PubMed Health⌕ Search

Biomedical subjects

A B Roberts

Publications and source records attributed to A B Roberts.

At least 19 recordsLinked to original sources
PubMed1995

Characterization of the promoter region of the human transforming growth factor-beta type II receptor gene.

Diminished cellular responsiveness to transforming growth factor-beta (TGF-beta) is frequently correlated with decreased transcription of the type II receptor for TGF-beta (TGF-beta RII). We have cloned and characterized the human TGF-beta RII promoter and, using S1 nuclease mapping and 5' rapid amplification of cDNA ends polymerase chain reaction, have identified five alternative transcription start sites within the region -33 to +57. DNA transfection experiments and electrophoretic mobility shift assays have revealed the existence of five distinct regulatory regions including two positive regulatory elements and two negative regulatory elements in addition to the core promoter region. The first positive regulatory element (-219 to -172) interacts with two distinct nuclear protein complexes, at least one of which appears to be a previously unidentified transcription factor. The second positive regulatory element (+1 to +35) also interacts with two separate protein complexes, both of which appear to be novel transcription factors. Deletion of either positive regulatory element markedly decreased expression of the target gene, suggesting that both positive regulatory elements are necessary for basal expression levels of TGF-beta RII.

Base Sequence↗
PubMed1995

Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions.

Aberrant expression of transforming growth factor beta 1 (TGF-beta 1) has been implicated in a number of disease processes, particularly those involving fibrotic and inflammatory lesions. To determine the in vivo effects of overexpression of TGF-beta 1 on the function and structure of hepatic as well as extrahepatic tissues, transgenic mice were generated containing a fusion gene (Alb/TGF-beta 1) consisting of modified porcine TGF-beta 1 cDNA under the control of the regulatory elements of the mouse albumin gene. Five transgenic lines were developed, all of which expressed the Alb/TGF-beta 1 transgene selectively in hepatocytes. The transgenic line 25 expressing the highest level of the transgene in the liver also had high (> 10-fold over control) plasma levels of TGF-beta 1. Hepatic fibrosis and apoptotic death of hepatocytes developed in all the transgenic lines but was more pronounced in line 25. The fibrotic process was characterized by deposition of collagen around individual hepatocytes and within the space of Disse in a radiating linear pattern. Several extrahepatic lesions developed in line 25, including glomerulonephritis and renal failure, arteritis and myocarditis, as well as atrophic changes in pancreas and testis. The results from this transgenic model strongly support the proposed etiological role for TGF-beta 1 in a variety of fibrotic and inflammatory disorders. The transgenic model may also provide an appropriate paradigm for testing therapeutic interventions aimed at neutralizing the detrimental effects of this important cytokine.

Animals↗
PubMed1995

Specific and sensitive quantitation of transforming growth factor beta 3 by sandwich enzyme-linked immunosorbent assay.

Transforming growth factors beta (TGF-beta) consist of a highly homologous family of 25 kDa dimers involved in a diverse array of biological functions. Progress in understanding the biology of the third isoform (TGF-beta 3) of this family has been limited by the absence of a quantitative assay for TGF-beta 3. Here we report the development of a sensitive and specific sandwich enzyme-linked immunosorbent assay (SELISA), which is based on an anti-TGF-beta mouse monoclonal IgG as the capture antibody and chicken anti-recombinant-hTGF-beta 3 IgY as the secondary antibody. This assay can quantitate TGF-beta 3 in complex biological fluids, with a detection limit of 2 pg and no cross-reactivity or interference with as high as 1000-fold molar excesses of either TGF-beta s 1, 2 or 1.2. This TGF-beta 3 SELISA is the first reported assay for the direct and sensitive quantitation of TGF-beta 3 in complex biological fluids.

Animals↗
PubMed1995

Pulmonary hypoplasia and fetal breathing in preterm premature rupture of membranes.

Forty-eight patients with premature rupture of membranes prior to 24 weeks gestation (PPROM) were studied to investigate whether there was any difference in fetal breathing movements in those fetuses that did or did not develop pulmonary hypoplasia. The diagnosis of pulmonary hypoplasia was made on the basis of ultrasound measurement of fetal lung length. A control group of fetuses with intact membranes and normal pregnancies at the same gestation was also studied. The group that developed pulmonary hypoplasia had significantly less breathing movements than those that did not develop pulmonary hypoplasia and controls. This reduction in breathing movements may contribute to the development of pulmonary hypoplasia.

Female↗
PubMed1994

Enhanced access to rare brain cDNAs by prescreening libraries: 207 new mouse brain ESTs.

