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Biomedical subjects

M C Schmidt

Publications and source records attributed to M C Schmidt.

At least 37 records · Page 2Linked to original sources
PubMed1998

Identification of a calcineurin-independent pathway required for sodium ion stress response in Saccharomyces cerevisiae.

The calcium-dependent protein phosphatase calcineurin plays an essential role in ion homeostasis in yeast. In this study, we identify a parallel ion stress response pathway that is independent of the calcineurin signaling pathway. Cells with null alleles in both STD1 and its homologue, MTH1, manifest numerous phenotypes observed in calcineurin mutants, including sodium, lithium, manganese, and hydroxyl ion sensitivity, as well as alpha factor toxicity. Furthermore, increased gene dosage of STD1 suppresses the ion stress phenotypes in calcineurin mutants and confers halotolerance in wild-type cells. However, Std1p functions in a calcineurin-independent ion stress response pathway, since a std1 mth1 mutant is FK506 sensitive under conditions of ion stress. Mutations in other genes known to regulate gene expression in response to changes in glucose concentration, including SNF3, RGT2, and SNF5, also affect cell growth under ion stress conditions. Gene expression studies indicate that the regulation of HAL1 and PMR2 expression is affected by STD1 gene dosage. Taken together, our data demonstrate that response to ion stress requires the participation of both calcineurin-dependent and -independent pathways.

Adaptation, Physiological↗
PubMed1997

Assessment of myocardial perfusion by magnetic resonance imaging.

Magnetic resonance imaging (MRI) has proven useful for anatomic and functional evaluation of the heart. However, until recently assessment of myocardial perfusion has not been possible by MRI. Using newly developed ultrafast imaging sequences, images can be acquired rapidly with a high temporal resolution, which is a prerequisite for imaging the initial passage of a bolus of MR-contrast medium through the myocardium. Only gadolinium chelates, which rapidly diffuse out of vascular space, are currently approved for clinical use. The first pass of a bolus of one of these agents through hypoperfused myocardium distal to a coronary artery stenosis enhances this area less as compared to normally perfused areas. This different myocardial enhancement is often visible when looking at the series of MR images. However, intensity differences are rapidly decreasing as MR-contrast media are diluted in the systemio circulation after the first pass and diffuse to the interstitium. Therefore, only the first pass is of interest for MR-perfusion imaging. Additional and often more precise information can be derived by measuring parameters of the signal intensity time curve such as mean transit time, maximum signal intensity increase, upslope, downslope, and delay before reaching maximum signal intensity. Temporal resolution is the crucial factor in MR-perfusion imaging because it takes only 20 to 60 seconds for the contrast medium to pass through the myocardium. Therefore, this dynamic process must be imaged with a high temporal resolution. Moreover, image acquisition must be fast enough to minimize motion artefacts and to maximize the spatial coverage of the ventricle. Ultrafast gradient echo techniques and echo planar imaging are in principle capable to fulfill these demands. While ultrafast gradient echo sequences enable one to acquire a maximum of 2 slices per heartbeat, echo planar sequences need only 30 to 50 msec to completely acquire one image and are thus able to image the entire ventricle within one heartbeat. However, they are also more susceptible to image artefacts. As gradients capable of producing high quality echo planar images are not widely available, ultrafast gradient echo techniques are commonly used for MR-perfusion imaging. A good correlation between quantitative estimates of myocardial perfusion by MRI after injection of an intravascular contrast agent and microsphere measurements has been shown in animal experiments but quantitative MR perfusion measurements have not yet been performed in humans. Clinical studies have until now focused on visual and parametric analysis of signal intensity time curves. From these studies, sensitivities and specifities in the range of 60 to 90% as compared to x-ray coronary angiography and scintigraphy were reported despite the fact that only parts of the left ventricular myocardium could be assessed. However, a generally accepted method of acquiring and analysing MR perfusion images does not yet exist. Therefore, future improvements of hardware and pulse-sequences as well as the development of new blood pool contrast agents are necessary before MR-perfusion imaging will become a widely accepted and clinically useful diagnostic procedure.

