The tight-skin (Tsk) mutation is closely linked to B2m on mouse chromosome 2.
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Alleles↗
Biomedical subjects
S E Harris
Publications and source records attributed to S E Harris.
Placental cell release of transferrin: analysis by reverse haemolytic plaque assay.
Recent demonstrations of transferrin (TF) mRNA in placental tissue raised the possibility that the placenta may serve as an extra-hepatic source of this iron-binding protein during development. In this study, we first confirmed these findings using cRNA probes for TF, and then adapted the TF reverse haemolytic plaque assay for use with rat placental cells to identify the location and functional characteristics of cells secreting this product. We found TF releasing cells in placentae (day 19-21) with greater proportions present in cultures from basal than labyrinth zone regions. These cells appeared quite stable as evidenced by observations that fresh populations and 24, 48 and 72 h cultures from the same placental regions all contained similar percentages of secretors. The rate of TF plaque formation was greatly enhanced in the presence of tumour necrosis factor (0.1 ng/ml) for basal zone and yolk sac cells, but not for labyrinth zone cells, suggesting a potent but regionally specific modulation of TF release. When taken together, these findings demonstrate clearly that TF is released from cells of the placenta. Moreover, the regional differences in frequency and modulation of these cells suggests that the release of placental TF is a dynamic and carefully controlled process.
Animals↗
A survey of rural parents' attitudes toward sexuality education.
Although local attitudes and values influence a community's support for sexuality education, educators rarely assess local views in a systematic way. A survey of community parents provides a simple, relatively inexpensive way to assess support for sexuality education and strengthen the school's leadership in implementing an effective curriculum. This paper describes the use of a parent survey for building a comprehensive sexuality education program in a rural community. Most respondents supported sexuality education even for the elementary grades. Compared to a survey conducted 10 years earlier, respondents were significantly less confident of themselves and other parents as sexuality educators. Attitudes varied by demographic characteristics. Using these findings, the paper illustrates how the strategic use of a parent survey can strengthen school-based sexuality education by minimizing potential controversy and building a broader base of active community support and involvement for sexuality education.
Adolescent↗
Expression of the BMP 2 gene during bone cell differentiation.
Bone morphogenetic protein 2 (BMP 2) and transforming growth factor beta (TGF beta) are actively involved in bone formation and remodeling. TGF beta, a powerful stimulant in the early stage of bone cell growth and matrix formation, inhibits differentiation and in vitro mineralized nodule formation in primary fetal rat calvarial osteoblast system. TGF beta also negatively regulates BMP 2 expression at the transcriptional level. BMP 2 gene expression is controlled by a battery of transcriptional factors, some known and some yet to be identified. Immortalized osteoblast cell lines generated from a transgenic mouse carrying BMP 2 promoter-driven SV40 large T antigen transgene are described as powerful tools for studying regulation of BMP 2 gene expression and bone cell differentiation at the molecular level.
Animals↗
Synthesis of novel tellurium containing analogues of choline and acetylcholine and their quantitation by pyrolysis-gas chromatography-mass spectrometry.
Methods for the synthesis and quantitation of the novel choline analogues, telluronium choline and acetyltelluronium choline, are described. An assay procedure utilizing pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) with cold trapping was developed with [2H4]telluronium choline and [2H4]acetyltelluronium choline as internal standards. The telluronium compounds were ion-pair extracted from tissue with dipicrylamine, washed with 2-butanone, and pyrolyzed prior to GC-MS analysis. The compounds were monitored using selected ion monitoring at m/z 232 and m/z 190 for acetyltelluronium and telluronium choline, respectively, and at m/z 236 and m/z 194 for the analogous deuterated internal standards. The assay was linear over a range of 20 pmol-20 nmol of compound taken through the assay.
Gas Chromatography-Mass Spectrometry↗
Cloning and sequence of bone morphogenetic protein 4 cDNA from fetal rat calvarial cell.
A cDNA clone encoding bone morphogenetic protein 4 (BMP 4) has been isolated from a primary fetal rat calvarial cell cDNA library. Sequencing of this clone has revealed a single open reading frame encoding a 408 amino acid protein. Comparison of 5' noncoding exon 1 portion of this cDNA with that of human bone and prostate BMP4 cDNA shows that BMP4 gene expression may possess tissue specificity.
Amino Acid Sequence↗
Cloning of two novel forms of human acidic fibroblast growth factor (aFGF) mRNA.
