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Biomedical subjects

D Bu

Publications and source records attributed to D Bu.

At least 19 recordsLinked to original sources
PubMed2004

DNA-based prenatal diagnosis in a Chinese family with xeroderma pigmentosum group A.

BACKGROUND: Xeroderma pigmentosum (XP) is a group of autosomal recessive diseases characterized by hypersensitivity to ultraviolet rays. Among its eight complementation groups, XP group A (XPA) is the most severe type. The XPAC gene has been identified as the defective gene in XPA patients. OBJECTIVES: To examine genomic DNA from a Chinese family with XPA, to determine the XPAC mutation and, after genetic counselling, to undertake DNA-based prenatal diagnosis in a subsequent pregnancy. METHODS: Fetal DNA was extracted from amniotic fluid and used to amplify exon 5 of XPAC containing the potential mutation. Direct sequencing and restriction endonuclease digestion were used for prenatal diagnosis. RESULTS: We identified a homozygous nonsense XPAC mutation of 631C-->T, which results in an R211X mutation in XPA protein, in the proband. Both her parents are heterozygous. Prenatal diagnosis demonstrated a heterozygous sequence predicting an unaffected child, and a healthy girl was born. CONCLUSIONS: These data provide the first example of a DNA-based prenatal test for genodermatosis in China.

Child, Preschool↗
PubMed2004

Mutations in SCN9A, encoding a sodium channel alpha subunit, in patients with primary erythermalgia.

Primary erythermalgia is a rare autosomal dominant disease characterised by intermittent burning pain with redness and heat in the extremities. A previous study established the linkage of primary erythermalgia to a 7.94 cM interval on chromosome 2q, but the causative gene was not identified. We performed linkage analysis in a Chinese family with primary erythermalgia, and screened the mutations in the two candidate genes, SCN9A and GCA, in the family and a sporadic patient. Linkage analysis yielded a maximum lod score of 2.11 for both markers D2S2370 and D2S2330. Based on critical recombination events in two patients in the family, we further limited the genetic region to 5.98 cM between D2S2370 and D2S2345. We then identified two missense mutations in SCN9A in the family (T2573A) and the sporadic patient (T2543C). Our data suggest that mutations in SCN9A cause primary erythermalgia. SCN9A, encoding a voltage-gated sodium channel alpha subunit predominantly expressed in sensory and sympathetic neurones, may play an important role in nociception and vasomotor regulation.

Base Sequence↗
PubMed2002

Immunobiology and long-term graft function in a transplant heterotopic renal rat model.

BACKGROUND: The Th1-Th2 paradigm proposes clonal expansion of Th2 lymphocytes as the basis of allograft tolerance. The Th2 cells have been found to be present in recipients with long-term allograft survival. However, the presence of Th2 cells and tolerance is not a uniform finding. Previously we have shown that pre-engraftment single dose rapamycin and a 7-d course of cyclosporin induce transplantation tolerance to 120 d. In the present study, we investigated the immunobiology of grafts in a long-term follow-up (>350 d). METHODS: Kidney allografts (n = 7), isografts (n = 5) and single nephrectomy (n = 3) groups were followed for 350 +/- 87 d. Heterotopic kidney transplant was performed by the same surgeon in the allograft group (ACI-Lewis) and the isograft group (Lewis-Lewis). The left kidney was removed in the single nephrectomy group. The allograft group was treated with pre-engraftment single dose rapamycin and a 7-d course of cyclosporin. A kidney biopsy was performed at midpoint time for histological study and tissue was frozen for measuring intragraft cytokine expression (IL-4, IL-10) in all animals. Prior to biopsy, serum blood urea nitrogen (BUN) and creatinine (Cr) levels were studied. Serum BUN, Cr levels, plus 24-h urinary protein (PRO) were measured prior to sacrifice. Randomly, four allograft rats received skin grafts (ACI, Lewis and Buffalo skin donors) after kidney biopsy. Skin grafts were studied for a mean of 6 weeks for signs of acceptance or rejection. Analysis of variance (ANOVA) with Tukey's test was used; p < 0.05 was considered statistically significant. RESULTS: The mean follow up was 352 +/- 87 d. BUN and Cr levels at biopsy time (mean 214 d) were not statistically different between the three groups (p = 0.19 and p = 0.66). At sacrifice (mean 352 d), BUN, Cr and PRO were statistically different between allograft and isograft groups (p = 0.013), and between allograft and single nephrectomy groups (p = 0.027). Functional and histological signs of graft loss occurred in three of seven (42.8%) of the allografts at 352 d. Using BANFF criteria, three allografts at biopsy time and seven allografts (100%) and four isografts (80%) at sacrifice time developed chronic histologic changes. Intragraft overexpression of IL-4 and IL-10 was seen at biopsy and sacrifice time in six of seven allografts and one of five isografts. All donor specific skin grafts (ACI-Lewis) on allografts were accepted and third party (Buffalo) donor skin grafts were rejected in all animals (>95% skin necrosis). CONCLUSIONS: This highly stringent, functional, renal transplant model yields 100% normal renal function as compared with isografts at 120 d follow-up. With the follow-up extended to 350 d, 43% of the allografts loose function and develop a chronic allograft histology despite a demonstrated intragraft Th2 cytokine dominance and donor specific skin graft acceptance.