Artifacts↗
PubMed1996

Expression of human lysosomal alpha-mannosidase activity in transfected murine cells and human alpha-mannosidase deficient fibroblasts.

We studied the human lysosomal alpha-mannosidase (MANB) by expressing the putative cDNA in mammalian cells, using the eucaryotic expression vector pCDE. The construct pCDE-MANB and pSV2-Neo were cotransfected into human alpha-mannosidase deficient fibroblasts and into a murine cell line and selected by culture in the presence of G418. Six G418 resistant 3T3 clones had increased alpha-mannosidase activity 2 to 3 times above the controls. Two clones from transfected human fibroblasts showed a 2 fold increase in enzyme activity. The human MANB cDNA gene was demonstrated in the target cells by Southern blot analysis and the expression of the gene was shown by RT-PCR analysis. This study is the first to successfully express the MANB gene in a human and a murine cell line. The results confirm that the putative MANB cDNA encodes the full length of lysosomal alpha-mannosidase. Molecular characterization of mannosidosis and approaches to gene therapy are now possible using this cDNA.

3T3 Cells↗
PubMed1996

DNA-binding properties of the yeast SWI/SNF complex.

The SWI/SNF complex is required for the enhancement of transcription by many transcriptional activators in yeast. Genetic and biochemical studies indicate that the complex facilitates activator function by antagonizing chromatin-mediated transcriptional repression. The absence of known DNA-binding motifs in several SWI/SNF subunits and the failure to identify SWI/SNF-dependent DNA-binding activities in crude yeast extracts have led to the belief that the complex does not bind DNA. Here we show that the SWI/SNF complex has a high affinity for DNA and that its DNA-binding properties are similar to those of proteins containing HMG-box domains. The complex interacts with the minor groove of the DNA helix, binds synthetic four-way junction DNA, and introduces positive supercoils into relaxed plasmid DNA. These properties are likely to be important in the remodelling of chromatin structure by the SWI/SNF complex

Adenosine Triphosphatases↗
PubMed1996

Intracoronary Doppler flow velocity measurements in two patients with solitary coronary ostium: is there true obstruction?

BACKGROUND: Coronary anomalies in the absence of other major cardiac malformations are rare, with an incidence of less than 1-1.5%. Although most have been incidental findings without clinical relevance, a subgroup of anomalies has been identified that are associated with cardiac symptoms or even sudden cardiac death. This subgroup includes an ectopic origin and abnormal course of the left coronary artery from the right sinus of Valsalva. RESULTS: We examined two patients with this anomaly. In patient I the left main coronary artery took a deep intramural course anterior to the pulmonary trunk, and in patient II the left anterior descending coronary artery took its course between the pulmonary trunk and ascending aorta. Both anomalies were identified by coronary angiography and that in patient I was confirmed by surgery. Intracoronary Doppler flow velocity measurements during cardiac catheterization, including atrial pacing in patient II, demonstrated significant flow acceleration in both patients. This occurred within the intramural segment (in patient I) and in the segment between the pulmonary artery and ascending aorta (in patient II). CONCLUSIONS: These findings indicate that external vessel compression is one pathophysiological mechanism that leads to ischaemia and a longstanding history of cardiac symptoms in these patients. Invasive haemodynamic measurements such as these have not previously been available.

Blood Flow Velocity↗
PubMed1996

cis-acting elements involved in transcriptional regulation of the herpes simplex virus type 1 latency-associated promoter 1 (LAP1) in vitro and in vivo.

