphatics. Consequently, peak B is not a product of processes of the lymph system. 4) That in pigs intact PIIINP has a circulatory half-life of about 1 hour, and that it is degraded by at least two intermediary steps. The first step gives rises to peak B, which is found in an almost constant ratio to intact PIIINP. Peak B has a half-life of about 4 hours. Given steady state conditions peaks B and C (intact PIIINP) thus reflect the same process. In three different studies a fraction with an MW lower than that of PIIINP but higher than the col 1 domain, appeared during the degradation of intact PIIINP. This fraction (peak E) has not been described before. Furthermore, we did not observe the formation of peak D (proposed to be the col 1 domain of PIIINP) which indicates that this fraction does not originate from the metabolism of PIIINP. 5) That PIIINP rapidly distributed from the circulation to tissues, and that the liver and the kidneys are the organs mainly responsible for the degradation of PIIINP. The high hepatic clearance described previously is in part due to shunting of PIIINP directly to the lymph, but most of it is extracted by the liver for subsequent degradation or release of the intact molecule to the hepatic vein. Only a small part is irreversibly cleared and metabolised (about 5%). 6) That, given steady state conditions, the turnover of PIIINP is well reflected by changes in serum PIIINP, but also that this relation disappears when the body is in a catabolic state. Anabolic states give rise to increased serum concentrations of PIIINP as compared with normals states. The general conclusion is that serum PIIINP is a marker of type III collagen turnover under well-defined conditions. Serum PIIINP, mainly consisting of peaks B and C (intact PIIINP) may, owing to the disposal rate, reflect changes in type III collagen turnover over one day (6 half-lives). The liver and kidneys actively take part in the degradation of circulating PIIINP. Serum concentrations of PIIINP in the presence of changing body composition (weight loss) or during treatment with cytostatic drugs (cyclophosphamide) should be interpreted with caution.

Animals↗
PubMed1996

Diffusional transport of the aminoterminal propeptide of type III procollagen in the interstitium of the globally ischaemic cat myocardium.

Local repair after acute myocardial infarction appears to be reflected by levels in serum of the aminoterminal propeptide of type III procollagen (serum-PIIINP). Furthermore, serum-PIIINP has recently been reported to provide information on prognosis after acute myocardial infarction. However, no attention has yet been paid to the resistance to diffusion offered by the myocardial interstitium. We determined the diffusion coefficient of PIIINP in the interstitium of the globally ischaemic interstitium of the cat (D'37) by means of a "true transient diffusion' method, and compared with the free diffusion in water (D37). D'37 (in cm2 s-1.10(-5) was 0.0157 +/- 0.0005 (mean +/- SEM) (n = 13), and D37 was 0.0624 +/- 0.0024 (n = 12). The mean diffusive progression during 20 min of the concentration profile of [125I]PIIINP into the tissue was calculated to be 0.19 mm. The D'37 of albumin is practically identical to the D'37 of PIIINP, and the myocardium offers a similar resistance to diffusion of PIIINP and albumin, as expressed from the ratio D37/D'37 of approximately 4 for both molecules. PIIINP has a molecular weight of 42,000 Da, is rod shaped and has an overall negative charge. These characteristics explain the similarity in diffusion coefficients of PIIINP and albumin, which has a molecular weight of 69,000 Da. Albumin is known to pass the membrane of the continuous capillaries of the heart, making it very likely that direct exchange of PIIINP between interstitium and capillary plasma can also occur. During one hour of interstitial diffusion PIIINP will have traversed a distance calculated tp correspond to 15-20 capillaries. Therefore, the results support the concept of serum-PIIINP as a direct marker of events taking place locally in the myocardium following acute myocardial infarction.

Animals↗