We have previously isolated two different aFGF cDNA clones from kidney and brain. The two corresponding mRNA, designated aFGF 1.A and 1.B, are the predominant species in kidney and brain, respectively. During the characterization of aFGF mRNA in glioblastoma cells, we demonstrated that aFGF mRNA in U1242MG and D65MG glioblastoma cells contain 5'-untranslated sequences different from those of 1.A and 1.B. Through a strategy combining chromosome walking, identification and sequencing of evolutionarily conserved DNA regions, and a reverse transcription and polymerase chain reaction (RT-PCR)-based assay for RNA expression, we have isolated two novel aFGF cDNA clones. The cDNA clone representing aFGF mRNA 1.C was isolated from U1242MG cells; another aFGF cDNA, designated 1.D, was isolated from D65MG cells. Promoter 1C has extensive sequence homology to the hamster aFGF gene promoter that was shown to respond to testosterone stimulation by chloramphenicol acetyltransferase reporter gene assays. Using RT-PCR, we showed that normal, benign and cancerous prostate tissues do not express aFGF 1.C mRNA. In contrast, a prostate carcinoma cell line (PC-3) expresses 1.C mRNA. RT-PCR using 1.D-specific primers showed that kidney, brain and prostate do not express 1.D mRNA even though kidney and brain are the most abundant source for aFGF protein. RNase protection analysis further showed that 1.D mRNA is the predominant aFGF transcript in D65MG glioblastoma cells and in NFF-6 neonatal foreskin fibroblast cells. The genomic DNA corresponding to these two cDNA clones and the 5'-flanking regions were also isolated and their sequences determined. These DNA clones will provide important reagents for studying the regulatory elements of aFGF gene expression.
Animals↗
Acidic fibroblast growth factor gene 5' non-coding exon and flanking region from hamster DDT1 cells: identification of the promoter region and transcriptional regulation by testosterone and aFGF protein.
Selected clones of Syrian hamster DDT1-MF2 cells are responsive to testosterone for growth. Heparin binding growth factor 1 (HBGF-1) or acidic fibroblast growth factor (aFGF) can replace testosterone (T) in the stimulation of growth in these cells. This phenomena is correlated with testosterone's ability to elevate aFGF mRNA two- to threefold in DDT1 cells. To better understand the possible mechanisms of regulation of aFGF mRNA by steroids and other growth factors, we isolated the aFGF 5' non-coding exon and its flanking region from a EMBL3 DDT1 genomic library, using a 5' non-coding exon 69 bp DDT1 aFGF cDNA probe. Clones spanning 30 kb of genomic DNA were isolated. After restriction mapping and DNA sequence analysis, the clones were shown to contain all of the 5' non-coding exon included in the cDNA and approximately 10 kb of 5' flanking region. RNase protection and primer extension assays confirmed that the 5' non-coding exon is included in the DDT1 aFGF mRNA and that a major transcription start site is approximately 136 bp upstream of the 5' non-coding splice junction of this exon. The 5' flanking region DNA was inserted into pBLCAT3 reporter gene and transfected into DDT1 cells. Chloramphenicol acetyltransferase (CAT) assays demonstrated that there are promoter elements in the -1645/-392 and -392/+131 regions of the aFGF gene in the context of DDT1 cells. NIH 3T3 cells, on the other hand, show no CAT activity with these aFGF-CAT plasmids. CAT assays also demonstrated that addition of testosterone (T) or aFGF to DDT1 cells increased CAT activity threefold. This activity was mapped to -1645 to -4 bp region of this DDT1 aFGF gene promoter.
3T3 Cells↗
Mutations of human dihydrofolate reductase causing decreased inhibition by methotrexate.
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Amino Acid Sequence↗
Molecular cloning of a differentiation-related mRNA in the adipogenic cell line 1246.
The 1246 cell line is a C3H mouse teratoma-derived adipogenic cell line that can proliferate and differentiate in defined medium. We have constructed a recombinant phage library containing complementary DNAs (cDNAs) prepared from mRNA of differentiated 1246 cells. This library was screened using a differential hybridization technique. We have isolated five different cDNA clones corresponding to mRNAs that are induced during adipogenesis of 1246 cells and one cDNA clone corresponding to mRNA that is decreased during adipogenesis. Among the mRNAs expressed during adipose differentiation, some are not expressed in undifferentiated cells, whereas some are expressed at very low levels under these conditions. Moreover, the level of induction during differentiation and the temporal expression of the mRNAs corresponding to these cDNAs varied. Our results indicate that one of the cDNA clones isolated, called 154, which selects a 2.2-kilobase mRNA, was induced 100-fold at a very early time during the onset of the differentiation program in 1246 cells and also in adipocyte precursors in primary culture. Direct sequencing of 154 cDNA insert revealed no homology with sequences in GenBank and PIR protein databases. The expression of 154 mRNA was stimulated by accelerators of differentiation such as dexamethasone and isobutylmethylxanthine and inhibited by tumor necrosis factor alpha, transforming growth factor beta, and epidermal growth factor, which are known inhibitors of 1246 cell differentiation. In addition, 154 mRNA level in adipocytes was down-regulated by tumor necrosis factor alpha, but not by transforming growth factor beta or epidermal growth factor. These results suggest that the increase in 154 mRNA expression is related to the onset of adipose differentiation. Further analysis of this clone should allow characterization of a novel protein induced early during the process of differentiation.