Latency-associated promoter 1 (LAP1) of herpes simplex virus type 1 is required to generate a series of latency-associated transcripts (LATs) in sensory neurons of latently infected animals. Sequence analysis and DNA binding studies have suggested the existence of several cis-acting elements within LAP1 that are potentially important for promoter function, although their role in LAT gene expression during latency is largely unexplored. In this report, we present evidence that the LAP1 TATA box is essential for transcription initiation in vitro. A reduction in LAT synthesis measured by in situ hybridization and reverse transcription-PCR (RT-PCR) of rat brain tissue latently infected with a LAP1 TATA substitution virus demonstrated that this sequence was required for full LAP1 activity in vivo. Analysis of additional site-directed and 5'-deletion mutants of LAP1 by in vitro transcription-primer extension assays showed that upstream elements including the USF and cyclic AMP response element (CRE) site specifically contributed to LAP1 function and that sequences beginning at position -620 relative to the transcription start site were essential for full promoter activity. The combination of deleting USF, CRE, and TATA completely abolished LAT expression in the brain, identifying these as essential elements for the neuron-specific functioning of LAP1 during latency. Mutation of the transcription start site did not abolish transcription, suggesting the absence of an initiator element. However, one of the most exciting findings from this study is that the region downstream of the TATA box appears to contain a true enhancer that is not only essential for transcription, but also functional when positioned 1.6 kb downstream of the start site of transcription. It was concluded that (i) the TATA box was essential for full transcriptional activity from LAP1 both in vitro and in vivo, (ii) the USF element and CRE contribute to LAP1 function during latency in combination with the TATA element, (iii) multiple trans-acting factors besides the USF- and CRE-binding proteins were required for full promoter activity in vitro, and (iv) sequences downstream of the TATA box enhanced promoter activity in vitro.

Animals↗
PubMed1996

Involvement of a high-mobility-group protein in the transcriptional activity of herpes simplex virus latency-active promoter 2.

Latency-active promoter 2 (LAP 2) is a TATA-less promoter in herpes simplex virus type 1 (HSV-1) that can express genes during viral latency. Four regions of LAP2 are protected from DNase I digestion in vitro by either HeLa cell nuclear extracts or purified Sp1. Transient gene expression assays of LAP2 substitution mutants demonstrate that two of the regions protected by Sp1 and three other regions protected by nuclear extract are important for promoter function. The mutation causing the most significant reduction in expression alters a stretch of 23 thymidine residues (T23) that binds a protein with several properties common to high-mobility-group (HMG) proteins. The T23 binding activity is heat stable, can be inhibited by poly(dA-dT).poly(dA-dT), and is inhibited by minor-groove-binding drugs. Antiserum directed against HMG I(Y) blocked the formation of one of the DNA-protein complexes on the T23 oligonucleotide, suggesting that a protein antigenically related to HMG I(Y) binds to LAP2 in vitro. Direct evidence of HMG I(Y) involvement in LAP2 function is provided by the findings that recombinant HMG I(Y) protein facilitates Sp1 binding to LAP2 in mobility shift assays and that antisense HMG I(Y) RNA specifically inhibits LAP2 function in vivo. These results suggest that DNA structure may be an important determinant of the activity of a promoter that is capable of escaping the global shutoff of transcription that occurs during viral latency.

Animals↗
PubMed1996

In vivo transgene activation from an HSV-based gene therapy vector by GAL4:vp16.

Herpes simplex virus type 1 (HSV-1) has many attributes which make it attractive as a base for the development of vectors for the delivery of transgenes to the nervous system. In this report we describe the adaptation of the bipartite GAL4:VP16 transactivation system to replication-deficient HSV vectors. We demonstrate that the recombinant transactivator GAL4:VP16 produced from a replication-deficient HSV vector is capable of activating transcription of a reporter gene using a synthetic promoter consisting of GAL4 binding sites and the TATA box of the adenovirus E1b gene. Activation by vector produced GAL:VP16 was demonstrated with the recombinant promoter/reporter gene cassette in the infected cell chromosome, in the genome of a second virus infecting the same cells and with a single vector engineered to produce both GAL4:VP16 transactivator and to contain a recombinant promoter/reporter gene cassette. Furthermore, the double recombinant virus also produced the reporter gene product in neurons after direct intracranial inoculation into rat hippocampus. This system may be used to extend and improve promoter function in HSV gene transfer vectors in vivo.

