Automated laser-fluorescence sequencing.
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DNA↗
Biomedical subjects
M Freeman
Publications and source records attributed to M Freeman.
An ancient, highly conserved family of cysteine-rich protein domains revealed by cloning type I and type II murine macrophage scavenger receptors.
Scavenger receptors have been implicated in the development of atherosclerosis and other macrophage-associated functions. The bovine type I and type II scavenger receptors are multidomain transmembrane proteins that differ only by the presence in the type I receptor of an additional, extracellular cysteine-rich C-terminal domain. The isolation of type I and type II receptor cDNAs from a murine macrophage cell line, P388D1, establishes the presence of mRNAs encoding both receptor types in a single cell. Their sequences are highly similar to the bovine cDNAs. Receptor type-specific cDNA probes map to a common locus on murine chromosomes 8, suggesting that a single gene encodes both mRNAs. The type I-specific scavenger receptor cysteine-rich (SRCR) domain helps define a previously unrecognized family of remarkably well-conserved domains. Highly homologous SRCR domains (one, three, or four per polypeptide chain) are found in diverse secreted and cell-surface proteins from humans (e.g., CD5, complement factor I), mice (Ly-1), and sea urchins (speract receptor).
Amino Acid Sequence↗
Impact of childhood cancer on healthy school-age siblings.
One part of a study on the effects of childhood cancer on families focused on the emotional reactions of school-age siblings. The authors identify themes in the siblings' feelings taken from transcripts of interviews with them. Responses were categorized as experience of diagnosis and hospitalization; awareness of prognosis and mortality; need for information; continuing effect of disease or death on the well child; correct understanding of cancer; and hopes and desires for the future. The responses were grouped either in a sibling with a living child category or sibling with a deceased child category. From their analysis, the authors present implications for nurses' interactions with these children: children's need for information; their feelings of being displaced and unimportant; need for encouraging involvement in the life of the ill child. The authors also identify additional questions that could direct future nursing research.
Adult↗
Thyroid follicular cell function after non-lethal complement membrane attack.
Terminal complement complexes have been identified around thyroid follicles in Graves' disease and Hashimoto's thyroiditis, and the concentrations of such complexes are increased in the sera of these patients, suggesting a role for complement activation and membrane attack complexes (MAC) in autoimmune thyroiditis. This has been investigated further using cultured human and rat thyroid cells. Thyrocytes were resistant to lysis by homologous complement, in contrast to the effects of heterologous (rabbit) complement. The formation of non-lethal amounts of MAC, using reactive lysis or classical pathway activation, significantly reduced cAMP production by these cells in response to thyroid-stimulating hormone (TSH) (P less than 0.01); similar effects were seen with thyroid-stimulating antibodies. Thyroid cells were able to recover rapidly from complement attack after washing and incubation for 30 min. Non-lethal MAC formation also resulted in reactive oxygen metabolite production, detected by luminol-dependent chemiluminescence in three out of five thyroid cell preparations tested. Ionomycin, but not TSH, also stimulated reactive oxygen metabolite production. These results suggest that repeated or continuous sub-lethal complement attack on thyroid cells may exacerbate hypothyroidism in Hashimoto's thyroiditis, or partially counter the effects of thyroid-stimulating antibodies in Graves' disease. Furthermore, the production of reactive oxygen metabolites in these circumstances could increase the intra-thyroidal inflammatory response; oxygen radical scavenging by anti-thyroid drugs (which are concentrated by thyrocytes) may account in part for the amelioration of thyroiditis observed with such treatment.
Animals↗
Reversal and inhibition of cholera toxin-induced secretion in isolated rabbit ileum.
