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Biomedical subjects

A B Roberts

Publications and source records attributed to A B Roberts.

At least 127 records · Page 7Linked to original sources
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

Regulation of the transforming growth factor-beta 1 and -beta 3 promoters by transcription factor Sp1.

The promoter regions of the genes encoding the three mammalian transforming growth factors-beta (TGF-beta 1, -beta 2, and -beta 3) show little similarity in sequence, suggesting diverse transcriptional control. As a step towards understanding transcriptional regulation of the individual TGF-beta genes we tested each of the three TGF-beta promoter regions (pTGF-beta) for stimulation by the transcription factor Sp1, given that several possible Sp1-binding sites were identified by sequence analysis in pTGF-beta 1 and pTGF-beta 3. A Drosophila melanogaster cell culture system was employed to examine expression levels of pTGF-beta::cat constructs coexpressed with an Sp1 expression plasmid in a cell background devoid of any Sp1 homolog. While both pTGF-beta 1 and pTGF-beta 3 were strongly stimulated by Sp1, pTGF-beta 2 was completely unaffected. Promoter fragments of the TGF-beta 1 and TGF-beta 3 genes, but not TGF-beta 2 were able to compete for binding of Sp1 to DNA oligomers containing consensus Sp1-binding sites. Moreover, specific binding to pTGF-beta 1 and pTGF-beta 3 fragments was seen using pure Sp1 or nuclear protein extracts. Thus, TGF-beta 1 and TGF-beta 3 (but not TGF-beta 2) are regulated by the transcription factor Sp1, indicating differential transcriptional regulation of genes whose protein products are functionally very similar.

Acetylation↗
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↗
PubMed1993

Transforming growth factor beta 1: NMR signal assignments of the recombinant protein expressed and isotopically enriched using Chinese hamster ovary cells.

The transforming growth factor beta s are a homologous family of multifunctional cytokines that regulate cell growth and differentiation. As a prelude to studies of the solution structure and dynamics of TGF-beta 1, we report virtually complete assignment of 1H and 15N resonances for this 25-kDa homodimeric protein. Recombinant TGF-beta 1 was expressed in Chinese hamster ovary cells. The cells were grown either in a completely 15N-enriched medium or in a medium containing selectively 13C, 15N-labeled amino acids to obtain either uniformly or specifically labeled protein, respectively. Two- and three-dimensional heteronuclear edited magnetic resonance spectra of the uniformly 15N-labeled protein and three samples selectively labeled with 13C and 15N yielded assignments for 96% of the backbone amide and C alpha protons and 87% of the side chain protons. To our knowledge, this is the first report of the use of an animal cell expression system to obtain extensive isotopic enrichment in order to sequentially assign a protein. The methodology described herein for the isotopic enrichment and resonance assignments of TGF-beta 1 should be generally applicable to other eukaryotic proteins expressed by animal cells.

Amino Acid Sequence↗
PubMed1993

Transforming growth factor beta 1: secondary structure as determined by heteronuclear magnetic resonance spectroscopy.

Virtually complete backbone NMR signal assignments have been reported for transforming growth factor beta 1 (TGF-beta 1) [Archer et al. (1993) Biochemistry (preceding paper in this issue)]. Herein we report the secondary structure of the protein in solution on the basis of these assignments and proton NOE's observed in a variety of 2D and 3D heteronuclear NMR spectra. Regular elements of secondary structure derived from the NOE data consist of (a) three helices spanning residues Y58-H68, F24-G29, and N5-F8 and (b) several pairs of two-stranded antiparallel beta-sheets. The longest two-stranded sheet runs from residue L83 to V106 with a type II reverse turn at G93-R94 and a chain twist at residue N103-M104. These elements of regular structure were confirmed by hydrogen exchange, chemical shift, and coupling constant data. With the exception of residues G46-S53, which exhibit relatively few and weak intraresidue NOE's, residues in the rest of the protein adopt an irregular but well-defined structure. All peptide bonds are trans except for a cis peptide bond between Glu35 and Pro36. The structural characteristics observed for TGF-beta 1 in solution generally agree closely with the recently derived crystal structures of TGF-beta 2 [Daopin et al. (1992) Science 257, 369-374; Schlunegger & Grütter (1992) Nature 358, 430-434]. Several noteworthy differences were observed that may be related to function.

Amino Acid Sequence↗
PubMed1993

Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death.