Animals↗
PubMed1995

STD1 (MSN3) interacts directly with the TATA-binding protein and modulates transcription of the SUC2 gene of Saccharomyces cerevisiae.

STD1 (MSN3) was isolated independently as a multicopy suppressor of mutations in the TATA-binding protein and in SNF4, suggesting that STD1 might couple the SNF1 kinase signaling pathway to the transcriptional machinery. We report here a direct physical interaction between STD1 and the TATA-binding protein (TBP), observed in vivo by the two-hybrid system and in vitro by binding studies. STD1 bound both native TBP in yeast cell-free extracts and purified recombinant TBP. This interaction was altered when TBP delta 57 was used, suggesting a role for the non-conserved N-terminal domain of TBP in mediating protein-protein interactions. We also show that perturbation of STD1-TBP stoichiometry alters SUC2 expression in vivo and that this effect is dependent on the N-terminal domain of TBP. The activation of SUC2 expression by increased copy number of STD1 occurs at the level of mRNA accumulation and it requires the same TATA element and uses the same transcription start site as does activation of SUC2 by glucose limitation. Taken together, these results suggest that STD1 modulates SUC2 transcription through direct interactions with TBP.

Base Sequence↗
PubMed1995

Two herpes simplex virus type 1 latency-active promoters differ in their contributions to latency-associated transcript expression during lytic and latent infections.

Herpes simplex virus type 1 (HSV-1) establishes latency in human sensory ganglia, during which time the viral genome is transcriptionally silent with the exception of the latency-associated transcripts (LATs). The most abundant LAT is a 2-kb RNA whose biosynthesis is poorly characterized. The 2-kb LAT may be a primary transcript, or its synthesis may involve splicing and/or other forms of processing. Two potential RNA polymerase II promoters (LAP1 and LAP2) upstream of the 2-kb LAT 5' end have been identified. To investigate the role played by LAP1 and LAP2 in the synthesis of the 2-kb LAT under lytic and latent conditions, we analyzed HSV-1 mutants which contain deletions of one or both of these promoters. During lytic infection in cell culture, the cis elements critical for the normal accumulation of the 2-kb LAT were mapped to LAP2, while LAP1 sequences were largely dispensable. The 5' ends of the major 2-kb LATs produced by the wild-type and LAP deletion viruses were examined by primer extension analysis and were all found to be identical (+/- 2 bp). The accumulation of the 2-kb LAT during latent infections of murine trigeminal ganglia was examined by Northern (RNA) blot and by reverse transcription-PCR. In contrast to the results found in lytic infections, the critical cis elements needed for 2-kb LAT accumulation during latency were mapped to LAP1. Deletion of LAP1 resulted in a 500-fold reduction in 2-kb LAT accumulation, whereas deletion of LAP2 resulted in only a 2- to 3-fold reduction. Deletion of both LAP1 and LAP2 resulted in undetectable levels of the 2-kb LAT. Our results indicate that both LAP1 and LAP2 are critical for 2-kb LAT expression but under different conditions. LAP1 is essential for LAT expression during latency, while LAP2 is primarily responsible for LAT expression in lytic infections in cell culture. LAP1 and LAP2 may prove to be functionally independent promoter elements that control 2-kb LAT expression during different stages of HSV-1 infections.

Animals↗
PubMed1994

Human lysosomal alpha-mannosidase: isolation and nucleotide sequence of the full-length cDNA.

The amino acid sequence of the human lysosomal alpha-mannosidase precursor has been deduced by PCR mediated cloning and sequencing of the cDNA. The protein has 961 amino acids and a molecular weight of 107,644 Daltons. The amino acid sequence shows 38% identity to the Dictyostelium discoideum lysosomal alpha-mannosidase. The cDNA maps proximal to the centromere on chromosome 19q, the same locus as the MANB gene. Our results provide valuable information for the study of the lysosomal storage disease alpha-mannosidosis, an inherited disorder caused by mutations in the MANB gene which encodes the human lysosomal alpha-mannosidase.

