Cytologic evidence for three human X-chromosomal segments escaping inactivation

Summary Early replication of prometaphasic human sex chromosomes was studied with the bromodeoxyuridine (BrdU)-replication technique. The studies reveal that two distal segments of Xp, including bands Xp 22.13 and Xp 22.3, replicate early in S-phase and therefore may not be subject to random inactivation. Furthermore, the replication of these distal segments of Xp occurs synchronously with those of the short arm of the Y chromosome including bands Yp 11.2 and Yp 11.32. These segments of Xp and Yp correspond well to the pairing segment of the X and Y chromosomes where a synaptonemal complex forms at early pachytene of human spermatogenesis. The homologous early replication of Yp and the distal portion of Xp may be interpreted as a remnant left untouched by the differentiation of heteromorphic sex chromosomes from originally homomorphic autosomes. A third early replicating segment is situated on the long arm of the X chromosome and corresponds to band Xq 13.1. This segment may be correlated with the X-inactivation center postulated by Therman et al. (1979).     

The genetics of phenotypic plasticity. IV. Chromosomal localization

The contributions of each chromosome to the traits thorax size and plasticity of thorax size as affected by temperature in Drosophila melanogaster were measured. A composite stock was created from lines previously subjected to selection on thorax size or plasticity of thorax size. A chromosome extraction was performed against a uniform background lacking genetic variation, provided by a stock of marked balancer flies. With regard to amount of plasticity, chromosome I and the balancer stock showed no plasticity, the composite stock showed the greatest plasticity, and chromosomes II and III were intermediate. Chromosome I showed significant genetic variation for thorax size at both 19° C and 25° C, but not for plasticity, while chromosome II showed significant genetic variation for plasticity, but not for thorax size. Chromosome III showed significant genetic variation for both thorax size and plasticity. We tested the predictions of three models of the genetic basis of phenotypic plasticity: overdominance, pleiotropy, and epistasis. The results support the epistasis model, in agreement with earlier work. The amount of developmental noise was correlated with phenotypic plasticity at 25° C, in agreement with earlier work. A negative correlation was found at 19° C for chromosome II, contrary to earlier work.     

Expression of GTP-binding proteins and prostaglandin E2 receptors during human T cell activation.

GTP-binding proteins (G-proteins) are a family of closely related, yet structurally distinct signal transducing proteins. In this study the presence and relative abundance of several G-proteins and of their corresponding mRNAs were measured in resting and activated human T lymphocytes. We found that T lymphocytes contain RNA coding for Gs, Gi2, and Gi3. No Gi1- and Go-specific RNA could be detected. Membrane fractions of resting and activated lymphocytes were studied in immunoblot experiments. Again, Gs, Gi2, and Gi3, but not Gi1 and Go, were detected. Upon mitogenic activation, a relative increase in mRNA for Gs and Gi3, but not for Gi2 could be demonstrated in Northern blot experiments. Immunoblotting indicated an increase in Gs and Gi3 density in membrane fractions of T cells as well. Paralleling the increase in Gs, we found that activated T cells produce five to seven times more cAMP per cell in response to prostaglandin E2 (PGE2) than resting lymphocytes. Finally, PGE2 binding studies showed that the number of receptors for this hormone increased from 435 +/- 322 to 1035 +/- 357 per cell following in vitro stimulation. We propose that in vitro T cell activation is paralleled by an increase in sensitivity to PGE2-induced cAMP generation. This sensitization is accompanied by both an increase in cell surface PGE2 receptor numbers as well as by increased expression of the signal transducing protein Gs and may physiologically be important for limiting an immune response.     

