PubMed Health⌕ Search

Biomedical subjects

W Reith

Publications and source records attributed to W Reith.

At least 145 records · Page 8Linked to original sources
PubMed1996

Cloning and functional characterization of mammalian homologues of the COPII component Sec23.

We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa. hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p. Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p. Cytosolic hSec23Ap migrated with an apparent molecular weight of 350 kDa on a gel filtration column, suggesting that it is part of a protein complex. By immunoelectron microscopy, hSec23Ap was found essentially in the ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles. hSec23Ap is a functional homologue of the yeast Sec23p as the hSec23A isoform complemented the temperature sensitivity of the Saccharomyces cerevisiae sec23-1 mutation at a restrictive temperature of 34 degrees C. RNase protection assays indicated that both hSec23 isoforms are coexpressed in various human tissues, although at a variable ratio. Our data demonstrate that hSec23Ap is the functional human counterpart of the yeast COPII component Sec23p and suggest that it plays a similar role in mammalian protein export from the ER. The exact function of hSec23Bp remains to be determined.

Animals↗
PubMed1996

A consensus motif in the RFX DNA binding domain and binding domain mutants with altered specificity.

The RFX DNA binding domain is a novel motif that has been conserved in a growing number of dimeric DNA-binding proteins, having diverse regulatory functions, in eukaryotic organisms ranging from yeasts to humans. To characterize this novel motif, we have performed a detailed dissection of the site-specific DNA binding activity of RFX1, a prototypical member of the RFX family. First, we have performed a site selection procedure to define the consensus binding site of RFX1. Second, we have developed a new mutagenesis-selection procedure to derive a precise consensus motif, and to test the accuracy of a secondary structure prediction, for the RFX domain. Third, a modification of this procedure has allowed us to isolate altered-specificity RFX1 mutants. These results should facilitate the identification both of additional candidate genes controlled by RFX1 and of new members of the RFX family. Moreover, the altered-specificity RFX1 mutants represent valuable tools that will permit the function of RFX1 to be analyzed in vivo without interference from the ubiquitously expressed endogenous protein. Finally, the simplicity, efficiency, and versatility of the selection procedure we have developed make it of general value for the determination of consensus motifs, and for the isolation of mutants exhibiting altered functional properties, for large protein domains involved in protein-DNA as well as protein-protein interactions.

Amino Acid Sequence↗
PubMed1996

Regulation of MHC class II genes: lessons from a disease.

Precise regulation of major histocompatibility complex class II (MHC-II) gene expression plays a crucial role in the control of the immune response. A major breakthrough in the elucidation of the molecular mechanisms involved in MHC-II regulation has recently come from the study of patients that suffer from a primary immunodeficiency resulting from regulatory defects in MHC-II expression. A genetic complementation cloning approach has led to the isolation of CIITA and RFX5, two essential MHC-II gene transactivators. CIITA and RFX5 are mutated in these patients, and the wild-type genes are capable of correcting their defect in MHC-II expression. The identification of these regulatory factors has furthered our understanding of the molecular mechanisms that regulate MHC-II genes. CIITA was found to be a non-DNA binding transactivator that functions as a molecular switch controlling both constitutive and inducible MHC-II expression. The finding that RFX5 is a subunit of the nuclear RFX-complex has confirmed that a deficiency in the binding of this complex is indeed the molecular basis for MHC-II deficiency in the majority of patients. Furthermore, the study of RFX has demonstrated that MHC-II promoter activity is dependent on the binding of higher-order complexes that are formed by highly specific cooperative binding interactions between certain MHC-II promoter-binding proteins. Two of these proteins belong to families of which the other members, although capable of binding to the same DNA motifs, are probably not directly involved in the control of MHC-II expression. Finally, the facts that CIITA and RFX5 are both essential and highly specific for MHC-II genes make possible novel strategies designed to achieve immunomodulation via transcriptional intervention.

Animals↗
PubMed1996

Decompressive craniectomy in a rat model of "malignant" cerebral hemispheric stroke: experimental support for an aggressive therapeutic approach.

