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Biomedical subjects

W Reith

Publications and source records attributed to W Reith.

At least 109 records · Page 6Linked to original sources
PubMed1994

MRI in acute cerebral ischaemia: perfusion imaging with superparamagnetic iron oxide in a rat model.

An imaging technique capable of detecting ischaemic cerebral injury at an early stage could improve diagnosis in acute or transient cerebral ischaemia. We compared the ability of superparamagnetically contrast-enhanced MRI and conventional T2-weighted MRI to detect ischaemic injury early after unilateral occlusion of the middle cerebral artery in 12 male Wistar rats. Permanent vessel occlusion was achieved by a transvascular approach, which has the advantage of not requiring a craniectomy. At 45-60 min after the procedure, the animals had conventional T2-weighted MRI before and after administration of a superparamagnetic contrast agent (iron oxide particles). Unenhanced images were normal in all animals. After administration of iron oxide particles, the presumed ischaemic area was clearly visible, as relatively increased signal, in all animals; this high signal area corresponded to the area of ischaemic brain infarction seen on histological studies. Magnetic susceptibility effects of iron particles cause low signal in normally perfused cerebral tissue, whereas tissue with reduced or absent blood flow continues to give relatively high signal. Our results suggest that superparamagnetic iron particles may significantly reduce the interval between an ischaemic insult and the appearance of parenchymal changes on MRI.

Acute Disease↗
PubMed1994

Laser Doppler flowmetry of focal ischaemia and reperfusion in deep brain structures in rats.

Monitoring cerebral blood flow during focal ischaemia and reperfusion with established techniques such as hydrogen clearance and autoradiography is difficult. Laser Doppler flowmetry is a new technique, it allows one to continuously measure blood flow in small tissue samples. The objective of this study was to compare laser Doppler flowmetry with hydrogen clearance using a new single fiber probe to obtain measurements in deep brain structures and then to show the temporal profile of cerebral blood flow during focal ischaemia and after reperfusion. First, the single fiber laser Doppler method was compared with the hydrogen clearance method in ten Wistar rats. Second, focal cerebral ischaemia was induced in fifteen Wistar rats using a model of middle cerebral artery occlusion based on the intravascular insertion of a nylon suture; reperfusion occurred after withdrawal of the suture. The laser Doppler probe was placed in the lateral caudatoputamen, and local cerebral blood flow was measured continuously before and during occlusion as well as after reperfusion. The relative blood flow values obtained by the laser Doppler method and the hydrogen clearance method showed a good correlation (r = 0.76) and a linear relationship. A rapid decrease in laser Doppler flowmetry to 42 +/- 16% of former baseline values was seen with occlusion of the middle cerebral artery; during occlusion cerebral blood flow remained at this level. Reperfusion resulted in a heterogeneous pattern of cerebral blood flow as laser Doppler flowmetry values ranged from 25% to 134% of baseline values. The effects of middle cerebral artery occlusion and reperfusion on cerebral blood flow can be monitored on-line with laser Doppler flowmetry.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Functional complementation of major histocompatibility complex class II regulatory mutants by the purified X-box-binding protein RFX.

Major histocompatibility complex (MHC) class II deficiency, or bare lymphocyte syndrome (BLS), is a disease of gene regulation. Patients with BLS have been classified into at least three complementation groups (A, B, and C) believed to correspond to three distinct MHC class II regulatory genes. The elucidation of the molecular basis for this disease will thus clarify the mechanisms controlling the complex regulation of MHC class II genes. Complementation groups B and C are characterized by a lack of binding of RFX, a nuclear protein that normally binds specifically to the X box cis-acting element present in the promoters of all MHC class II genes. We have now purified RFX to near homogeneity by affinity chromatography. Using an in vitro transcription system based on the HLA-DRA promoter, we show here that extracts from RFX-deficient cells from patients with BLS (BLS cells) in groups B and C, which are transcriptionally inactive in this assay, can be complemented to full transcriptional activity by the purified RFX. As expected, purified RFX also restores a completely normal pattern of X box-binding complexes in these mutant extracts. This provides the first direct functional evidence that RFX is an activator of MHC class II gene transcription and that its absence is indeed responsible for the regulatory defect in MHC class II gene expression in patients with BLS.