Animals↗
PubMed2000

Defining hepatocellular chimerism in a liver failure patient bridged with hepatocyte infusion.

BACKGROUND: A practical method of monitoring engraftment by transplanted hepatocytes for the purpose of bridging human liver failure to native regeneration is described. METHODS: A previously healthy 37-year-old female with a 2-week history of a febrile illness presented with fulminant liver failure. Findings on admission included the following: illicit drug use, serum hepatitis B surface antigen positive, grade 1 encephalopathy, prothrombin time (pt) >100 sec, F7<1%, NH3 150 micromol/L, alanine aminotransferase 4079 U/L, total bilirubin level 11.4 mg/dl, and glucose 70 mg/dl (on IV D10). With immunosuppression, 8.8x10(8), 96% viable human hepatocytes were intraportally infused. Clinical chemistries, total sHLA class I, and ELISA to measure donor-specific sHLA-A1 and -B8 were recorded. Serial transjugular liver biopsies were performed and pooled for histological examination, DNA extraction, and HLA DNA typing. RESULTS: The patient fully recovered. At months 3 and 4 with donor biopsy specimen class I HLA DNA no longer detectable, immunosuppression was tapered off. The patient is clinically normal, serum hepatitis B surface antigen negative at 10 months of follow-up. CONCLUSIONS: Bridging liver failure with donor hepatocytes with HLA class I antigen disparate from recipients is clinically feasible, and allows for a marker, combined with serial graft histology, to safely wean immunosuppression when native liver regeneration succeeds.

Adult↗
PubMed2000

[A novel mutation of the ALAS2 gene in a family with X-linked sideroblastic anemia].

OBJECTIVE: To confirm the mutation of ALAS2 gene is the cause of sideroblastic anemia in a family. METHODS: Polymerase chain reaction (PCR) was used to amplify the microsatellite DXS 991, DXS 1199 in the chromosome Xp11.22 linked gene ALAS2 and haplotype analysis was performed in a kindred with 2 patients and 7 normal members. All cDNA encoded regions in the ALAS2 gene of the patients and their normal siblings were cloned, sequenced and compared. RESULT: Both brother patients had the same allele of ALAS2 and their normal siblings did not. The mutation in the patients' ALAS2 gene was exon 5 A523G, causing threonine to alanine; and exon 3 T372C, leucine to proline. The latter located in the splicing region, its significance is not clear. CONCLUSION: The pathogenesis of this kindred of X-linked sideroblastic anemia (XLSA) involved a novel mutation in ALAS2 exon 5.

5-Aminolevulinate Synthetase↗
PubMed1999

[Pathogenic gene linkage analysis and hemopoietic characteristics in a kindred with sideroblastic anemia].

