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Biomedical subjects

M Freeman

Publications and source records attributed to M Freeman.

At least 127 records · Page 7Linked to original sources
PubMed1989

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↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1988

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↗
PubMed1988

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↗
PubMed1987

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↗
PubMed1987

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↗
PubMed1987

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↗
PubMed1987

The gnu mutation of Drosophila causes inappropriate DNA synthesis in unfertilized and fertilized eggs.

Drosophila melanogaster embryos whose mothers are homozygous for the maternal effect lethal mutation gnu (GNU embryos) under DNA synthesis but no nuclear division; this leads to the formation of a small number of giant nuclei in the syncytial blastoderm. We have shown previously that many components of the mitotic apparatus are present and functional in GNU embryos, and the primary lesion of the gnu mutation has therefore remained obscure. Here, we report that fertilization is not necessary for GNU eggs to develop. Giant nuclei originate from the products of female meiosis, and we see autonomously replicating centrosomes that must be maternally derived. If GNU eggs are inseminated, however, the male pronucleus also undergoes DNA replication. Our observations suggest that the GNU cytoplasm permits DNA synthesis in a relatively unregulated manner. In embryos from females homozygous for gnu and the female sterile haploid mutation mh, we find replication of DNA derived from the male pronucleus. This contrasts with embryos from mothers homozygous for mh alone, in which this does not occur. We propose that the gnu gene product participates in the repression of DNA synthesis found in unfertilized eggs.

Animals↗
PubMed1987

Human preproparathyroid hormone synthesized in Escherichia coli is transported to the surface of the bacterial inner membrane but not processed to the mature hormone.

cDNA encoding human preproPTH (hpreproPTH) was expressed in Escherichia coli to study the processing of the precursor to hPTH and its secretion by the bacterial secretory apparatus. We first constructed hybrid genes that differed randomly in the distance between the E. coli lac promoter's ribosomal binding site and DNA encoding a fusion protein with beta-galactosidase activity and the prepro sequence of hpreproPTH on the aminoterminus. Starting with clones identified as efficient producers of beta-galactosidase on indicator agar plates, the coding sequence for hpreproPTH was reconstituted intact. In a different construction we placed the hpreproPTH coding sequence downstream from the lac promoter at a distance of 12 base pairs from the ribosomal binding site. PTH immunoreactive proteins from multiple clones were identified by protein gel electrophoresis and by protein microsequencing. PTH-related proteins encoded by different plasmids were shown to be hpreproPTH with amino-terminal extensions of either two or four amino acids and as authentic hpreproPTH. Two hPTH fragments, hPTH(3-84) and hPTH(8-84), were also observed. The trypsin accessibility of hpreproPTH and of the two hPTH fragments in pulse-chase, cell-fractionation experiments using intact and lysed spheroplasts lets us conclude that the mammalian signal sequence directs hpreproPTH to the surface of the spheroplast membrane but is not appropriately cleaved by the signal peptidase.

Animals↗
PubMed1987

The distribution of a 'mitosis-specific' antigen during Drosophila development.

We have used MPM-2, a monoclonal antibody raised against mitotic HeLa cells, to stain a Drosophila cell line, whole mounts of Drosophila embryos, and sectioned tissue from embryonic and larval stages of development. MPM-2 recognizes a major phosphoprotein of approximately 125 X 10(3) Mr in Drosophila tissue culture cells that, like the mammalian MPM-2 antigen, appears to be recognized only in mitotic cells. During early embryogenesis, when the embryonic nuclei divide as a syncytium with a very short nuclear division time, MPM-2 antigen is observed within the spindle compartment at all stages of the nuclear division cycle. Upon cellularization of the embryo and lengthening of the duration of the cycle, the antigen is predominantly seen in mitotic cells. Drosophila larvae contain both diploid and polytene tissues: in diploid tissue MPM-2 staining is specifically observed over mitotic cells, as expected from its distribution in cellularized embryos. Surprisingly, antigen is also detected in the nuclei of polytene cells that replicate their DNA but do not undergo mitosis.

Animals↗
PubMed1987

Concurrent validity of equilibrium tests in boys with learning disabilities with and without vestibular dysfunction.

Six equilibrium measures were administered to 50 boys with learning disabilities, 25 with and 25 without suspected vestibular system dysfunction. Pearson product moment correlations were computed between test scores for the total sample and for each subgroup to establish concurrent validity between tests. Four correlations for the total sample and three for each of the subgroups were statistically significant. However, only 3 of the 10 correlation coefficients mentioned were greater than r = 0.5. The relatively low magnitude of many of the correlations obtained demonstrates that different tests of equilibrium measure different balance-related competencies, and that competence in one area does not indicate competence in another. Therapists evaluating equilibrium should administer more than one test. The tilt test used in this investigation did not correlate significantly with any other test. This suggests that tilt tests should routinely be included in the evaluation of equilibrium.

Adolescent↗
PubMed1987

Eosinophilic fasciitis with atypical features.

Eosinophilic fasciitis has been reported to be a syndrome distinct from progressive systemic sclerosis, due to the absence of Raynaud's phenomenon, visceral disease, and autoantibodies as well as steroid responsiveness and an abnormal histopathologic appearance that primarily involves the lower subcutis and fascia. More recent studies, however, have noted considerable overlap in the clinical, pathologic, and laboratory features of these two entities. We report a case that further blurs the distinction between eosinophilic fasciitis and progressive systemic sclerosis.

Aged↗
PubMed1986

The dissociation of nuclear and centrosomal division in gnu, a mutation causing giant nuclei in Drosophila.

We describe a recessive, maternal-effect lethal mutation of Drosophila, gnu. gnu uncouples nuclear division from many cytoplasmic events of mitosis in the Drosophila embryo. Embryos from homozygous females are defective in nuclear division, but not in DNA replication, and therefore develop a small number of giant nuclei. Centrosomes divide independently of nuclear division and migrate to the surface of the syncytial blastoderm. There they nucleate microtubules into asters, which appear normal at first but become very large. Only later, when the giant nuclei begin to break down, are spindles sometimes formed. The cortical actin of these embryos develops into a characteristic network.

Actins↗
PubMed1986

Should the intent of testing influence its interpretation?

A test is often interpreted as "normal" or "abnormal" by a single criterion, regardless of the intent of testing. The discriminate accuracy of this convention was critically analyzed using information content (I), likelihood ratio and the area under the receiver-operating characteristic curve. Three ejection fraction variables were assessed--ejection fraction at rest, exercise ejection fraction and the change in ejection fraction from rest to exercise--each relative to three intentional goals: diagnosis of coronary artery disease in 929 patients without previous myocardial infarction, prediction of multivessel disease in these same 929 patients and prediction of multivessel disease in 507 patients with previous myocardial infarction. The information content of exercise ejection fraction (IEX) was higher than for ejection fraction at rest (IR) or for the change from rest to exercise (IEX-R), and was relatively constant regardless of the goal of testing. In contrast, neither IR nor IEX-R was constant. IR was lowest for diagnosis of coronary artery disease, whereas IEX-R was highest for this same goal. These empiric observations are consistent with the quantitative relation predicted by information theory: IEX = IR + IEX-R. Thus, ejection fraction at rest has little discriminate value relative to the diagnosis of coronary artery disease, but does have value in evaluating the extent of disease in patients after myocardial infarction. Exercise ejection fraction and change in ejection fraction are nearly equally useful for purposes of diagnosis, whereas the former is most useful for functional evaluation in postinfarction patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