To use single-pass cDNA sequencing to characterize low-frequency cDNA clones from a region of the brain that includes the primary site of neurodegeneration in human Parkinson disease, we have developed a prescreening procedure using single brain region first-strand cDNA probes. Selection of cDNA clones giving low hybridization signals allowed the elimination of clones resulting from abundant messages and enrichment for clones corresponding to low-copy messages. Comparative sequencing of standard and prescreened cDNA libraries (191 and 124 clones, respectively) showed that this procedure raised the frequency of novel sequences encountered from 54 to 81%. The increased proportion of novel ESTs justifies the labor of prescreening. Automation of this procedure will accelerate the molecular description of genes expressed in any brain region, or any tissue, and represents a way to maximize access to cDNA sequences for human and mouse genome characterization. In total, the comparative sequencing experiments generated 207 new mouse and 11 new rat brain ESTs.

Animals↗
PubMed1994

Poor transduction efficiency of human hematopoietic progenitor cells by a high-titer amphotropic retrovirus producer cell clone.

The transduction efficiency of human bone marrow CD34+ cells with supernatants from the retrovirus producer cell clone PA317/LGSN 16 was only one-fifth of that with supernatants from GP+ envAm12/LGSN 15, even though both producers had similar infection titers on 3T3 cells. PA317/LGSN 16-conditioned medium inhibited the proliferation of the bone marrow CD34+ cells, and this inhibitory effect was partially blocked by anti-transforming growth factor beta antibodies. These studies suggest that cytokine secretion plays a role in the suppression of retrovirus transduction of human CD34+ cells.

3T3 Cells↗
PubMed1994

Oversaturation of color may obscure small intraluminal partial occlusions in color Doppler imaging.

Color Doppler imaging has been reported to have a low degree of accuracy in diagnosing nonocclusive deep venous thrombosis. In this modality the color saturation while diagnosing deep vein thrombosis depends on the blood flow velocity and the sensitivity setting of the color image. To determine the effect of color saturation on the detection of thrombi, an in vitro experiment was performed using a closed-loop tube circulation system with a simulated small intraluminal partial occlusion. Heparinized blood was circulated at three different velocities (2, 5.5, and 9 cm/sec) and the vessels were scanned longitudinally and transversely with color Doppler imaging at three color sensitivity settings (low, medium, and high sensitivity). In appropriate color sensitivity settings (i.e., the low, medium, and high sensitivity for the velocity of 9,5.5 and 2 cm/sec, respectively), the color was saturated adequately in the tube lumen so that the partial occlusion was delineated as a color filling defect in the color field. In undersaturated color conditions, the partial occlusion was depicted owing to its echogenicity, but not as a color filling defect. In over-saturated color conditions, such as the high color sensitivity at the high velocity, the partial occlusion was obscured by the excessive amount of color. The over saturation of color may be one of the reasons color Doppler imaging fails to detect deep vein thrombosis, particularly small intraluminal thrombi.

Blood Flow Velocity↗
PubMed1993

Ultrasound detection of fetal structural abnormalities in Auckland 1988-9.

AIMS: To conduct an audit of ultrasound screening for fetal abnormalities in central Auckland 1988-89. METHOD: All babies born at National Women's Hospital in the time period were examined and fetal abnormalities coded on to a computer database. All terminations of pregnancy for fetal abnormality in the area were similarly audited. RESULTS: There were 12,909 births. 218 babies had a congenital abnormality of whom 88% were scanned antenatally. 48% of abnormalities were detected (27% before 24 weeks). In fetuses that were scanned the detection rate was 52% (44% before 24 weeks). The range of detection of specific abnormalities included neural tube defects 95% and congenital heart defects 16%. CONCLUSIONS: 1. Major abnormalities of the central nervous system, renal tract and abdominal wall have a high detection rate. 2. Cardiovascular, facial and gastrointestinal abnormalities are either not detectable or difficult to detect. 3. Detection rates could be improved by routine scanning, better ultrasound equipment and training, and by the introduction of maternal biochemical screening tests. 4. The detection rate is similar to other overseas surveys.

Abortion, Therapeutic↗
PubMed1993

Characterization of distinct functional domains of transforming growth factor beta.

Three distinct isoforms of transforming growth factor beta (TGF-beta) are expressed in mammalian cells. Although many cells respond equivalently to all three isoforms, certain cells respond selectively. Using chimeric proteins in which selected regions of the different isoforms were interchanged, we have identified two distinct functional domains of TGF-beta involved in determining the biological potencies and functions of the molecule. The first domain is important for determining whether TGF-beta can be sequestered by alpha 2-macroglobulin. By replacing aa 45 and 47 of TGF-beta 2 with the corresponding amino acids of TGF-beta 1, sequestration of the TGF-beta molecule by alpha 2-macroglobulin was markedly reduced. The second domain is functionally different from the alpha 2-macroglobulin sequestration site and is important for determining the potency of TGF-beta to inhibit growth of the LS513 human colorectal cancer cell line. Neither the TGF-beta 2/beta 1-(40-47) replacement construct nor a chimera containing aa 1-39 of TGF-beta 2, aa 40-82 of TGF-beta 1, and aa 83-112 of TGF-beta 2 was equivalent to TGF-beta 1 in inhibiting growth of LS513 cells. This fact suggests that additional amino acids outside of the aa 40-82 region are required to specify TGF-beta 1 activity with these cells.