1. Cholera toxin (1 microgram/ml) abolished net fluid absorption by everted sacs of rabbit ileum, leading to net fluid secretion. This action occurred via the toxin-catalysed ADP ribosylation of the stimulatory GTP-binding protein Gs which is linked to adenylate cyclase. Nicotinamide (10 mM), a reaction product of ADP ribosylation, reversed cholera toxin-induced secretion, restoring absorption. Lower concentrations of nicotinamide induced partial reversal. 2. Nicotinamide (1 mM) blocked the secretory action of cholera toxin applied to ileal sacs. This inhibitory action was more effective in the presence of methionine (1 mM). 3. Other inhibitors of ADP ribosylation, benzamide and adenine, blocked the secretory action of cholera toxin. Hypoxanthine, an analogue and metabolite of adenine, was similarly effective. 4. Nicotinamide was not, however, effective in blocking or reversing the secretory action of theophylline (10 mM) or of heat-stable E. coli enterotoxin STa. This indicates that nicotinamide has a highly specific action against ADP ribosylating toxins. 5. It is proposed that nicotinamide reverses the secretory action of cholera toxin by reversing ADP ribosylation, simply by the law of mass action. This counters the established idea that the effects of cholera and other ADP-ribosylating toxins are irreversible under physiological conditions.
Adenine↗
Regulation of interleukin-6 release by human thyrocytes.
Autoimmune thyroiditis is characterized by lymphocytic accumulation within the thyroid which may be the result, in part, of immunomodulatory cytokine secretion by thyrocytes. We have tested human thyroid cell cultures (n = 9) for interleukin-6 (IL-6) release using two bioassays. IL-6 was detected in all culture supernatants under basal conditions and was increased by gamma-interferon, tumour necrosis factor and TSH in a dose-dependent manner. The bioactivity was confirmed as IL-6 by immunoblotting experiments and could not be accounted for by contamination of the thyroid cell cultures with fibroblasts, lymphocytes or monocytes. Circulating IL-6 levels were not raised in patients with Graves' hyperthyroidism. Exogenous recombinant IL-6 reduced cyclic AMP production in response to TSH when added to thyroid cell cultures. Since IL-6 plays a major role in B cell differentiation and T cell activation, release of IL-6 by thyrocytes may increase the intrathyroidal autoimmune response in Graves' disease and Hashimoto's thyroditis. Our results also suggest that IL-6 may modulate thyroid cell function.
Adenoma↗
Breast Cancer Screening Project in Northeast Florida.
The American Cancer Society--Duval Unit, in June, 1987, helped organize a community demonstration screening project involving all hospitals and institutions with mammography units in the area. A Northeast Florida Cooperative Breast Cancer Screening Group was formed comprised of physicians and administrators from each institution. A total of 1,200 women agreed to participate in the project and each underwent complete screening including education, instruction in self-examination, physical examination by a physician and mammography as indicated according to ACS guidelines. Of the study group, 1,032 women were eligible for mammography at a participating center, and 628 (61%) underwent a mammogram at no cost to them as instructed. Twenty four (4%) had definite abnormalities which led to biopsy and seven (1%) of them had malignant lesions. The medical community organized to provide breast cancer screening and follow-up with low-cost mammography.
Adult↗
Translocation to rat liver mitochondria of phosphatidate phosphohydrolase.
When a particle-free supernatant fraction from rat liver was incubated at 37 degrees C with mitochondria and oleate, some of the enzyme phosphatidate phosphohydrolase (PAP), initially present in the particle-free supernatant, was recovered, after the incubation, bound to mitochondria. This translocation of PAP from cytosol to mitochondria was stimulated by oleate or palmitate in a similar fashion to the stimulation of translocation of PAP to endoplasmic reticulum [Martin-Sanz, Hopewell & Brindley (1984) FEBS Lett. 175, 284-288]. Translocation of PAP from particle-free supernatant to a partially purified mitochondrial-outer-membrane preparation was also stimulated by oleate. More PAP was bound to a mitochondrial-outer-membrane fraction washed in 0.5 M-NaCl before resuspension in sucrose than to a sucrose-washed mitochondrial-outer-membrane preparation. In contrast, washing of microsomal membranes in 0.5 M-NaCl did not enhance the binding of PAP to these membranes. PAP also binds to phosphatidate-loaded mitochondria or microsomes (microsomal fractions). In the experimental system employed, more PAP bound to mitochondria loaded with phosphatidate than to microsomes loaded with phosphatidate. The results are discussed in relation to the role of mitochondrial phosphatidate in liver lipid metabolism.