1-Methyl-3-isobutylxanthine↗
Cloning of a mu-class glutathione S-transferase complementary DNA and characterization of its glucocorticoid inducibility in a smooth muscle tumor cell line.
A cDNA (designated hGSTYBX) encompassing the complete coding sequence of a hamster mu-class glutathione S-transferase (GST) subunit was cloned from a lambda ZAP library constructed with mRNA isolated from triamcinolone acetonide-treated smooth muscle tumor cells (DDT1 MF-2). Analysis of its nucleotide and deduced amino acid sequences demonstrated highest homology to the rat mu-class GST YB2 subunit. In proliferating subconfluent cells, in which constitutive expression of hGSTYBX mRNA was undetectable, glucocorticoid treatment induced hGSTYBX expression after a time lag of 3 h, and maximal induction occurred at 10 h. Nuclear run-on analysis showed that glucocorticoid induction resulted at least in part from an increased rate of transcription. Simultaneous treatment with glucocorticoid and cycloheximide prevented glucocorticoid induction, but had little effect on basal expression in confluent cells. In contrast, cycloheximide treatment 3 h after glucocorticoid treatment resulted in nearly full induction. These results taken together suggest that hGSTYBX induction may be a secondary glucocorticoid response.
Amino Acid Sequence↗
Military policies regarding homosexual behavior: an international survey.
The military attachés of 110 non-Communist embassies in Washington, DC, were surveyed from 1982-1984 regarding their countries' military policies about homosexuality. Of the 57 that responded, 37% reported policies excluding persons who engage in homosexual behavior from military service, 14% reported policies that accept homosexual and bisexual persons, and 49% reported having no policies about homosexual behavior. Cultural factors appear to be related to the military policies.
Culture↗
Structural characterization of the rat seminal vesicle secretion II protein and gene.
The gene encoding rat seminal vesicle secretion II (SVS II) protein has been cloned from a rat genomic DNA library using a cDNA probe generated from rat dorsal prostate androgen-dependent mRNA. The cloned 7.3-kilobase pair genomic fragment contains approximately 5000 base pairs (bp) of the 5'-flanking region and the entire coding region of the SVS II protein within two exons. A sequence of 4156 bp of the rat SVS II gene has been determined, including 2037 bp of the 5'-flanking region, exon 1 (95 bp), intron 1 (236 bp), exon 2 (1171 bp), and 614 bp of the 3'-flanking region. The 5'-flanking region contains three conserved elements found in other seminal vesicle secretion genes (SVS IV-VI proteins) within 250 bp of the transcription start site as well as a glucocorticoid response element at position -314 in the SVS II gene. The first exon encodes a 22-amino acid leader peptide plus the first 2 amino acids of the secreted protein. The second exon encodes the remaining amino acids in the SVS II protein sequence. The mature protein contains 392 residues and has an Mr of 43,116. Concomitant with the gene analysis, the rat SVS II protein was purified to homogeneity, and 333 residues (85%) of the amino acid sequence were determined by automated Edman degradation. The DNA-deduced sequence and that determined by direct analysis of the protein are in complete agreement. The blocked NH2-terminal amino acid was identified as pyroglutamic acid by mass spectrometry and aminopeptidase digestion. A 13-residue structure with the consensus sequence GSQLKSFGQVKSS is repeated 13 times within the SVS II protein and appears to be involved in the formation of the rat copulatory plug via a transglutaminase reaction cross-linking glutamine and lysine residues. Overall, the SVS II protein sequence exhibits little structural relatedness to any other known protein sequence; however, some similarity can be found between the 13-residue repeat and another repeating structure and apparent transglutaminase substrate in the guinea pig seminal vesicle clotting protein.
Amino Acid Sequence↗
Characterization of the hamster DDT-1 cell aFGF/HGBF-I gene and cDNA and its modulation by steroids.