Amino Acid Sequence↗
PubMed1993

Effect of the non-conserved N-terminus on the DNA binding activity of the yeast TATA binding protein.

We have studied the DNA binding activity of recombinant yeast TATA Binding Protein (TBP) with particular interest in the role played by the non-conserved N-terminal domain. By comparing the DNA binding activity of wild type yeast TBP with a mutant form of TBP that lacks the non-conserved N-terminal domain (TBP delta 57), we have determined that the N-terminus of TBP alters both the shape and the stability of the TBP-DNA complex. Measurements of the DNA bending angle indicate that the N-terminus enhances the bending of the DNA that is induced by TBP binding and greatly destabilizes the TBP-DNA complex during native gel electrophoresis. In solution, the N-terminus has only a slight effect on the equilibrium dissociation constant and the dissociation rate constant. However, the N-terminal domain reduces the association rate constant in a temperature dependent manner and increases the apparent activation energy of the TBP-DNA complex formation by 3 kcal/mole. These data suggest that a conformational change involving the N-terminus of TBP may be one of the isomerization steps in the formation of a stable TBP-DNA complex.

Base Sequence↗
PubMed1993

Isolation of STD1, a high-copy-number suppressor of a dominant negative mutation in the yeast TATA-binding protein.

The TATA-binding protein (TBP) is an essential component of the transcriptional machinery of all three nuclear RNA polymerase enzymes. Comparison of the amino acid sequence of TBPs from a number of species reveals a highly conserved 180-residue C-terminal domain. In contrast, the N terminus is variable in both size and amino acid sequence. Overexpression of a TBP protein with a deletion of the nonconserved N terminus (TBP delta 57) in Saccharomyces cerevisiae results in a dominant negative phenotype of extremely slow growth. Associated with the slow-growth phenotype are defects in RNA polymerase II transcription in vivo. We have screened a high-copy-number yeast genomic library for suppression of the slow-growth phenotype and have isolated plasmids which encode suppressors of TBP delta 57 overexpression. Here we report the sequence and initial characterization of one suppressor, designated STD1 for suppressor of TBP deletion. The STD1 gene contains a single continuous open reading frame with the potential to encode a 50.2-kDa protein. Disruption of the STD1 gene indicates that it is not essential for vegetative growth, mating, or sporulation. High-copy-number suppression by the STD1 gene is not the result of a decrease in TBP delta 57 protein accumulation or DNA-binding activity; instead, STD1 suppression is coincident with the elimination of TBP delta 57-induced RNA polymerase II defects in both uninduced and induced transcription in vivo.

Amino Acid Sequence↗
PubMed1991

Requirement for acidic amino acid residues immediately N-terminal to the conserved domain of Saccharomyces cerevisiae TFIID.

TFIID binds to TATA boxes and initiates the assembly of general transcription factors and pol II on promoters. TFIID proteins from various species consist of a highly conserved carboxy terminal domain and very divergent amino terminal domains. We investigated the function of the non-conserved amino terminal domain (residues 1-60) of Saccharomyces cerevisiae TFIID (YIID, 240 residues) by testing the ability of a series of YIID amino terminal deletion mutants to complement a YIID deficient yeast strain. Mutants with deletions up to amino acid 48 restored the YIID deficient yeast strain to an apparently wild type phenotype. However, deletion up to position 57 or 60 produced yeast strains which formed extremely small colonies. Moreover, overexpression of YIID delta 2-57 or YIID delta 3-60 protein in the presence of wild type YIID resulted in a dominant-negative inhibition of growth. No difference between the basal transcriptional activity of wild type YIID and these amino terminal deletion mutants was observed in vitro. However, transcriptional activation in vivo of promoter-lacZ fusions showed that the YIID delta 2-57 deletion affects the ability of certain promoters (CUP1 and an HSP UAS-CYC1 promoter hybrid promoter) to respond to upstream factor stimulation. At least one inducible promoter, PHO5, was not affected by this deletion. The defect produced by YIID delta 2-57 was due to the deletion of several acidic residues present between residues 48 and 57. The results show that the conserved carboxy terminal domain of YIID is sufficient for cell viability. However, an acidic region just amino terminal to the conserved domain is required for normal growth and transcription control in most yeast strains.