Animals↗
Responses of left and right ventricular ejection fractions to aerobic and anaerobic phases of upright and supine exercise in normal subjects.
The effects of aerobic and anaerobic exercise on ventricular performance were studied in 13 normal subjects who underwent simultaneous pulmonary gas exchange evaluation and exercise radionuclide ventriculography in the supine and upright postures. Right and left ventricular ejection fraction was measured serially at 2-minute intervals during exercise. The anaerobic threshold occurred at 74% and 80% of maximum heart rate, respectively, during upright and supine exercise. Left and right ventricular ejection fractions rose from rest to the anaerobic threshold (p less than 0.01, p less than 0.01, respectively) and there was a further increase between the anaerobic threshold and maximum exercise (p less than 0.01, p less than 0.01, respectively). The rate of rise of ejection fraction beyond the anaerobic threshold was slightly blunted compared with the rise prior to attaining the anaerobic threshold. There was no significant difference in ventricular performance between supine and upright exercise. The data demonstrate that ventricular performance increases steadily during exercise and is not limited by the conversion of aerobic to anaerobic metabolism.
Adult↗
The influence of oocyte maturity and embryo quality on pregnancy rate in a program for in vitro fertilization-embryo transfer.
An important factor influencing the pregnancy rate after in vitro fertilization-embryo transfer (IVF-ET) appears to be the number of embryos transferred to the uterus. In this study, the influence of oocyte maturity and embryo quality on pregnancy rate was assessed in patients undergoing IVF-ET. Ovarian hyperstimulation was performed by human menopausal gonadotropin (hMG [n = 29]), clomiphene citrate (CC)/hMG (n = 81), and hMG/follicle-stimulating hormone (FSH [n = 13]) protocols. Oocyte maturity was graded on a scale from 1 to 5 based on the morphology of the ooplasm, cumulus mass, corona radiata, and membrana granulosa cells. Embryos were graded according to the symmetry of the blastomeres and the presence or absence of fragmentation. Mature preovulatory oocytes yielded the highest fertilization rates. No differences were found among the protocols in terms of fertilization rate, embryo quality, or pregnancy rate. When all protocols were combined, patients who conceived had a significantly higher number of embryos transferred than those who did not conceive (3.6 +/- 0.1 [mean = SEM] versus 2.7 +/- 0.1). When embryo quality was compared, there was no difference in the number of "B" embryos transferred between patients who conceived and those who did not (1.2 +/- 0.2 versus 1.2 +/- 0.1), but the patients who conceived had significantly more "A" embryos transferred (1.6 +/- 0.3 versus 0.8 +/- 0.1). These data suggest that the treatment protocol did not determine embryo quality. Furthermore, the increase in pregnancy rates seen with an increase in embryos transferred is the result of the transfer of more "A" embryos.
Blastomeres↗
Functional analysis of NTF-1, a developmentally regulated Drosophila transcription factor that binds neuronal cis elements.
In an effort to characterize sequence-specific transcription factors that regulate gene expression during Drosophila development, we identified and purified a novel DNA-binding activity (NTF-1). The purified protein consists of several polypeptides that bind selectively to a functionally important cis-control element of the Ultrabithorax (Ubx) promoter and to the neurogenic elements of both the dopa decarboxylase (Ddc) and fushi tarazu (ftz) promoter/enhancer regions. Purified NTF-1 activates transcription in vitro in a binding site-dependent manner through upstream sequences of the Ubx promoter. A cDNA clone encoding the open reading frame of NTF-1 was isolated, and the deduced primary amino acid sequence of NTF-1 includes a glutamine-rich region reminiscent of the transcriptional activation domains found in Sp1 but no recognizable DNA-binding domain. NTF-1 expression is temporally regulated during embryonic development. In addition, in situ hybridization experiments revealed that NTF-1 is transcribed in a spatially restricted pattern in the embryo, with the highest level of expression observed in the epidermis and a subset of cells in the CNS. Expression of the NTF-1 cDNA in mammalian cells yields a protein that displays DNA-binding and transcriptional activities indistinguishable from that of the collection of proteins isolated from Drosophila embryos. These findings suggest that NTF-1 is a member of a family of developmentally regulated transcription factors that may be involved in directing the expression of genes such as Ubx, Ddc, and ftz in neuronal cells.