Syrian hamster DDT-1 cells are derived from smooth muscle of the ductus deferens. DDT-1 cell growth is increased by the addition of testosterone (T). Acidic fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF) also known as heparin binding growth factor I and II (HBGF-I and HBGF-II) can replace T in the stimulation of growth in these cells. This phenomenon is correlated with testosterone's ability to elevate aFGF/HBGF-I mRNA. The increase steady-state levels of aFGF/HBGF-I mRNA were documented by northern blots and by in situ hybridization. Using a 520 bp human aFGF/HBGF-I cDNA probe, a genomic clone with a 38 kb DNA insert was isolated from a cosmid library. By restriction enzyme analysis and southern hybridization, it was determined that there are three coding exons. DNA sequence analysis showed all of the coding region and 3' noncoding sequences were on this clone. A 5' noncoding exon not in the 38 kb insert is indicated, based on the cDNA sequences and genomic sequences of aFGF/HBGF-I's from hamster DDT-1 cells and several other species. The cDNA for hamster aFGF/HBGF-I was isolated from a DDT-1 lambda gt11 library and sequenced. Comparison of the coding region of aFGF/HBGF-I from four species shows a greater than 90% conservation of amino acid sequence.
Amino Acid Sequence↗
Patient selection and outcomes for out-of-hospital births in one family practice.
This paper reports a study of the pregnancies followed to delivery in one family medicine group practice that offered a choice of childbirth settings. Those choosing out-of-hospital birth (OHB) were a self-selected group of highly motivated couples interested in natural childbirth or desiring to minimize the cost of pregnancy care. Of 790 singleton pregnancies followed to term, 71 (9.0%) planned home births, 510 (64.6%) planned clinic births, and 209 (26.5%) planned hospital births. Of those planning clinic or home birth, 73% gave birth outside the hospital as planned (44 at home and 379 in the clinic), 81 (14%) changed plans prenatally and gave birth in a local hospital, 46 (8%) were transferred to the local hospital intrapartum, and 29 (5%) were referred to tertiary care. Primiparas who initially chose OHB were more likely than multiparas to give birth in a hospital (46% vs 16%). Controlling retrospectively for obstetric risk and parity, there were few differences in outcome between local hospital and out-of-hospital births. The observed rates of serious complications for OHB were low, but overall, 27% of those initially considered candidates for birth outside the hospital required a change to a higher level of care. For primiparas initially planning clinic or home birth, discriminant analysis revealed five variables that together might have improved the prediction of the eventual decision for hospital delivery in 46%. Clinical pelvimetry was the most powerful variable, in keeping with the finding that most intrapartum transfers were for arrests of labor. The results also suggest that financial factors and other features of the physician-patient relationship influenced clinical decision making.
Adult↗
Androgens and glucocorticoids modulate heparin-binding growth factor I mRNA accumulation in DDT1 cells as analyzed by in situ hybridization.
The ductus deferens smooth muscle tumor cell line (DDT1-MF-2) is very sensitive to steroids. Treatment with 10 nM testosterone accelerates the growth of DDT1 cells in the absence of serum. Glucocorticoids in the presence or absence of androgens inhibits growth. Stimulation of growth of DDT1 cells by testosterone can be replaced by the addition of heparin-binding growth factor I and II (HBGF). Addition of testosterone plus HBGF growth factors results in a further increase in cell number. By in situ hybridization, accumulation of HBGF-I mRNA is significantly increased by testosterone treatment of low density cultures. Testosterone treatment of high density cultures results in no stimulation of HBGF-I mRNA accumulation. Glucocorticoids alone, which block growth of DDT1 cells, have no effect of HBGF-I mRNA accumulation. However, the simultaneous addition of glucocorticoid and androgens to DDT1 cells results in a rapid accumulation of HBGF-I mRNA by 12 h, although growth is inhibited by the presence of both steroid analogs.
Animals↗
Cloning of the cDNAs encoding the cellular retinaldehyde-binding protein from bovine and human retina and comparison of the protein structures.
A 1173-base pair cDNA encoding bovine cellular retinaldehyde-binding protein (CRALBP) was cloned from a bovine retinal cDNA expression library using as probes both anti-CRALBP polyclonal and monoclonal antibodies. The amino acid sequence deduced from the cDNA corresponds exactly to that determined by direct analysis of NH2-terminally acetylated bovine CRALBP (Crabb, J. W., Johnson, C. M., Carr, S. A., Armes, L. G., and Saari, J. C. (1988) J. Biol. Chem. 263, 18678-18687). Nick-translated bovine CRALBP cDNA probes were then used to clone from a human retinal cDNA library a 1317-base pair cDNA encoding human CRALBP. Bovine and human CRALBP are 92% identical in amino acid sequence and not related to any other known protein sequence. Both the bovine and human proteins contain 316 residues and have calculated molecular weights of 36,378 and 36,347, respectively, exclusive of the NH2-terminal blocking groups. The CRALBP cDNA clones should prove valuable as tools for studying the physiological role of the protein in vision and visual disorders.
Animals↗
Aversion therapy for homosexuality.
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Aversive Therapy↗