Base Sequence↗
PubMed1994

RFX1, a transactivator of hepatitis B virus enhancer I, belongs to a novel family of homodimeric and heterodimeric DNA-binding proteins.

RFX1 is a transactivator of human hepatitis B virus enhancer I. We show here that RFX1 belongs to a previously unidentified family of DNA-binding proteins of which we have cloned three members, RFX1, RFX2, and RFX3, from humans and mice. Members of the RFX family constitute the nuclear complexes that have been referred to previously as enhancer factor C, EP, methylation-dependent DNA-binding protein, or rpL30 alpha. RFX proteins share five strongly conserved regions which include the two domains required for DNA binding and dimerization. They have very similar DNA-binding specificities and heterodimerize both in vitro and in vivo. mRNA levels for all three genes, particularly RFX2, are elevated in testis. In other cell lines and tissues, RFX mRNA levels are variable, particularly for RFX2 and RFX3. RFX proteins share several novel features, including new DNA-binding and dimerization motifs and a peculiar dependence on methylated CpG dinucleotides at certain sites.

Alternative Splicing↗
PubMed1994

[Radiology of cerebrovascular malformations].

Cerebral vascular malformations are, after hypertension, the second most frequent cause of non-traumatic, intracerebral hemorrhage. A reliable intravital diagnosis only became possible with radiological diagnostic methods which also contributed significantly to the development of therapeutic concepts. Diagnostic and therapeutic possibilities for the various forms of cerebral vascular malformations are surveyed in the present review. Since radiology occupies a central position in the diagnosis and (in the meantime also) therapy of cerebral vascular malformations, epidemiological and prognostic factors are also discussed in detail.

Brain Neoplasms↗
PubMed1993

Clathrin-coated pit-mediated receptor internalization. Role of internalization signals and receptor mobility.

Most signals controlling receptor-mediated endocytosis have been identified by alteration of sequences present in receptors normally internalized via clathrin-coated pits. In the present work we have reconsidered the factors that control internalization the other way around: i.e. by introducing potential internalization sequences in complement receptor 1 (CR1) which does not preferentially associate with clathrin-coated pits. The analysis of the internalization efficiency of NPxY related motifs generated by substituting His2010 and/or Glu2015 by either Phe or Tyr indicates that FxNPxY is the stronger promoter of endocytosis and that the signal efficiency depends on the presence of aromatic residues (including a tyrosine) at both ends of the -xNPx- motif. Moreover, CR1-tyr (substitution of Glu2015 for Tyr) internalization was superposable to that of a receptor composed of the extracellular and transmembrane domains of CR1 fused to the intracytoplasmic tail of the low density lipoprotein (LDL) receptor (including the FxNPxY motif) (CR1-LDL). When analyzed by fluorescence recovery after photobleaching, the surface mobility of CR1-LDL was decreased as compared with that of either CR1-tyr or CR1-wt, despite a similar association with clathrin-coated pits. The role of receptor mobility in internalization was confirmed by the observation that CR1-tl, with a deletion of the cytoplasmic tail, was more mobile and more efficiently internalized than CR1-wt.

Amino Acid Sequence↗
PubMed1993

The different level of expression of HLA-DRB1 and -DRB3 genes is controlled by conserved isotypic differences in promoter sequence.

HLA-DRB1 and -DRB3 are two genes encoding two distinct HLA-DR beta chains in the DRw52 family of haplotypes (DR3, DR5, DR13, and DR14). These beta chains determine the structural and functional identity of the two kinds of HLA-DR molecules expressed. The highly polymorphic HLA-DRB1 locus is always expressed at a higher level than the HLA-DRB3 locus, and functional assays indicate that the proximal promoter of DRB1 is indeed more active than that of DRB3. The DNA sequence of the two promoters in nine different DRw52 haplotypes has revealed a stricking allelic conservation as well as characteristic, isotype-specific, conserved-sequence motifs. These isotype-specific differences concern the functionally essential X and Y box motifs of HLA class II promoters, and they do indeed affect binding of specific nuclear factors to the X and Y boxes of DRB1 or DRB3 promoters. Finally, analysis of the activity of various normal and mutated DRB1 or DRB3 promoters indicates that the X box region of these promoters plays a dominant role in controlling the relative levels of HLA-DRB1 and -DRB3 gene expression.