3T3 Cells↗
PubMed1993

Inhibition of skin development by overexpression of transforming growth factor beta 1 in the epidermis of transgenic mice.

To assess the effect of transforming growth factor beta 1 on the skin in vivo, we have targeted its expression to the epidermis of transgenic mice. To ensure that active TGF-beta 1 was expressed, we used a porcine TGF-beta 1 cDNA with mutations of Cys-223-->Ser and Cys-225-->Ser, which allow constitutive activation. Mice expressing the mutant transforming growth factor beta 1 transgene exhibited a marked phenotype at birth. The skin was very shiny and tautly stretched. These animals were rigid and appeared to be restricted in their ability to move and breathe; death occurred within 24 hr. Histologically, the most prominent features of the skin were a compact orthohyperkeratosis and a reduction in the number of hair follicles. Pulse-labeling studies with 5-bromodeoxyuridine demonstrated a marked reduction in the number of replicating cells in the epidermis and hair follicles. Thus, the macro- and microscopic appearance of these mice, as well as their neonatal lethality, most likely result from inhibition of normal skin development and suppression of epithelial cell proliferation by the overexpression of transforming growth factor beta 1.

Amino Acid Sequence↗
PubMed1992

Identification of an activating transcription factor (ATF) binding site in the human transforming growth factor-beta 2 promoter.

Transforming growth factor TGF-beta 2 is encoded by multiple mRNA transcripts of 5.8, 5.1, 4.0, 3.8, and 2.8 kilobase pairs (kb) that are expressed in various human and monkey cells. Northern blot analysis using genomic fragments of DNA was used to demonstrate that some of this size heterogeneity is due to differences in the length of the 5'-untranslated region. Probes that were colinear with the first 600 nucleotides of the 5'-untranslated region detected only the 5.8-, 4.0-, and 3.8-kb transcripts. In order to identify DNA elements that regulate the transcription of these mRNA transcripts, deletion constructs of 5'-flanking DNA were ligated to the coding region for chloramphenicol acetyltransferase (CAT) and analyzed for promoter activity in several cell lines. Sequences responsible for putative enhancer and silencer regions were identified between -778 and -40 relative to the transcription initiation site. Addition of a cyclic AMP-responsive element/activating transcription factor-like element at -74 resulted in a 5-10-fold increase in CAT activity over that expressed with a construct that contained only the TATA box. This increase in CAT activity was suppressed by the addition of DNA sequences between -257 and -187, whereas sequences between -778 and -257 stimulated CAT activity. Point mutations within the ATF binding site at -74 resulted in a marked decrease in CAT expression. Cotransfection with ATF-1 or ATF-2 expression plasmids resulted in both dose-dependent stimulatory and inhibitory activities that were cell type-dependent. These studies identify multiple transcription initiation sites for TGF-beta 2 and demonstrate that transcription from one of these promoters is dependent upon an ATF binding site located 5' of the TATA box.

Activating Transcription Factors↗
PubMed1992

Inhibition of mitogen-induced c-fos expression in melanoma cells by retinoic acid involves the serum response element.

To investigate the mechanism(s) by which all-transretinoic acid (RA) inhibits cell growth, we studied its effect on the expression of c-fos and c-jun in B16 melanoma cells. RA differentially inhibited proto-oncogene induction by mitogens, such as phorbol 12-myristate 13-acetate and serum. Suppression of c-fos was achieved with doses of RA as low as 10(-10) M and required pretreatment of cells with RA for a minimum of 2 h. In contrast, inhibition of c-jun required pretreatment for greater than 16 h with at least 10(-8) M RA and coincided with the observed decrease in cell growth. RA blocked c-fos induction by inhibiting transcription. This inhibition of transcription occurs through the serum response element (SRE), since the SRE alone was sufficient to confer down-regulation by RA to a minimal c-fos promoter construct. Thus, the SRE plays a critical role in the suppression of c-fos transcription by RA.

Animals↗
PubMed1992

Autocrine secretion--10 years later.

The concept of autocrine secretion, its subsequent modifications, its application for understanding pathogenesis of disease, and its potential for developing new approaches to prevention and treatment are reviewed. Peptide growth factors (cytokines) act as local autocrine and paracrine mediators of tissue homeostasis. Many diseases, including cancer, atherosclerosis, rheumatoid arthritis, and other fibrotic diseases characterized by chronic inflammation, are associated with aberrant expression and cellular coordination of the homeostatic action of these regulatory molecules. Modern biotechnology and pharmacology offer unique opportunities for the therapeutic prevention and treatment of these molecular and cellular lesions, using either cytokines or other agents that modify their synthesis and activity.