OBJECTIVE: Analysis of pathogenic gene linkage and hemopoietic characteristics in a kindred with sideroblastic anemia. METHODS: PCR amplification of the microsatellites DXS991,DXS1199 in chromosome Xp11.22 linked gene ALAS2 and of the microsatellite DXS1226 in Xp22. 13 linked another irrelevant gene and analysis of gene linkage in a kindred with 2 patients and 7 normal persons. The bone marrow hemopoietic cells from 2 patients were cultured in condition culture matrix with various cytokines added in and the CFU-E, CFU-GM and CFU-Meg formations were observed at different times. RESULTS: The kindred study revealed that pathogenic gene linked with DXS991 and DXS1199 but did not link with DXS1226.Hemopoietic cell culture showed that erythroid colonies of the two patients grew more vigorously than controls and they could grew in the absence of Epo except in common condition matrix. The erythroid colonies withered after a week and were smaller than the controls after 13 days. CONCLUSION: The kindred is subject to an X-linked sideroblastic anemia(XLSA) with the pathogenic gene ALAS2 involved. In XLSA,the function of stem cells is primarily normal before erythropoiesis, then the erythroid progenitors become dysplasia.

Adult↗
PubMed1999

Intragraft cytokine expression and tolerance induction in rat renal allografts.

BACKGROUND: Intragraft cytokine expression was evaluated in a model of renal transplantation. ACI and Lewis rats were used as donors and recipients, respectively, for heterotopic renal transplantation. METHODS: Treated allograft rats (n=10) received a preoperative dose of rapamycin and cyclosporine, followed by 7 days of cyclosporine postoperatively. Isograft rats (n=5) and control allograft rats (n=4) received no immunosuppression. Sacrifice was performed after 120 days. Expression of interleukin (IL)-4, IL-10, and interferon-gamma (IFN-gamma) transcripts was determined with semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: All treated allograft rats had normal function with 50% histologic rejection. All isografts had normal function. IL-4 and IL-10 were in greater density in allografts with normal histology, whereas IFN-gamma was only seen in allografts with cellular rejection. No IL-10 was seen in isografts, but IL-4 was detected in 3/5 isografts. CONCLUSIONS: We conclude that the lymphocyte population's elaboration of IL-4 and IL-10 is associated with tolerance, whereas the production of IFN-gamma and absence of IL-4 is associated with histology suggestive of acute cellular rejection.

Animals↗
PubMed1999

Spatial-specific TGF-beta1 adenoviral expression determines morphogenetic phenotypes in embryonic mouse lung.

The precise spatial-temporal role that expression and activation of transforming growth factor (TGF)-beta plays in mammalian organ morphogenesis remains incompletely understood. Using replication deficient adenoviral vectors containing engineered TGF-beta1 cDNAs, we studied the spatial effects of locally over-expressing either latent or mutated, constitutively active TGF-beta1 protein during embryonic mouse lung branching morphogenesis in culture. Transfer of exogenous genes into lung epithelium was achieved by intra-tracheal micro-injection of recombinant adenovirus, while submerging lungs in virus resulted in gene transfer into the pleura and subjacent mesenchymal cells, as revealed by cytochemical staining for beta-galactosidase. Only lungs transfected with active, but not latent TGF-beta1 gene, showed elevated levels of active TGF-beta. Epithelial over-expression of active, but not latent TGF-beta1, via intra-tracheal micro-injection inhibited lung branching morphogenesis by 36 %. In contrast, lungs submerged with either active or latent TGF-beta1 recombinant virus did not demonstrate an inhibitory effect upon branching. Pulmonary gene regulation was assayed by competitive polymerase chain reaction coupled with reverse transcription. Direct respiratory tract micro-injection of adenovirus over-expressing active TGF-beta1 resulted in a dose-dependent inhibition of epithelial surfactant protein (SP)-C and SP-B mRNA levels by up to 76 % and 70 %, respectively, while in contrast, fibronectin and matrix Gla protein (MGP) mRNA levels remained stable. However, lungs that had been submerged in adenovirus expressing active TGF-beta1 demonstrated a concentration-dependent induction of both fibronectin and MGP mRNA levels up to 4.3- and 4.7-fold respectively in the presence of 1 x 10(11) pfu/ml active TGF-beta1 virus. On the other hand, lungs treated with adenovirus expressing latent TGF-beta1 either by micro-injection or submerging failed to demonstrate any regulatory effect either upon epithelial or mesenchymal gene expression. We conclude that adenovector-mediated over-expression of activated TGF-beta1 in specific spatial compartments results respectively in either inhibition of branching morphogenesis and epithelium-specific gene expression, or in induction of matrix gene expression without affecting morphogenesis or epithelium-specific gene expression, depending on the route of administration. Also, the lack of effect of latent TGF-beta1 over-expression strongly suggests that TGF-beta activation per se provides an important locus of fine regulation of the spatial effects of TGF-beta signaling during embryonic lung branching morphogenesis.