Alkaline Phosphatase↗
PubMed1993

RFX1 is identical to enhancer factor C and functions as a transactivator of the hepatitis B virus enhancer.

Hepatitis B virus gene expression is to a large extent under the control of enhancer I (EnhI). The activity of EnhI is strictly dependent on the enhancer factor C (EF-C) site, an inverted repeat that is bound by a ubiquitous nuclear protein known as EF-C. Here we report the unexpected finding that EF-C is in fact identical to RFX1, a novel transcription factor previously cloned by virtue of its affinity for the HLA class II X-box promoter element. This finding has allowed us to provide direct evidence that RFX1 (EF-C) is crucial for EnhI function in HepG2 hepatoma cells; RFX1-specific antisense oligonucleotides appear to inhibit EnhI-driven expression of the hepatitis B virus major surface antigen gene, and in transfection assays, RFX1 behaves as a potent transactivator of EnhI. Interestingly, transactivation of EnhI by RFX1 (EF-C) is not observed in cell lines that are not of liver origin, suggesting that the ubiquitous RFX1 protein cooperates with liver-specific factors.

Animals↗
PubMed1993

[Radiology of stroke].

Today radiologic methods play an important role in the diagnosis of cerebral stroke. The aim of the radiologist, however, should not only be to classify the stroke into the four main categories (ischemic stroke, intracerebral bleeding, subarachnoid hemorrhage, sinovenous thrombosis), but also to interpret the findings with regard to the etiology of the disease. The pattern of lesions gives information about the etiology of ischemic stroke; the correct interpretation of these lesion patterns allows one to optimize therapeutic decisions. This paper additionally focuses on the differential diagnosis of intracerebral hemorrhage and the CT and MR signs of sinovenous thrombosis. New developed concepts in the field of stroke therapy and prophylaxis call for authority and continuous education of the radiologist on this topic. Pure descriptions of radiologic findings without an understanding of the pathogenesis of the disease will be ignored by the clinician. On the other hand, the radiologist can turn the therapeutic decisions in the right direction by combining morphological descriptions with pathogenetic orientated interpretations. In this way, the radiologist can contribute to the reduction of costs in the public health system.

Cerebrovascular Disorders↗
PubMed1992

The genes for MHC class II regulatory factors RFX1 and RFX2 are located on the short arm of chromosome 19.

RFX1 is a transacting DNA-binding regulatory factor involved in the control of MHC class II gene expression. RFX2 is a structurally very similar protein with identical DNA binding features. A member of the family of RFX factors is affected in an autosomal recessive disease, MHC class II deficient combined immunodeficiency (CID), caused by a defect in a trans-acting regulatory factor controlling MHC class II gene expression. In situ hybridization with 3H-labeled RFX1 cDNA has allowed us to identify two distinct targets on the short arm of chromosome 19 (19p13.1 and 19p13.2-p13.3). With the use of biotinylated genomic cosmid clones specific for RFX1 and RFX2, respectively, it was then possible to localize RFX1 at 19p13.1 and RFX2 at 19p13.2-p13.3. These two regulatory genes are thus assigned to a region of high gene density and RFX1 is close to another DNA-binding factor, LYL1.

Chromosome Mapping↗
PubMed1992

The DNA-binding defect observed in major histocompatibility complex class II regulatory mutants concerns only one member of a family of complexes binding to the X boxes of class II promoters.

The X box of major histocompatibility complex class II promoters is essential for proper expression of class II genes. Here we show that two distinct protein-DNA complexes (A and B), which exhibit similar binding characteristics and identical contact points on the X box, can be formed. This suggests the existence of a family of related X box-binding factors. Complex B (and not complex A) is specifically affected in primary combined immunodeficiency, a congenital defect in class II gene regulation. RFX1, the first X box-binding protein cloned, encodes a functionally relevant factor present in complex A and not in complex B as originally suspected. This report also illustrates the need for caution in correlating specific cloned proteins with nuclear factors identified by DNA-binding assays, particularly when dealing with families of related proteins.