Prebiotic sugar synthesis: Hexose and hydroxy acid synthesis from glyceraldehyde catalyzed by iron(III) hydroxide oxide

Summary Iron(III) hydroxide oxide [Fe(OH)O] efficiently catalyzed the condensation of 25 MM dl-glyceraldehyde to ketohexoses at 25°C (pH 5–6). At 16 days the yields were sorbose (15.2%), fructose (12.9%), psicose (6.1%), tagatose (5.6%), and dendroketose (2.5%) with 19.6% of triose unreacted. Analysis at 96 days showed no decomposition of hexoses. Under these conditions Fe(OH)O also catalyzed the isomerization and rearrangement of glyceraldehyde to dihydroxyacetone and lactic acid, respectively. In these reactions, about 10% of the glyceraldehyde was oxidized to glyceric acid with concurrent reduction of the iron(III) to iron(II). The partial reduction of Fe(OH)O did not noticeably reduce its ability to catalyze hexose synthesis. The relationship of these results to prebiotic sugar synthesis is discussed.     

On the location and function of tyrosine beta 331 in the catalytic site of Escherichia coli F1-ATPase.

1) Using a combination of site-directed mutagenesis and fluorescence spectroscopy we have studied the location and function of residue beta Y331 in the catalytic site of Escherichia coli F1-ATPase. The fluorescent analog lin-benzo-ADP was used as a catalytic-site probe, and was found to bind to three sites in normal F1, with Kd1 = 0.20 microM and Kd2,3 = 5.5 microM. lin-Benzo-ATP was a good substrate for hydrolysis. 2) The mutants investigated were beta Y331F, L, A and E. kcat/KM for ATP hydrolysis in purified F1 was reduced according to the series Y greater than or equal to F greater than L greater than A greater than E, with E being severely impaired; concomitant decreases in binding affinity for lin-benzo-ADP were seen. 3) Fluorescence properties of lin-benzo-ADP bound to F1 differed widely, depending on the residue present at position beta 331. Red shifts of excitation and emission spectra occurred with F and L residues, but not with Y, A, or E. There was strong quenching of fluorescence with wild-type (Y), partial quenching with A, and no quenching with F, L, or E. 4) We conclude that (a) the environment around the bound adenine moiety in the catalytic site is nonpolar, (b) residue beta 331 is part of the adenine-binding subdomain and when tyrosine is the residue, the phenolic hydroxyl makes direct interaction with the fluorophore, (c) an aromatic residue is not absolutely required at position beta 331 for catalytic function, but an increase in polarity leads to functional impairment, and (d) in terms of fluorescence response of bound lin-benzo-ADP all three catalytic sites behaved the same. 5) F1 from mutant beta Y297F bound lin-benzo-ADP with the same fluorescence and binding characteristics as normal F1, and catalytic properties were similar to normal. Therefore, there was no reason to conclude that residue beta Y297 is involved in binding the adenine moiety of ATP.     

Adipogenic cell line TA1: a suitable model to study the effect of beta-adrenergic agonists on lipid metabolism.

The stable adipogenic cell line TA1 was investigated as a potential in vitro system to examine effects of beta-adrenergic agonists on lipid metabolism at the cellular level. Initial experiments were conducted to establish whether dexamethasone, indomethacin, or both in combination induce rapid differentiation of TA1 preadipocytes to adipocytes. Based on activity of fatty acid synthase, dexamethasone and indomethacin, individually and in combination, were observed to induce differentiation in TA1 cells at different rates (dexamethasone/indomethacin greater than indomethacin greater than dexamethasone). Dexamethasone/indomethacin induced complete differentiation in TA1 cells 4 days after confluence, as indicated by increased activity of fatty acid synthase, glycerol-3-phosphate dehydrogenase, and malic enzyme. Finally, mature TA1 adipocytes were treated with various concentrations of isoproterenol and ractopamine to determine the responsiveness of TA1 adipocytes to a beta-adrenergic challenge. Glycerol release was increased and fatty acid synthase activity was decreased in a dose-dependent manner for both isoproterenol and ractopamine. These results indicate that fully differentiated TA1 adipocytes may be useful to study direct cellular effects of lipolytic and lipogenic agents on lipid metabolism.