To delineate specific developmental roles of transforming growth factor beta 1 (TGF-beta 1) we have disrupted its cognate gene in mouse embryonic stem cells by homologous recombination to generate TGF-beta 1 null mice. These mice do not produce detectable amounts of either TGF-beta 1 RNA or protein. After normal growth for the first 2 weeks they develop a rapid wasting syndrome and die by 3-4 weeks of age. Pathological examination revealed an excessive inflammatory response with massive infiltration of lymphocytes and macrophages in many organs, but primarily in heart and lungs. Many lesions resembled those found in autoimmune disorders, graft-vs.-host disease, or certain viral diseases. This phenotype suggests a prominent role for TGF-beta 1 in homeostatic regulation of immune cell proliferation and extravasation into tissues.

Alleles↗
PubMed1993

Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.

Transforming growth factor-beta 1 (TGF-beta 1) possesses highly potent, diverse and often opposing cell-specific activities, and has been implicated in the regulation of a variety of physiologic and developmental processes. To determine the effects of in vivo overexpression of TGF-beta 1 on mammary gland function, transgenic mice were generated harboring a fusion gene consisting of the porcine TGF-beta 1 cDNA placed under the control of regulatory elements of the pregnancy-responsive mouse whey-acidic protein (WAP) gene. Females from two of four transgenic lines were unable to lactate due to inhibition of the formation of lobuloalveolar structures and suppression of production of endogenous milk protein. In contrast, ductal development of the mammary glands was not overtly impaired. There was a complete concordance in transgenic mice between manifestation of the lactation-deficient phenotype and expression of RNA from the WAP/TGF-beta 1 transgene, which was present at low levels in the virgin gland, but was greatly induced at mid-pregnancy. TGF-beta 1 was localized to numerous alveoli and to the periductal extracellular matrix in the mammary gland of transgenic females late in pregnancy by immunohistochemical analysis. Glands reconstituted from cultured transgenic mammary epithelial cells duplicated the inhibition of lobuloalveolar development observed in situ in the mammary glands of pregnant transgenic mice. Results from this transgenic model strongly support the hypothesis that TGF-beta 1 plays an important in vivo role in regulating the development and function of the mammary gland.

Animals↗
PubMed1993

TGF-beta 1 prevents hypertrophy of epiphyseal chondrocytes: regulation of gene expression for cartilage matrix proteins and metalloproteases.

Using an in vitro model of rat epiphyseal chondrocyte differentiation in which cells are maintained in a three-dimensional cell pellet, we show that exogenous TGF-beta 1 reversibly prevents terminal differentiation of epiphyseal chondrocytes into hypertrophic cells. Through maintenance of gene expression for the cartilage matrix proteins type II collagen and aggrecan core protein, and with coordinate inhibition of expression of genes encoding the metalloproteases collagenase and stromelysin, TGF-beta 1 stabilizes the phenotype of the prehypertrophic epiphyseal chondrocyte. This ability of TGF-beta 1 to stabilize the cartilage phenotype is critically dependent on culture conditions. Epiphyseal chondrocytes cultured as a subconfluent monolayer of cells dedifferentiate (reduce type II collagen and aggrecan core protein expression, increase metalloprotease expression, and acquire a spindled morphology) in response to short-term TGF-beta 1 treatment. Increasing the initial seeding density and allowing the cells to become multilayered prior to the addition of growth factor reverse the effects of TGF-beta 1 on type II collagen and transin/stromelysin gene expression and maintain a rounded cellular morphology. This finding emphasizes the importance of considering cell density and environmental context in the analysis of the regulatory action of peptide growth factors in general and of the TGF-beta s in particular. We propose that one function of TGF-beta 1 during endochondral ossification is regulation of chondrocyte growth and differentiation through modulation of the relative expression of cartilage matrix proteins and metalloproteases. This function of TGF-beta 1 helps illustrate how the regulation of diverse cellular processes such as matrix synthesis, matrix degradation, and cell growth and differentiation may be coordinated at the molecular level by a single peptide growth factor.

Animals↗
PubMed1993

Giant splenic artery aneurysm.

Giant aneurysms of the splenic artery are uncommon. The mean size of splenic artery aneurysms is reported to be 2.1 cm; they are rarely larger than 3 cm. We present two cases in which the splenic artery aneurysm was larger than 8 cm, discuss the management and operative approach to these infrequently encountered entities, and provide a brief review of the relevant literature.

