Enzyme immunoassay of oestrogen and progesterone receptors in uterine and intrauterine tissue during human pregnancy and labour

Oestrogen and progesterone receptors were studied in the non-pregnant state, in early pregnancy and at term using monoclonal antibody enzyme immunoassays. Receptors for both steroids were found in tissues from non-pregnant patients and patients in early pregnancy. At term oestrogen receptors were undetectable in all tissues studied. Progesterone receptors were undetectable in chorion, amnion and placenta at term, while present in extremely low concentrations in decidua and myometrium.     

M-07e human leukemic factor-dependent cell line provides a rapid and sensitive bioassay for the human cytokines GM-CSF and IL-3

We have isolated a subline of the M-07 human megakaryoblastic leukemia cell line, designated M-07e, that requires either interleukin-3 (IL-3) or granulocyte macrophage colony-stimulating factor (GM-CSF) for growth, even in the presence of fetal calf serum. This cell line will not grow long term in any other cytokine although it responds slightly to IL-2, IL-4, IL-6, IL-9, and interferon-gamma. We have used the M-07e subline to develop a quantitative bioassay for the measurement of levels of either GM-CSF or IL-3. This assay is as sensitive to either factor as the human bone marrow colony assay (CFU-GM) or the chronic myelogeneous leukemic (CML) blast cell proliferation assay for these factors and is much more convenient and reliable than either. With this assay, as little as 25-50 pg/ml of either IL-3 or GM-CSF can be detected, a level that should render the assay useful for analysis of these molecules in samples from patients undergoing colony-stimulating factor therapy and from conditioned media from natural sources of the factors. In these cases, neutralizing antisera to each cytokine are required to demonstrate the specificity of the assay. This assay, in combination with quantitative immunoassays, should greatly facilitate the analysis of the roles of IL-3 and GM-CSF in regulating hematopoiesis both in patients and in natural sources of the cytokines.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Biochemical and immunological studies of three genetic variants of 3-phosphoglycerate kinase 2 from the mouse

Three electrophoretic variants of 3-phosphoglycerate kinase 2 (PGK-2A, PGK-2B, and PGK-2C) were purified from DBA/2J, C3H/HeJ, and C57L/J mice, respectively. PGK-2C exhibits only 2% of the specific activity of PGK-2A and PGK-2B in the reaction leading to the formation of 1,3-diphosphoglycerate. Compared to PGK-2A and PGK-2B, PGK-2C exhibits broader coenzyme specificity and lower K_ms for substrate and coenzymes. Incubation at 45C revealed that PGK-2B is more heat stable than either PGK-2A or PGK-2C. Enzyme immunoinactivation and double immunodiffusion studies showed that mice carrying any one of these three PGK-2 alleles have similar amounts of proteins for PGK-1 and PGK-2 in testes. The results of these studies suggest that low PGK-2C activity in C57L/J mice is a result of a structural rather than a regulatory gene mutation.     

Purification and characterization of two isozymes of 3-phosphoglycerate kinase from the mouse.

Two isozymes of 3-phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3), designated PGK-A and PGK-B, were purified from separate extracts of muscle and testicular tissue of DBA/2J mice, respectively. A similar procedure was used to purify the corresponding isozymes from C57BL/6J mice in order to make inter-strain comparisons. The purification involved the use of affinity chromatography with an 8-(6-aminohexyl)amino-ATP-Sepharose column and DEAE-Sephadex chromatography. Lactate dehydrogenase isozyme LDH-X was also co-purified from extract of mouse testes by this two-step procedure. The same isozyme isolated from either mouse strain was found to be identical in physical and biochemical properties. Both isozymes are monomeric as determined by gel filtration chromatography and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Furthermore, the isozymes have similar molecular weights, of 47 000 +/- 2000 and exhibit similar Km values for both coenzymes and substrate, as well as temperature dependence of enzyme activity. However, it was observed that the B isozyme is more labile than the A isozyme by denaturation at high temperature, urea and acidic pH.     

The FSHD2 Gene SMCHD1 Is a Modifier of Disease Severity in Families Affected by FSHD1

Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is caused by contraction of the D4Z4 repeat array on chromosome 4 to a size of 1–10 units. The residual number of D4Z4 units inversely correlates with clinical severity, but significant clinical variability exists. Each unit contains a copy of the DUX4 retrogene. Repeat contractions are associated with changes in D4Z4 chromatin structure that increase the likelihood of DUX4 expression in skeletal muscle, but only when the repeat resides in a genetic background that contains a DUX4 polyadenylation signal. Mutations in the structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) gene, encoding a chromatin modifier of D4Z4, also result in the increased likelihood of DUX4 expression in individuals with a rare form of FSHD (FSHD2). Because SMCHD1 directly binds to D4Z4 and suppresses somatic expression of DUX4, we hypothesized that SMCHD1 may act as a genetic modifier in FSHD1. We describe three unrelated individuals with FSHD1 presenting an unusual high clinical severity based on their upper-sized FSHD1 repeat array of nine units. Each of these individuals also carries a mutation in the SMCHD1 gene. Familial carriers of the FSHD1 allele without the SMCHD1 mutation were only mildly affected, suggesting a modifier effect of the SMCHD1 mutation. Knocking down SMCHD1 in FSHD1 myotubes increased DUX4 expression, lending molecular support to a modifier role for SMCHD1 in FSHD1. We conclude that FSHD1 and FSHD2 share a common pathophysiological pathway in which the FSHD2 gene can act as modifier for disease severity in families affected by FSHD1.