Acute ischemia in the complete territory of the carotid artery may lead to massive cerebral edema with raised intracranial pressure and progression to coma and death due to uncal, cingulate, or tonsillar herniation. Although clinical data suggest that patients benefit from undergoing decompressive surgery for acute ischemia, little data about the effect of this procedure on experimental ischemia are available. In this article the authors present results of an experimental study on the effects of decompressive craniectomy performed at various time points after endovascular middle cerebral artery (MCA) occlusion in rats. Focal cerebral ischemia was induced in 68 rats using an endovascular occlusion technique focused on the MCA. Decompressive craniectomy was performed in 48 animals (in groups of 12 rats each) 4, 12, 24, or 36 hours after vessel occlusion. Twenty animals (control group) were not treated by decompressive craniectomy. The authors used the infarct volume and neurological performance at Day 7 as study endpoints. Although the mortality rate in the untreated group was 35%, none of the animals treated by decompressive craniectomy died (mortality 0%). Neurological behavior was significantly better in all animals treated by decompressive craniectomy, regardless of whether they were treated early or late. Neurological behavior and infarction size were significantly better in animals treated very early by decompressive craniectomy (4 hours) after endovascular MCA occlusion (p < 0.01); surgery performed at later time points did not significantly reduce infarction size. The results suggest that use of decompressive craniectomy in treating cerebral ischemia reduces mortality and significantly improves outcome. If performed early after vessel occlusion, it also significantly reduces infarction size. By performing decompressive craniectomy neurosurgeons will play a major role in the management of stroke patients.

Animals↗
PubMed1995

Purified X2 binding protein (X2BP) cooperatively binds the class II MHC X box region in the presence of purified RFX, the X box factor deficient in the bare lymphocyte syndrome.

The conserved X2 box sequence of MHC class II promoters is homologous to TRE/CRE elements, and is required for B cell expression and IFN-gamma induction of MHC class II genes. The X2 binding protein (X2BP) was initially identified as a DNA-binding activity that specifically interacts with the conserved X2 box sequence in both the MHC HLA-DRA and HLA-DRB promoters. To begin to demonstrate that X2BP is the X2 box factor responsible for class II expression in B cells, we have purified X2BP to homogeneity from B cell nuclear extracts using DNA-affinity chromatography. X-box DNA-affinity purification indicates that X2BP is most likely composed of two polypeptides of 120 kDa and 46 kDa. The 120-kDa protein was specifically cross-linked to an X-box probe by exposure to UV irradiation. The 46-kDa subunit of X2BP cross-reacted with anti-rat CREB polyclonal Abs but not to anti-human CREB Abs in Western analysis and supershift assays, indicating that it may be a novel member of the ATF/CREB family. Purified X2BP interacted with purified RFX, a factor that binds to the adjacent X1 box and is absent in some cell lines that are mutant for MHC class II transcription. This interaction increases the DNA-binding half-life of RFX from 5 to at least 60 min, suggesting that X2BP functions in class II MHC gene expression by forming a stable complex with RFX.

Base Sequence↗
PubMed1995

A novel DNA-binding regulatory factor is mutated in primary MHC class II deficiency (bare lymphocyte syndrome).

Regulation of MHC class II gene expression is an essential aspect of the control of the immune response. Primary MHC class II deficiency is a genetically heterogeneous disease of gene regulation that offers the unique opportunity of a genetic approach for the identification of the functionally relevant regulatory genes and factors. Most patients exhibit a characteristic defect in the binding of a nuclear complex, RFX, to the X box motif of MHC class II promoters. Genetic complementation of a B-lymphocyte cell line from such a patient with a cDNA expression library has allowed us to isolate RFX5, the regulatory gene responsible for the MHC class II deficiency. This gene encodes a novel DNA-binding protein that is indeed a subunit of the RFX complex. Mutations in the RFX5 gene have been characterized in two patients. Transfection of the patient's cells with the RFX5 cDNA repairs the binding defect and fully restores expression of all the endogenous MHC class II genes in vivo.

Amino Acid Sequence↗
PubMed1995

Molecular defects in the bare lymphocyte syndrome and regulation of MHC class II genes.

The complex pattern of expression of major histocompatibility complex (MHC) class II molecules plays an essential role in the control of the immune response. Our understanding of the molecular mechanisms controlling this expression has benefited greatly from the identification of the regulatory factors defective in two forms of a hereditary disease of MHC class II regulation: bare lymphocyte syndrome. This has also provided new tools for the experimental modulation of MHC class II expression.

Gene Expression Regulation↗
PubMed1995

Decompressive craniectomy for cerebral infarction. An experimental study in rats.