Adenoviridae↗
PubMed1999

Intragraft cytokine expression in tolerant rat renal allografts with rapamycin and cyclosporin immunosuppression.

The Th-1/Th-2 paradigm proposes clonal expansion of Th-2 lymphocytes as the basis of tolerance towards allografts. Intragraft cytokine expression was evaluated in a highly stringent model of renal transplantation. ACI and Lewis rats were used as donors and recipients, respectively, for heterotopic renal transplantation. Group A (n = 8) received a single dose of rapamycin and cyclosporin 12 h prior to engraftment, followed by 7 d of cyclosporin post-operatively. Isografts (Group B, n = 5) and control allografts (Group C, n = 4) received no immunosuppression. Sacrifice was performed after 120 d. Intragraft expression of IL-10, IL-4, and IFN-gamma was determined using qualitative reverse transcriptase-polymerase chain reaction (RT-PCR). All groups had functionally normal grafts at sacrifice, with 50% histological tolerance among Group A animals. No isografts showed evidence of cellular infiltrate, and all control allografts showed severe rejection. IL-10 was only detected in the tolerant animals (p < 0.001). Similarly, IL-4 was detected predominantly in the tolerant allografts (p < 0.05). IFN-gamma was only isolated in rejected allografts, whether treated or untreated (p < 0.001). We conclude that the expansion of Th-2 cells is associated with tolerance, while the expansion of Th-1 cell is associated with acute cellular rejection.

Animals↗
PubMed1997

Impaired lung branching morphogenesis in the absence of functional EGF receptor.

The mammalian lung develops through branching morphogenesis which is controlled by growth factors, hormones, and extracellular matrix proteins. We have evaluated the role of EGF-receptor signaling in lung morphogenesis by analyzing the developmental phenotype of lungs in mice with an inactivated the EGF-receptor gene both in vivo and in organ culture. Neonatal EGF-receptor-deficient mice often show evidence of lung immaturity which can result in visible respiratory distress. The lungs of these mutant mice had impaired branching and deficient alveolization and septation, resulting in a 50% reduction in alveolar volume and, thus, a markedly reduced surface for gas exchange. The EGF-receptor inactivation also resulted in type II pneumocyte immaturity, which was apparent from their increased glycogen content and a reduced number of lamellar bodies. The defective branching was already evident at Day 12 of embryonic development. When explants of embryonic lungs from Day 12 embryos were cultured under defined conditions, the branching defect in EGF-receptor-deficient lungs was even more pronounced, with only half as many terminal buds as normal lungs. EGF treatment stimulated the expression of surfactant protein C and thyroid transcription factor-1 in cultured normal lungs, but not in EGF-receptor-deficient lungs, suggesting that EGF-receptor signaling regulates the expression of these marker genes during type II pneumocyte maturation. Taken together, our data indicate that signal transduction through the EGF receptor plays a major role in lung development and that its inactivation leads to a respiratory distress-like syndrome.

Animals↗
PubMed1996

Abrogation of transforming growth factor-beta type II receptor stimulates embryonic mouse lung branching morphogenesis in culture.