Amino Acid Sequence↗
PubMed1992

Chorionic villus sampling in Auckland 1989-90.

AIMS: to perform an audit of all 197 chorionic villus sampling procedures performed in Auckland in 1989-90. METHOD: an attempt to trace the outcome in all 197 cases and this was possible in 188 cases. RESULTS: 89% of chorionic villus samples were performed transvaginally and 11% transabdominally. The total fetal loss rate was 9.6% (18) with six spontaneous miscarriages before 20 weeks gestation (3.2%), one intermediate fetal death and one neonatal death. There were 10 elective terminations of pregnancy following abnormal results so the spontaneous loss rate was 4.3%. CONCLUSIONS: the loss rate was lower than that reported in the European and Canadian multicentre trials. The sampling success rate, however, was poor with 13.7% failed procedures. This improved in the second year of the service to 10%. There were no failure cultures or examples of mosaicism.

Abortion, Spontaneous↗
PubMed1992

Inhibition of protein phosphatases blocks myogenesis by first altering MyoD binding activity.

To examine the role of protein phosphatases in skeletal muscle differentiation, C2C12 myoblasts were treated with okadaic acid, a potent in vitro inhibitor of protein phosphatases 1 and 2A which regulate various cellular events in intact cells. We now show that okadaic acid treatment of the mouse myoblast C2C12 cell line reversibly altered the morphology of the cells and blocked differentiation. At a molecular level, it extinguished expression of the myogenic determination genes, MyoD1 and myogenin, but induced the expression of an inhibitor of differentiation, Id. Analysis of the MyoD1 promoter showed that inhibition of MyoD1 expression by okadaic acid occurs at the transcriptional level. These changes occur 10-20 h after okadaic acid treatment. However, within 1 h of treatment the ability of muscle extracts to support a specific MyoD-dependent gel mobility shift using a MyoD DNA binding site is lost. These data suggest that protein phosphatases play an important role during myogenic differentiation.

Animals↗
PubMed1992

Identification of a structural domain that distinguishes the actions of the type 1 and 2 isoforms of transforming growth factor beta on endothelial cells.

A chimeric transforming growth factor beta (TGF-beta) molecule has been synthesized to map the amino acids responsible for the substantially greater activity of TGF-beta 1 than TGF-beta 2 on growth and migration of endothelial cells. This chimera consists of a dimer of a monomeric unit composed of amino acids 1-39 of TGF-beta 2, 40-82 of TGF-beta 1, and 83-112 of TGF-beta 2. Structural identity of the purified recombinant protein has been confirmed by immunoblotting and NH2-terminal sequencing. The biological potency of the TGF-beta 2-1-2 chimera was equal to that of TGF-beta 1 in inhibition of growth of both fetal bovine heart endothelial cells and rat epididymal fat pad microvascular endothelial cells. Similarly, the TGF-beta 2-1-2 chimera was nearly equivalent to TGF-beta 1 and at least 10-fold more active than TGF-beta 2 in inhibiting migration of bovine aortic endothelial cells. These results identify the sequence between amino acids 40-82 as an important region within TGF-beta that functions to specify a TGF-beta 1- or TGF-beta 2-like activity.

Animals↗
PubMed1992

Post-transcriptional regulation of the human transforming growth factor-beta 1 gene.

Since many lines of evidence suggest that expression of the transforming growth factor-beta 1 (TGF-beta 1) gene may be regulated post-transcriptionally, we examined the effect of the 5'-untranslated region (UTR) of this gene on TGF-beta 1 expression. For this purpose, fragments of the 840-nucleotide highly GC-rich TGF-beta 1 5'-UTR were inserted into the 5'-UTR of the structural gene for human growth hormone driven by the simian virus 40 early promoter. A portion of the 5'-UTR of TGF-beta 1 mRNA spanning the sequences from +11 to +147 was shown to inhibit growth hormone expression by as much as 22-fold. This effect was cell-specific; growth hormone production was inhibited in PC-3 human prostate adenocarcinoma and A-549 human lung adenocarcinoma cells, while no effect was seen in rat pheochromocytoma PC12 cells, which show efficient translation of endogenous TGF-beta 1 mRNA. Computer analysis showed that this region of the 5'-UTR contained a stable secondary stem-loop structure spanning sequences +49 to +76. This stem-loop region alone is sufficient to inhibit expression of the growth hormone gene, suggesting that it plays an important role in post-transcriptional regulation of TGF-beta 1 gene expression.

Animals↗