Amino Acid Sequence↗
PubMed1991

Two distinct domains in the yeast transcription factor IID and evidence for a TATA box-induced conformational change.

Transcription factor IID from Saccharomyces cerevisiae (YIID) binds the TATA box element present in most RNA polymerase II promoters. In this work, partial proteolysis was used as a biochemical probe of YIID structure. YIID consists of a protease-sensitive amino terminus and a highly stable, protease-resistant carboxy-terminal core. The cleavage sites of the predominant chymotrypsin- and trypsin-derived fragments were mapped to amino acid residues 40 to 41 and 48 to 49, respectively, by amino-terminal peptide sequencing. Removal of the amino terminus resulted in a dramatic increase in the ability of YIID to form a stable complex with DNA during gel electrophoresis mobility shift assays and a two- to fourfold increase in DNA-binding affinity, as assayed by DNase I footprinting analysis. The carboxy-terminal 190-amino-acid core was competent for transcription in vitro and was similar in activity to native YIID. DNA containing a TATA element induced hypersensitive sites in the amino-terminal domain and stabilized the core domain to further proteolytic attack. Native YIID did not bind to a TATA box at 0 degrees C, whereas the carboxy-terminal DNA-binding domain did. These results suggest that YIID undergoes a conformational change upon binding to a TATA box. Southern blotting showed that the carboxy-terminal domain is highly conserved, while the amino-terminal domain diverged rapidly in evolution, even between closely related budding yeasts.

Binding Sites↗
PubMed1990

Cloning of a transcriptionally active human TATA binding factor.

Transcription factor IID (TFIID) binds to the TATA box promoter element and regulates the expression of most eukaryotic genes transcribed by RNA polymerase II. Complementary DNA (cDNA) encoding a human TFIID protein has been cloned. The human TFIID polypeptide has 339 amino acids and a molecular size of 37,745 daltons. The carboxyl-terminal 181 amino acids of the human TFIID protein shares 80% identity with the TFIID protein from Saccharomyces cerevisiae. The amino terminus contains an unusual repeat of 38 consecutive glutamine residues and an X-Thr-Pro repeat. Expression of DNA in reticulocyte lysates or in Escherichia coli yielded a protein that was competent for both DNA binding and transcription activation.

Amino Acid Sequence↗
PubMed1990

Transcriptional activation by Sp1 as directed through TATA or initiator: specific requirement for mammalian transcription factor IID.

Transcription of mammalian genes by RNA polymerase II often begins at a specific nucleotide, whose location is determined either by an upstream DNA element known as a TATA box or by an element positioned at the transcription start site called an initiator (Inr). By in vitro analysis of synthetic promoters, we demonstrate here that the TATA and Inr elements are functionally similar and that the Inr is contained between nucleotides -3 and +5 relative to the initiation site. Moreover, we found that a mammalian transcription factor IID (TFIID) protein fraction is required for transcriptional stimulation by an Sp1-dependent activating element placed upstream of either TATA or Inr elements. However, in these assays, the yeast TATA-binding protein, which previously was shown to function similarly to mammalian TFIID, could not efficiently substitute for the mammalian TFIID fraction. These results demonstrate that mammalian TFIID is functionally distinct from the yeast TATA-binding protein and may contain additional subunits or domains that are important for transcriptional activation from some promoters.

Adenovirus Early Proteins↗