TGF-beta1 is a known inhibitor of branching morphogenesis when added exogenously to mouse embryonic lungs in culture. However, the issue of whether endogenous TGF-beta signaling has a function in the process of lung organogenesis is not completely resolved. We utilized immunoperturbation and antisense oligodeoxynucleotide inhibitory strategies to abrogate TGF-beta type II receptor function in embryonic mouse lungs undergoing branching morphogenesis in serumless explant culture. Antisera directed against a TGF-beta type II receptor N-terminal peptide that perturbs TGF-beta ligand-receptor binding increased branching by 70%. Similarly, antisense TGF-beta type II receptor oligodeoxynucleotides (40 microM) resulted in a 58% increase in branching, compared to scrambled and mismatched sequence controls, while TGF-beta, type II receptor mRNA and its protein expression levels were suppressed by 95 and 84%, respectively. Addition of exogenous TGF-beta1 did not overcome the stimulatory effects either of TGF-beta type II receptor immunoperturbation or of antisense oligodeoxynucleotide treatment on lung branching morphogenesis. Using in situ hybridization and immunohistochemistry, both TGF-beta type II receptor mRNA and protein were localized to the epithelium lining the developing airways, and to the surrounding mesenchyme, indicating that TGF-beta type II receptor is an important regulator of epithelial-mesenchymal interaction. Exogenous TGF-beta1 decreased cyclin A mRNA levels in control embryonic lung explants, while TGF-beta type II receptor antisense oligodeoxynucleotides prevented the downregulation of cyclin A mRNA expression by exogenous TGF-beta1. In addition, PCNA immunostaining of the primitive bronchial epithelium was increased in the presence of TGF-beta type II receptor antisense oligodeoxynucleotides either alone or together with exogenous TGF-beta1, whereas TGF-beta1 alone decreased PCNA staining. Thus, abrogation of TGF-beta type II receptor expression prevented TGF-beta1-induced epithelial cell G1 arrest. These results demonstrate, for the first time, that abrogation of the TGF-beta type II receptor stimulates embryonic lung organogenesis in culture and reverses the negative influence of endogenous TGF-beta signaling upon epithelial cell cycle progression.

Animals↗
PubMed1996

Cytokine-stimulated astrocytes damage human neurons via a nitric oxide mechanism.

Astrocytes have been reported to play a neuropathogenic role within the brain, although little is known about the mechanism underlying astrocyte-mediated neuronal injury. We investigated the hypothesis that cytokine-stimulated astrocytes adversely affect neuronal cell survival via generation of the free radical nitric oxide (NO). Primary human astrocytes produced substantial amounts of NO in response to interleukin (IL)-1 alpha or IL-1 beta, which was blocked by the NO synthase inhibitor NG-mono-methyl-L-arginine (NMMA). IL-1 beta-induced NO production was markedly potentiated by interferon (IFN)-gamma. IL-1 receptor agonist protein (IRAP) totally blocked NO generation by cytokine-stimulated astrocytes. Using reverse transcription-polymerase chain reaction and sequencing analyses of the astrocyte NO synthase gene, we found a single band encoding for a 615 bp product that was identical to the corresponding sequence reported for human hepatocytes. Treatment of human fetal brain cell cultures with IL-1 beta plus IFN-gamma resulted in marked neuronal loss, as assessed by microscopic analysis and measurement of lactate dehydrogenase release. This cytokine-induced neuronal damage was blocked by simultaneous treatment of the brain cell cultures with NMMA or IRAP, suggesting a critical role of IL-1. These findings indicate that cytokine-stimulated astrocytes are neurotoxic via a NO-mediated mechanism and point to potential new therapies for neurodegenerative disorders that involve cytokines and reactive astrocytes.

Astrocytes↗
PubMed1995

TTF-1 regulates lung epithelial morphogenesis.

TTF-1 is a homeodomain transcriptional factor expressed in thyroid, lung, and parts of the brain. In vitro, TTF-1 can activate the promoter of thyroid- and pulmonary-specific genes. We postulated that TTF-1 not only is essential for the activation of tissue-specific genes, but also may directly participate in epithelial cell morphogenesis. To test this postulate, we used an antisense oligonucleotide inhibitory strategy in an in vitro model of embryonic mouse lung branching morphogenesis. This strategy suppressed TTF-1 translation and inhibited lung branching morphogenesis. The resulting abnormal phenotype was characterized by hyperplastic and unorganized proliferation of epithelial cells in the airways. The mesenchymal compartment of the lung appeared to be unaffected. These results demonstrate, for the first time, that the expression of a homeoprotein transcriptional regulator is necessary for lung epithelial morphogenesis.

