Uridine enhances expression of the fragile X chromosome in human lymphocytes

Summary We have studied the effect of uridine on the expression of fragile X (fra[X]) in lymphocyte cultures established in the folate and thymidine deficient medium TC199. The results indicate that uridine enhances the expression of fra(X) and gives a higher mitotic rate. The excess of uridine during DNA synthesis might further promote the previously suggested cycle of misincorporation and removal of deoxyuridine monophosphate when the pool of deoxythymidine triphosphate is continuously depleted.     

Spring development of a Chlamydomonas population in Lake Nimetön,a small humic forest lake in southern Finland

Biomass development and vertical distribution of a Chlamydomonas population in a small humic forest lake was followed by daily sampling in May-June, 1984. Chlamydomonas dominated the phytoplankton spring bloom, forming 71% of the maximum phytoplankton biomass on 18 May. In early May the outflow rate was high and during the 24 hour period when the maximum rate of surface runoff was recorded (8–9 May), 43% of the Chlamydomonas biomass was flushed out of the lake, which delayed the onset of biomass increase. When surface runoff had slowed down Chlamydomonas biomass started increasing and during wax of the population most cells were < 10 µm in diameter. Population maximum lasted for one day (18 May) and there-after Chlamydomonas biomass decreased towards the end of the study. During wane of the population most cells were > 10 µm in diameter.     

Inhibition of bilirubin UDPglucuronosyltransferase activity by triphenylacetic acid and related compounds

Bilirubin UDPglucuronosyltransferase of rat or human liver microsomes was inhibited, in vitro, by triphenylacetic acid and by structurally related arylcarboxylic acids. This inhibition appeared to be competitive towards bilirubin, and mixed-type towards UDPglucuronic acid. A decrease in the number of phenyl rings or the absence of the carboxyl group in the molecule gave structures which did not affect enzyme activity, showing that both the triphenyl moiety and the carboxyl group were necessary for the inhibition. On the other hand, successive additions of methylene groups in the aliphatic chain progressively increased inhibitory potency. Kappi,bilirubin for triphenylacetic acid was 96 microM compared with 5 microM for 7,7,7-triphenylheptanoic acid. The inhibition of bilirubin UDPglucuronosyltransferase was not due to displacement of bilirubin from albumin. On the basis of these results an attempt was made to delineate the molecular events leading to glucuronidation of bilirubin.     

Regular and irregular divisions of Lepidoptera spermatocytes as related to the speed of meiotic prophase

Lepidoptera males bear two kinds of meiotic divisions. One is regular (eupyrene) and leads to nucleate, fertilizing spermatozoa. The other (apyrene) shows metaphase I chromosomes clumping together into irregular masses which later split forming daughter cells with unbalanced sets of chromosomes which are eventually extruded from the cells; hence, the spermatids develop into anucleate spermatozoa of unknown function. The apyrene divisions are induced by a haemolymph factor which becomes functional towards pupation. Using incorporation of tritiated thymidine at the premeiotic S-phase as a marker for timing, it was found that the prophase of the apyrene spermatocyte is shorter than that of the eupyrene spermatocyte. It is proposed that meiosis-specific proteins cannot be synthesized during the shortened apyrene prophase and that this is correlated with the irregular chromosome behaviour during the subsequent metaphase-telophase of these spermatocytes.     

Vascular smooth muscle caldesmon

Caldesmon, a major actin- and calmodulin-binding protein, has been identified in diverse bovine tissues, including smooth and striated muscles and various nonmuscle tissues, by denaturing polyacrylamide gel electrophoresis of tissue homogenates and immunoblotting using rabbit anti-chicken gizzard caldesmon. Caldesmon was purified from vascular smooth muscle (bovine aorta) by heat treatment of a tissue homogenate, ion-exchange chromatography, and affinity chromatography on a column of immobilized calmodulin. The isolated protein shared many properties in common with chicken gizzard caldesmon: immunological cross-reactivity, Ca2+-dependent interaction with calmodulin, Ca2+-independent interaction with F-actin, competition between actin and calmodulin for caldesmon binding only in the presence of Ca2+, and inhibition of the actin-activated Mg2+-ATPase activity of smooth muscle myosin without affecting the phosphorylation state of myosin. Maximal binding of aorta caldesmon to actin occurred at 1 mol of caldesmon: 9-10 mol of actin, and binding was unaffected by tropomyosin. Half-maximal inhibition of the actin-activated myosin Mg2+-ATPase occurred at approximately 1 mol of caldesmon: 12 mol of actin. This inhibition was also unaffected by tropomyosin. Caldesmon had no effect on the Mg2+-ATPase activity of smooth muscle myosin in the absence of actin. Bovine aorta and chicken gizzard caldesmons differed in several respects: Mr (149,000 for bovine aorta caldesmon and 141,000 for chicken gizzard caldesmon), extinction coefficient (E1%280nm = 19.5 and 5.0 for bovine aorta and chicken gizzard caldesmon, respectively), amino acid composition, and one-dimensional peptide maps obtained by limited chymotryptic and Staphylococcus aureus V8 protease digestion. In a competitive enzyme-linked immunosorbent assay, using anti-chicken gizzard caldesmon, a 174-fold molar excess of bovine aorta caldesmon relative to chicken gizzard caldesmon was required for half-maximal inhibition. These studies establish the widespread tissue and species distribution of caldesmon and indicate that vascular smooth muscle caldesmon exhibits physicochemical differences yet structural and functional similarities to caldesmon isolated from chicken gizzard.     