BACKGROUND AND PURPOSE: Acute ischemia in the territory of the carotid artery can lead to massive cerebral edema with raised intracranial pressure and progression to coma and death due to uncal, cingulate, or tonsillar herniation. Thus far, only anecdotal experience with supratentorial ischemia treated by decompressive craniectomy has been reported; and there are no published experimental data dealing with this kind of therapy in acute supratentorial stroke. In this study, we present our results on the effect of decompressive craniectomy in an endovascular model of cerebral infarction in rats. METHODS: Focal cerebral ischemia was induced in 50 rats using an endovascular occlusion technique of the middle cerebral artery. Decompressive craniectomy was performed in 30 animals: in 15 animals after 1 hour and in the remaining 15 animals 24 hours after vessel occlusion. Twenty animals were not treated by decompressive craniectomy (control group). RESULTS: Mortality in the nontreated group was 35%, whereas none of the animals treated by decompressive craniectomy died. Neurological behavior, weight loss, and infarction size were all significantly better in the animals treated by decompressive craniectomy, regardless of whether they had been treated after 1 or 24 hours (P < .01). CONCLUSIONS: Our results suggest that decompressive craniectomy for cerebral ischemia not only reduces mortality but also significantly improves outcome and reduces infarction size, probably because of increased perfusion pressure through leptomeningeal collaterals. This experimental study suggests that a controlled study of decompressive craniectomy in patients with acute internal carotid or middle cerebral artery occlusion would be worthwhile. By performing decompressive craniectomy in a small, selected group of patients, neurosurgeons may play an important role in the management of these patients.

Acute Disease↗
PubMed1995

Multislice diffusion mapping for 3-D evolution of cerebral ischemia in a rat stroke model.

Diffusion-weighted magnetic resonance imaging (DWI) can quantitatively demonstrate cerebral ischemia within minutes after the onset of ischemia. The use of a DWI echo-planar multislice technique in this study and the mapping of the apparent diffusion coefficient (ADC) of water, a reliable indicator of ischemic regions, allow for the detection of the three-dimensional (3-D) evolution of ischemia in a rat stroke model. We evaluated 13 time points from 5 to 180 minutes after occlusion of the middle cerebral artery (MCA) and monitored the 3-D spread of ischemia. Within 5 minutes after the onset of ischemia, regions with reduced ADC values occurred. The core of the lesion, with the lowest absolute ADC values, first appeared in the lateral caudoputamen and frontoparietal cortex, then spread to adjacent areas. The volume of ischemic tissue was 224 +/- 48.5 mm3 (mean +/- SEM) after 180 minutes, ranging from 92 to 320 mm3, and this correlated well with the corrected infarct volume at postmortem (194 +/- 23.1 mm3, r = 0.72, p < 0.05). This experiment demonstrated that 3-D multislice diffusion mapping can detect ischemic regions noninvasively 5 minutes after MCA occlusion and follow the development of ischemia. The distribution of changes in absolute ADC values within the ischemic region can be followed over time, giving important information about the evolution of focal ischemia.

Animals↗
PubMed1995

Early MR detection of experimentally induced cerebral ischemia using magnetic susceptibility contrast agents: comparison between gadopentetate dimeglumine and iron oxide particles.

PURPOSE: To evaluate early patterns of MR changes in a rat model of cerebral ischemia using the first pass of two magnetic susceptibility contrast agents. METHODS: One hours after endovascular middle cerebral artery occlusion, all animals were examined in an experimental MR unit. After bolus application of gadopentetate dimeglumine and, 10 minutes later, of iron oxide particles, the MR changes of the first pass of these contrast agents were followed using a T2*-weighted fast low-angle shot sequence. Time-density curves of both contrast agents were analyzed and compared. RESULTS: After bolus injection of either (paramagnetic) gadopentetate dimeglumine or superparamagnetic particles, nonischemic brain parenchyma decreased markedly in signal, whereas the ischemic brain area remained relatively hyperintense (and thus became clearly delineated). Only after application of gadopentetate dimeglumine did a mild reduction in signal occur in the ischemic hemisphere, although the main artery was occluded. An explantation for this phenomenon might be residual capillary perfusion (plasma flow), which is detectable only when the smaller (paramagnetic) contrast molecules are being used. CONCLUSIONS: Cerebral perfusion deficits can be detected 1 hour after vascular occlusion with T2*-weighted fast low-angle shot sequences and bolus injection of paramagnetic or superparamagnetic MR contrast agents. Gadopentetate dimeglumine may be used as a marker of microcirculatory plasma flow.

Animals↗
PubMed1994

Developmental extinction of major histocompatibility complex class II gene expression in plasmocytes is mediated by silencing of the transactivator gene CIITA.