Animals↗
PubMed1995

Characterization and response to interleukin 1 and tumor necrosis factor of immortalized murine biliary epithelial cells.

BACKGROUND & AIMS: Biliary epithelial cells are the target of numerous immune-mediated liver diseases, yet their role in pathogenesis remains unclear because of difficulties in obtaining pure preparations. The aim of this study was to establish pure clones of immortalized murine intrahepatic biliary epithelial cells. METHODS: The transgenic mouse harboring the SV40 thermosensitive immortalizing mutant gene TsA58 under the control of the major histocompatibility complex class I promoter was used to establish conditionally immortalized intrahepatic bile duct cells by countercurrent centrifugal elutriation and clonal dilution. RESULTS: Immortalized clones of cells expressing cytokeratin 19, which organized themselves into ductlike structures, were obtained. On electron-microscopic sections, cells were well differentiated and polarized. Cells proliferate in response to epidermal growth factor, interleukin 1 alpha, and tumor necrosis factor alpha. Using the reverse-transcriptase polymerase chain reaction technique, these cells were found to contain messenger RNA, which encodes for the interleukin 1 and tumor necrosis factor receptors. CONCLUSIONS: The availability of unlimited numbers of pure bile duct cells that behave in an identical fashion to biliary epithelial cells from "normal" mice will allow for more rigorous studies of the behavior and function of this epithelium.

Animals↗
PubMed1995

Abnormal lung development and cleft palate in mice lacking TGF-beta 3 indicates defects of epithelial-mesenchymal interaction.

A broad spectrum of biological activities has been proposed for transforming growth factor-beta 3 (TGF-beta 3). To study TGF-beta 3 function in development, TGF-beta 3 null mutant mice were generated by gene-targeting. Within 20 hours of birth, homozygous TGF-beta 3-/- mice die with unique and consistent phenotypic features including delayed pulmonary development and defective palatogenesis. Unlike other null mutants with cleft palate, TGF-beta 3-/- mice lack other concomitant craniofacial abnormalities. This study demonstrates an essential function for TGF-beta 3 in the normal morphogenesis of palate and lung, and directly implicates this cytokine in mechanisms of epithelial-mesenchymal interaction.

Animals↗
PubMed1993

Biophysical characterization of one-, two-, and three-tandem repeats of human mucin (muc-1) protein core.

Until recently mucin tandem repeat protein cores were believed to exist in random-coil conformations and to attain structure solely by the addition of carbohydrates to serine and threonine residues. Matsushima et al. (Proteins Struct. Funct. Genet., 7: 125-155, 1990) recently proposed a model of the secondary structure of proline rich tandem repeat proteins that has challenged this idea, especially for the case of the human polymorphic epithelial mucin encoded by the muc-1 gene. We report here results of structural analyses of the muc-1 protein core by using synthetic peptide analogues. Synthetic peptides were prepared to correspond to one-, two-, and three-tandem repeats of muc-1. Results of one- and two-dimensional 1H NMR correlation spectroscopy on these peptides confirm that the muc-1 protein core is not a random-coil secondary structure. Long-lived amide protons are protected in D2O, and increasing spectral complexity in the region of the beta-protons of Asp2 and His 15 reveals that structural changes are occurring as the number of repeats increases. The greatest changes occur when the number of repeats increases from one to two. These results are supported by the reactivity of a panel of monoclonal antibodies raised against tumor associated muc-1 with these synthetic peptides in enzyme-linked immunosorbent assay. The primary immunodominant mucin epitope, PDTRP, does not appear to attain a native conformation in the single repeat peptide (20 amino acids, starting with P), but is expressed on peptides with multiple repeats. Intrinsic viscosity measurements of the peptide containing three repeats indicate that an ordered structure present in solution is rod shaped. The circular dichroism spectrum of the same peptide is dominated by proline in the trans conformation. These results are all consistent with the prediction that the muc-1 tandem repeat polypeptide core forms a polyproline beta-turn helix.

Amino Acid Sequence↗