Immunocytochemical demonstration of tissue-type plasminogen activator in endocrine cells of the rat pituitary gland

We immunocytochemically stained rat pituitary glands using antibodies against plasminogen activators of the tissue type (t-PA) and the urokinase type (u-PA). A large population of endocrine cells in the anterior lobe of the gland displayed intense cytoplasmic immunoreactivity with anti-t-PA. In some areas of the intermediate lobe we found a weak staining, and we observed weakly staining granular structures in the posterior lobe. Controls included absorption of the antibodies with highly purified t-PA. In addition, SDS PAGE followed by immunoblotting of pituitary gland extracts revealed only one band with an electrophoretic mobility similar to that of t-PA when stained with anti-t-PA IgG. No u-PA immunoreactivity was detected in the rat pituitary gland. Sequential staining experiments using antibodies against growth hormone and t-PA demonstrated that the t-PA-immunoreactive cells constitute a large subpopulation of the growth hormone-containing cells. These findings represent the first direct evidence for the presence of t-PA in cell types other than endothelial cells in the intact normal organism. In this article we discuss the implications of the results for a possible role of t-PA in the posttranslational processing of prohormones.     

An immunological approach to enrich a mitotic stimulator and to reveal G2-phase-specific proteins in Physarum polycephalum

Purified antibodies from an antiserum against S-phase proteins of the myxomycete Physarum polycephalum were attached to protein-A-Sepharose CL-4B. A late G2-phase extract that contained a mitosis-stimulating protein was applied to this immunoadsorbent, and the mitosis-stimulating protein was enriched by a factor of ten. This protein, which is present in the cell in low amounts, is synthesized in late G2 phase and obviously degraded in a later stage of the cycle. Immunoadsorption of a G2-phase extract with anti-S-antibodies decreased the 700 main proteins to 20 as demonstrated by two-dimensional gel electrophoresis. No difference in protein pattern could be observed on two-dimensional gels between S-phase and G2-phase extracts before and after immunoadsorption with anti-S-antibodies. This indicates that there are no G2-phase-specific proteins among the 700 most abundant proteins of Physarum polycephalum.     

A plasmodial alpha-tubulin cDNA from Physarum polycephalum. Nucleotide sequence and comparative analysis

As the first step towards correlating structure and function of tubulin in the slime mold Physarum polycephalum we have elucidated the nucleotide sequence of a cDNA that appears to code for all but the last 25 to 30 C-terminal amino acids of a plasmodial alpha-tubulin. Differences in amino acid sequence from those of other alpha-tubulins are distributed fairly evenly throughout the sequence, although a relatively extensive conserved region is found in position 396 to 426 near the C terminus. A small region in position 298 to 307 contains a cluster of amino acid residues unique to Physarum alpha-tubulin. The sequence is 70% homologous to two yeast alpha-tubulins and about 83% homologous to five animal alpha-tubulins. A comparison of the homologies of all the known alpha-tubulins indicates that a large decrease in the accepted point mutation rate has occurred during the evolution of the metazoa, suggesting a major functional specialization of microtubules.     

Comparison of goose-type,chicken-type,and phage-type lysozymes illustrates the changes that occur in both amino acid sequence and three-dimensional structure during evolution

Summary The three-dimensional structure of goose-type lysozyme (GEWL), determined by x-ray crystallography and refined at high resolution, has similarities to the structures of hen (chicken) eggwhite lysozyme (HEWL) and bacteriophage T4 lysozyme (T4L). The nature of the structural correspondence suggests that all three classes of lysozyme diverged from a common evolutionary precursor, even though their amino acid sequences appear to be unrelated (Grütter et al. 1983).In this paper we make detailed comparisons of goose-type, chicken-type, and phage-type lysozymes. The lysozymes have undergone conformational changes at both the blobal and the local level. As in the globins, there are corresponding -helices that have rigid-body displacements relative to each other, but in some cases corresponding helices have increased or decreased in length, and in other cases there are helices in one structure that have no counterpart in another.Independent of the overall structural correspondence among the three lysozyme backbones is another, distinct correspondence between a set of three consecutive -helices in GEWL and three consecutive -helices in T4L. This structural correspondence could be due, in part, to a common energetically favorable contact between the first and the third helices.There are similarities in the active sites of the three lysozymes, but also one striking difference. Glu 73 (GEWL) spatially corresponds to Glu 35 (HEWL) and to Glu 11 (T4L). On the other hand, there are two aspartates in the GEWL active site, Asp 86 and Asp 97, neither of which corresponds exactly to Asp 52 (HEWL) or Asp 20 (T4L). (The discrepancy in the location of the carboxyl groups is about 10 Å for Asp 86 and 4 Å for Asp 97.) This lack of structural correspondence may reflect some differences in the mechanisms of action of three lysozymes. When the amino acid sequences of the three lysozyme types are aligned according to their structural correspondence, there is still no apparent relationship between the sequences except for possible weak matching in the vicinity of the active sites.     

A comparison between dopamine-stimulated adenylate cyclase and 3H-SCH 23390 binding in rat striatum

Methods for measuring 3H-SCH 23390 binding and dopamine (DA) stimulated adenylate cyclase (AC) were established in identical tissue preparations and under similar experimental conditions. Pharmacological characterization revealed that both assays involved interaction with the D1 receptor or closely associated sites. In order to investigate whether the binding sites for 3H-SCH 23390 and DA in fact are identical, the antagonistic effects of a variety of pharmacologically active compounds were examined. Surprisingly, the Ki-values obtained from Schild-plot analysis of the antagonism of DA-stimulated AC, were 80-240 times higher than the Ki-values obtained from competition curves of 3H-SCH 23390 binding. Since both assays were performed under identical conditions, the differences in Ki-values indicate the possibility of different binding sites for DA and 3H-SCH 23390 or, that DA and 3H-SCH 23390 label different states of the same receptor.