Base Sequence↗
PubMed1991

Regulatory factor-X binding to mutant HLA-DRA promoter sequences.

The class II genes of the major histocompatibility complex (MHC) encode a family of cell surface glycoproteins that present processed antigen to the T cell receptor. Class II genes are regulated coordinately, responding to both immunologic and developmental signals. Conserved sequence elements 5' to class II genes have been shown to be important in transcriptional control. One of these sequences, the X box, is a specific target for the binding of the factor RF-X. In the hereditary HLA class II deficiency, a form of primary immunodeficiency, a regulatory defect in expression of class II genes is associated with a defect in the binding of RF-X. To determine the basepairs that are important for this binding interaction, a series of single basepair substitutions spanning the X box motif of the DRA gene was constructed and tested for binding of RF-X by gel electrophoresis mobility shift assays (EMSAs). Several, but not all, of the mutants severely affected binding of RF-X. In addition, the binding of both the natural and the recombinant form of RF-X was affected with the same specificity. A comparison of X box basepair positions important for RF-X binding to DRA with sequences conserved between X boxes of other class II alpha chain genes suggests that high affinity RF-X binding is important for a high level of expression and may explain differences in the levels of class II expression of different class II alpha chains.

Animals↗
PubMed1991

Hyperexpression of interferon-gamma-induced MHC class II genes associated with reorganization of the cytoskeleton.

Class I and class II major histocompatibility complex (MHC) gene products are key recognition units in the induction and regulation of the immune response. Expression of class I and class II may be constitutive or inducible by cytokines such as interferon-gamma (IFN-gamma). A key step in the induction of MHC genes is recognition of IFN-gamma by its membrane receptor. The work described here examines the regulation of the occupied IFN-gamma receptor by the cytoskeleton. To do this the authors have used the fungal metabolites dihydrocytochalasin B (DHCB) and cytochalasin D (CD), substances that bind to actin filaments and thereby disrupt the cytoskeleton. The authors have studied the effect of DHCB and CD on IFN-gamma-induced MHC gene expression in 143 B cells, a human osteosarcoma-derived cell line. Herein the authors demonstrate that alterations in the cytoskeleton induced by DHCB and CD can lead to increases in IFN-gamma-induced MHC gene expression. Dihydrocytochalasin B added up to 3 hours after IFN-gamma results in a threefold to sixfold increase in levels of class II mRNA while producing minimal enhancement of class I gene expression. In contrast, glyceraldehyde-3-phosphate dehydrogenase mRNA expression was unaltered by IFN-gamma or by the cytochalasins. The increased amount of class II mRNA can be accounted for by a concomitant increase in transcription rate of this gene. Studies using 125I-IFN-gamma demonstrate that the occupied IFN-gamma receptor associates with a Triton X-100 insoluble fraction of 143 B cells and that DHCB and CD markedly inhibit this association. The results described here provide evidence that is consistent with the hypothesis that the activity of the occupied IFN-gamma receptor may be modulated by interactions with the cytoskeleton of the cell. This receptor may be one of a group of plasma membrane receptors that are sensitive to the action of cytochalasins after ligand binding.

B-Lymphocytes↗
PubMed1990

Significance of transcranial Doppler CO2. Reactivity measurements for the diagnosis of hemodynamically relevant carotid obstructions.

Transcranial Doppler ultrasonography can be used to determine CO2 reactivity in the large basal cerebral arteries. CO2 reactivity is expressed as percentage increase of mean flow velocity above one volume percent of CO2, using a reference value of 40 mmHg pCO2 normalized autoregulatory reserve. A normalized autoregulatory value of 15 as the lower limit of the normal range clearly separates patients with internal carotid artery obstructions greater than or equal to 70% from the control group. Stenoses of the ipsilateral internal carotid artery of greater than or equal to 70% result in a significant decrease of normalized autoregulatory reserve in the middle cerebral artery, which can be normalized by removal of the upstream flow obstacle using a carotid thromboendarterectomy. The scatter of normalized autoregulatory values in severe internal carotid obstructions indicates the variability of collateral circulation A retrospective comparison of normalized autoregulatory reserve and ipsilateral ischemic symptoms in the supply area of the internal carotid artery reveals a significant correlation between clinical symptoms and reduced normalized autoregulatory reserve.