Constitutive major histocompatibility complex (MHC) class II gene expression is tightly restricted to antigen presenting cells and is under developmental control. Cells of the B cell lineage acquire the capacity to express MHC class II genes early during ontogeny and lose this property during terminal differentiation into plasma cells. Cell fusion experiments have suggested that the extinction of MHC class II expression in plasma cells is due to a dominant repression, but the underlying mechanisms are not understood. CIITA was recently identified as an MHC class II transactivator that is essential for MHC class II expression in B lymphocytes. We show here that inactivation of MHC class II genes in plasmocytes is associated with silencing of the CIITA gene. Moreover, experimentally induced expression of CIITA in plasmocytes leads to reexpression of MHC class II molecules to the same level as that observed on B lymphocytes. We therefore conclude that the loss of MHC class II expression observed upon terminal differentiation of B lymphocytes into plasmocytes results from silencing of the transactivator gene CIITA.

B-Lymphocytes↗
PubMed1994

Cooperative binding between factors RFX and X2bp to the X and X2 boxes of MHC class II promoters.

Transcription of major histocompatibility complex (MHC) class II genes is controlled primarily by the promoter, which contains several conserved cis-acting elements, including the X, X2, and Y boxes. We show here that RFX, the X box-binding protein that is deficient in certain MHC class II regulatory mutants, binds cooperatively with an X2 box-binding protein (X2bp) to form an RFX.X2bp.DNA complex in which the interaction of the two factors with their target sites is strongly stabilized. A functional role of this RFX.X2bp complex is consistent with mutational analysis of the X and X2 boxes of the DRA and DRB1 class II promoters. Together with previous results demonstrating cooperative binding between RFX and the Y box-binding protein NF-Y, our results indicate that RFX plays a central role in promoting cooperative binding interactions required for stable occupation of the MHC class II promoter. This may explain why the highly specific defect in binding of RFX observed in certain MHC class II regulatory mutants is associated in vivo with a bare promoter in which all of the cis-acting elements, including the X, X2, and Y boxes, are unoccupied.

Base Sequence↗
PubMed1994

Function of major histocompatibility complex class II promoters requires cooperative binding between factors RFX and NF-Y.

Transcription of major histocompatibility complex (MHC) class II genes is controlled largely by the conserved promoter elements called the X and Y boxes. We show here that RFX, the X box-binding protein deficient in certain MHC class II-deficient immunodeficiency patients (CID), and the Y box-binding protein NF-Y bind cooperatively. Functional relevance of this protein-protein interaction is suggested by the fact that promoter activity correlates with cooperative binding of RFX and NF-Y rather than with binding of RFX or NF-Y alone. Stability of the RFX/NF-Y complex is affected by alterations in X-Y box spacing. These results are consistent with the fact that MHC class II promoter function is dependent on correct stereospecific alignment of the X and Y boxes. Cooperative binding involving RFX, NF-Y, and perhaps other MHC class II promoter-binding proteins may explain why the highly specific defect in binding of RFX observed in CID cells is associated in vivo with a bare promoter in which all of the cis-acting elements, including the X and Y boxes, are unoccupied.

Base Sequence↗
PubMed1994

MRI monitoring of experimental cerebral ischaemia: comparison of two models.

In experimental stroke research one of the key questions is whether animals models truly reflect human disease. One important reason for modelling stroke is to produce tissue changes that can be used to validate the correlation between the results of advanced radiological imaging and pathology. The aim of this study was to compare two minimally invasive rat models of focal ischaemic stroke as to their ability to simulate clinical stroke reproducibly. In one model a focal infarct was induced using the photochemical Rose Bengal technique. The second model was based on transvascular occlusion of the middle cerebral artery using a cervical approach. In all animals we performed MRI at different times with two different paramagnetic contrast agents. The time course of blood-brain-barrier disruption in the Rose Bengal model differed entirely from that in human brain ischaemia. The experimental stroke showed marked contrast enhancement in the first hour after the onset of ischaemia. On the other hand, the MRI changes in the suture occlusion model were very similar to the changes observed in human brain ischaemia: no early disruption of the blood-brain-barrier and increased T2-signal 4-6 h after the onset of stroke. We hope that the suture occlusion model will be used for research not only by basic and clinical scientists but also by radiologists, who, using modern imaging methods, may greatly influence the diagnosis as well as the management of this devastating disease.

Animals↗
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↗