Aged↗
PubMed1993

Thickness of tissues intervening between the transducer and fetus and models for fetal exposure calculations in transvaginal sonography.

In transvaginal scanning the tissues intervening between the fetus and transducer are relatively thin. The average thickness in the first trimester is 25 mm and reduces to 15 mm by the third trimester; the minimum thicknesses are 14 and 8 mm, respectively. Two models are proposed for calculating exposure in transvaginal scanning. The models are considered in terms of the overlying tissues and the target tissues. A fixed path, constant attenuation of 0.3 dB/MHz describes the properties of the overlying tissues throughout pregnancy. In the model used in first-trimester scanning, the target tissues of the embryo/fetus are considered to have properties similar to those of soft tissues, and they attenuate the energy at the same rate as the overlying tissues. In the model used in second- and third-trimester scanning, the bony structures of the fetus are the target tissues. These reflect 30% of the incident energy and attenuate all of the transmitted energy at their surface.

Extraembryonic Membranes↗
PubMed1993

TGF beta suppresses casein synthesis in mouse mammary explants and may play a role in controlling milk levels during pregnancy.

Mammary explants from 14-15-d-pregnant mice synthesize and secrete milk proteins in culture in response to insulin, hydrocortisone, and prolactin. Here we demonstrate that transforming growth factor beta (TGF beta) treatment suppresses, in a dose dependent and reversible manner, the ability of explants to synthesize and secrete milk caseins. TGF beta does not affect the level of casein mRNA within explants but inhibits casein synthesis posttranscriptionally. We also show increased expression of TGF beta 2 and TGF beta 3 in intact mammary gland as pregnancy progresses, with reduced expression of all three TGF betas at the onset of lactation. These findings suggest that endogenously produced TGF beta may limit the accumulation of milk caseins that are produced in the mammary gland during pregnancy.

Animals↗
PubMed1993

An element of the transforming growth factor-beta 1 5'-untranslated region represses translation and specifically binds a cytosolic factor.

In many cell types, there is a discrepancy between transforming growth factor-beta 1 (TGF-beta 1) mRNA and TGF-beta 1 protein, suggesting that expression of TGF-beta 1 is regulated posttranscriptionally. We have previously shown that a 137-nucleotide (nt) region of the TGF-beta 1 5'-untranslated region (UTR) potently inhibits the expression of a heterologous reporter gene, suggesting a role for this region in the posttranscriptional inhibition of TGF-beta 1 expression. To study the mechanism of inhibition, a chimeric plasmid containing this region of the TGF-beta 1 5'-UTR and the reading frame of the human GH gene was stably transfected into C2C12 myoblastic cells. Our results show that the TGF-beta 1 5'-UTR inhibits GH expression by inhibiting GH mRNA translation. In vitro gel retardation and cross-linking assays using a radiolabelled RNA probe transcribed from this region of the TGF-beta 1 5'-UTR demonstrate the specific binding of a cytosolic factor. Deletion of a potential stem-loop-forming region abolishes binding of this factor and partially restores GH production. These results suggest that posttranscriptional inhibition of TGF-beta 1 expression is at the level of mRNA translation and that a cytosolic factor may regulate TGF-beta 1 mRNA translation.

Animals↗
PubMed1993

Synergistic increase of phorbol ester-induced c-fos mRNA expression by retinoic acid through stabilization of the c-fos message.

Retinoic acid (RA) has been shown to be able to antagonize or synergize with phorbol 12-myristate 13-acetate (PMA). In contrast to its antagonistic effects on PMA-dependent gene expression, no molecular target or mechanism of synergism has been characterized yet. We now report, that RA synergistically enhances the induction of c-fos, but not c-jun mRNA by PMA in cells whose growth was stimulated by RA alone. The responding cells were hybrids of tumor cell lines whose growth and PMA-dependent c-fos mRNA expression remained unaffected by RA. The increase in PMA-dependent c-fos expression required pretreatment of cells with RA for at least 2-4 h and was achieved at doses as low as 10(-10) M. Nuclear run-on experiments and transient transfection assays using a chimeric reporter gene construct with sequences from the c-fos promoter indicated that RA did not affect PMA-dependent c-fos transcription. Instead, RA stabilized the c-fos message after induction by PMA as assessed by measuring the half-life of c-fos mRNA in actinomycin D-treated cells. This post-transcriptional regulation provides a mechanism whereby RA can synergistically enhance gene expression by PMA.

Dose-Response Relationship, Drug↗
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↗