Amino Acid Sequence↗
Mitosis in Drosophila development.
Many aspects of the mitotic cycle can take place independently in syncytial Drosophila embryos. Embryos from females homozygous for the mutation gnu undergo rounds of DNA synthesis without nuclear division to produce giant nuclei, and at the same time show many cycles of centrosome replication (Freeman et al. 1986). S phase can be inhibited in wild-type Drosophila embryos by injecting aphidicolin, in which case not only do centrosomes replicate, but chromosomes continue to condense and decondense, the nuclear envelope undergoes cycles of breakdown and reformation, and cycles of budding activity continue at the cortex of the embryo (Raff and Glover, 1988). If aphidicolin is injected when nuclei are in the interior of the embryo, centrosomes dissociate from the nuclei and can migrate to the cortex. Pole cells without nuclei then form around those centrosomes that reach the posterior pole (Raff and Glover, 1989); the centrosomes presumably must interact with polar granules, the maternally-provided determinants for pole cell formation. The pole cells form the germ-line of the developing organism, and as such may have specific requirements for mitotic cell division. This is suggested by our finding that a specific class of cyclin mRNAs, the products of the cyclin B gene, accumulate in pole cells during embryogenesis (Whitfield et al. 1989). Other genes that are essential for mitosis in early embryogenesis and in later development are discussed.
Animals↗
Effect of the MHC on the immunogenicity of vascular allografts in miniature swine.
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Animals↗
Primary structure of apolipophorin-III from the migratory locust, Locusta migratoria. Potential amphipathic structures and molecular evolution of an insect apolipoprotein.
The amino acid sequence of an insect apolipoprotein, apolipophorin-III from Locusta migratoria, has been deduced from the sequence of its cloned cDNA. The mature hemolymph protein consists of 161 amino acids. Optimized alignments of this protein with apolipophorin-III from the tobacco hornworm, Manduca sexta, disclosed an overall sequence identity of only 29%, even though the two proteins are functionally equivalent. The L. migratoria sequence is composed of 12 repeating peptides that are variable in length. Six amphipathic helical segments of varying length were identified in each protein using a newly described algorithm for detecting such secondary structures. The degree of sequence identity between the two insect apoproteins is considerably less than that observed among orthologous mammalian apolipoproteins. However, calculation of the rates of synonymous and nonsynonymous nucleotide substitutions indicates that the insect genes may be evolving at rates similar to the mammalian apolipoprotein genes. Further comparative analyses of insect and mammalian apolipoproteins should provide insights about the limits of sequence diversity tolerated by their predicted amphipathic helical domains.
Amino Acid Sequence↗
Direct chromosome analysis from neonatal cord blood.
Direct chromosome preparations of neonatal cord blood provides the unique opportunity for rapid chromosome analysis (turnaround time; 6 hr), without the necessity of bone marrow aspiration. Based on 42 samples we confirm the finding of Garnham and Sutherland [1987] for suitability of cord blood for direct chromosome preparation. Procedural modifications are provided for higher yield of cells for chromosome analysis. The procedure may well be of major significance for rapid diagnosis of neonates who suffer from aneusomy.
Azure Stains↗
Primary structure and comparative sequence analysis of an insect apolipoprotein. Apolipophorin-III from Manduca sexta.