Adult↗
PubMed1990

MHC class II regulatory factor RFX has a novel DNA-binding domain and a functionally independent dimerization domain.

The regulation of MHC class II gene expression controls T-cell activation and, hence, the immune response. Among the nuclear factors observed to bind to conserved DNA sequences in human leukocyte antigen (HLA) class II gene promoters, RFX is of special interest: Its binding is defective in congenital HLA class II deficiency, a disease of class II gene regulation. The cloning of an RFX cDNA has allowed us to show by transfection of a plasmid directing the synthesis of antisense RFX RNA that RFX is a class II gene regulatory factor. RFX is a novel 979-amino-acid DNA-binding protein that contains three structurally and functionally separate domains. The 91-amino-acid DNA-binding domain is distinct from other known DNA-binding motifs but may be distantly related to the helix-loop-helix motif. The most striking property of RFX is that it can bind stably to the class II X box as either a monomer or a homodimer and that the domain responsible for dimerization is distant from and functionally independent of the DNA-binding domain. This distinguishes RFX from other known dimeric DNA-binding proteins. It also implies that an RFX homodimer has two potential DNA-binding sites. We therefore speculate that RFX could form a DNA loop by cross-linking the two X-box sequences found far apart upstream of MHC class II genes.

Amino Acid Sequence↗
PubMed1990

Two DNA-binding proteins discriminate between the promoters of different members of the major histocompatibility complex class II multigene family.

The regulation of major histocompatibility complex (MHC) class II gene expression is a key feature of the control of normal and abnormal immune responses. In humans, class II alpha - and beta-chain genes are organized in a multigene family with three distinct subregions, HLA-DR, -DQ, and -DP. The regulation of these genes is generally coordinated, and their promoters contain highly conserved motifs, in particular the X and Y boxes. We have identified five distinct proteins that bind to specific DNA sequences within the first 145 base pairs of the HLA-DR promoter, a segment known to be functionally essential for class II gene regulation. Among these, RF-X is of special interest, since mutants affected in the regulation of MHC class II gene expression have a specific defect in RF-X binding. Unexpectedly, RF-X displays a characteristic gradient of binding affinities for the X boxes of three alpha-chain genes (DRA greater than DPA much greater than DQA). The same observation was made with recombinant RF-X. We also describe a novel factor, NF-S, which bound to the spacer region between the X and Y boxes of class II promoters. NF-S exhibited a reverse gradient of affinity compared with RF-X (DQA greater than DPA much greater than DRA). As expected, RF-X bound well to the mouse IE alpha promoter, while NF-S bound well to IA alpha. The drastic differences in the binding of RF-X and NF-S to different MHC class II promoters contrasts with the coordinate regulation of HLA-DR, -DQ, and -DP genes.

Animals↗
PubMed1989

Induction of HLA class II genes by IFN-gamma is transcriptional and requires a trans-acting protein.

HLA class II Ag are encoded by a family of related genes clustered in the HLA-D region of the MHC. The expression of this multi-gene family is highly regulated and this regulation is essential for the control of the immune response. Class II gene expression is constitutive in a limited number of cell types and can be induced by IFN-gamma in a number of class II negative cells. In this study, we have clarified two essential aspects of the regulation of HLA class II genes by IFN-gamma. 1) The induction mechanism operates at the level of transcription and there is a long lag phase in the signal transduction process. 2) The induction of class II genes requires the de novo synthesis of a new protein(s). On this basis, we propose that IFN-gamma regulates the transcription of HLA class II genes via the de novo synthesis of a trans-acting activator protein.

Antigens, Differentiation, B-Lymphocyte↗