The amino acid sequence of an insect apolipoprotein, apolipophorin-III from Manduca sexta, was determined by a combination of cDNA and protein sequencing. The mature hemolymph protein consists of 166 amino acids. The cDNA also encodes for an amino-terminal extension of 23 amino acids which is not represented in the mature hemolymph protein. The existence of a precursor protein was confirmed by in vitro translation of fat body mRNA. Computer-assisted comparative sequence analysis revealed the following points: 1) the protein is composed of tandemly repeating tetradecapeptide units with a high potential for forming amphiphilic helical structures. Compared to mammalian apolipoproteins the repeat units in the insect apolipoprotein show considerable length variability; 2) the sequence has a striking resemblance to several human apolipoproteins including apoE, AIV, AI, and CI. However, the homology seems to be entirely functional since, although the insect and mammalian apoproteins contain very similar types of amino acid residues, the actual degree of sequence identity is quite low. Whether the mammalian and insect apoproteins are derived from a common ancestral amphiphilic helix forming, lipid-binding protein, or arose by convergent evolution can not be determined at present. This represents the first complete amino acid sequence for an insect apolipoprotein.
Algorithms↗
Structure and expression of the human apolipoprotein A-IV gene.
We have isolated the human apolipoprotein (apo) A-IV gene from a cosmid library and determined its complete nucleotide sequence. The gene contains three exons of 162, 127, and 1180 nucleotides separated by two introns of 357 and 777 nucleotides. A sequence polymorphism has been identified in the 3' noncoding portion of the third exon. The human apoA-IV gene lacks an intron in the area encoding the 5' nontranslated region of its mRNA, which distinguishes it from all the other human apolipoprotein genes whose sequences are known. Comparison matrix analysis of the human apoA-IV gene sequence revealed evidence for an ancestral 11-nucleotide repeat unit that spans the third exon. These repeated sequences are much more highly conserved than those present in either rat apoA-IV or in any other human apolipoprotein. Optimal alignments of the 5' flanking regions of the rat and human apoA-IV genes disclosed multiple deletions in the rat sequence as well as a highly conserved region of 90 nucleotides (90% sequence identity) located within 170 nucleotides of the start site of transcription. The 5' flanking regions of the human and rat apoA-IV genes were ligated to the bacterial chloramphenicol acetyltransferase gene, then transfected into different cultured cells. The apoA-IV gene sequences elicited preferential expression of chloramphenicol acetyltransferase activity when introduced into intestinally derived Caco-2 cells and liver-derived Hep-G2 cells, consistent with the tissue specificity of the native gene. Analysis of deletion mutants of the human apoA-IV 5' flanking region indicated that regions from -293 to -233 and from -127 to -60 upstream of the transcription start site contain sequences required for maximum gene expression. These findings on the structure and expression of rat and human apoA-IV should prove useful in studying the control of the apoA-IV gene.
Amino Acid Sequence↗
Signal sequence of human preproparathyroid hormone is inactive in yeast.
The biosynthesis of human preproparathyroid hormone (hpreproPTH) and the processing to mature parathyroid hormone (hPTH) was investigated in yeast. Cells were transformed with a plasmid that carried a fusion gene made of the yeast pyruvate kinase promoter, complementary DNA (cDNA) encoding a slightly modified form of hpreproPTH and the transcription termination signal from yeast triosephosphate-isomerase. In transformed yeast cells we identified a protein that was recognized by a PTH antiserum and, on gel electrophoresis, comigrated with hpreproPTH marker. The amino-terminal sequence of the protein was consistent with that of hpreproPTH, indicating that the hormone precursor is not processed. It was localized inside the cell, when analyzed in pulse-chase experiments by trypsin accessibility in intact and lysed spheroplasts. In contrast, when mRNA from these yeast cells and from human parathyroid tissue was translated into preproPTH in a reticulocyte lysate supplemented with canine pancreatic microsomes, the preproPTHs from both mRNAs were transported and cleaved with identical efficiencies. We conclude that hpreproPTH is synthesized in yeast but not recognized and processed like a precursor of a secreted protein by the yeast secretory apparatus.
Animals↗