Differentiation of 2-cell and 8-cell mouse embryos arrested by cytoskeletal inhibitors

Mouse embryos at the 2-cell stage were cultured in the presence of cytochalasin B (CB), cytochalasin D (CD), colchicine (COL) or colcemid (COM) for up to 72 h. Cleavage was arrested in the 2-cell and 8-cell embryos cultured in CB or CD but the blastomeres continued to differentiate, since chromosome replication occurred in the blastomeres at approximately the same time as control embryos underwent cleavage; an increase in the incorporation of [³H]uridine into RNA was also detected. Furthermore, the cleavage-arrested embryos acquired the necessary information to undergo morphogenesis; these embryos when explanted to fresh medium after 48 h culture in CB or CD underwent compaction within 15–60 min and started to cavitate to produce trophoblastic vesicles within 5–6 h at the same time as when the control embryos were undergoing compaction and beginning to form blastocoelic cavities. In contrast, the embryos arrested in the presence of COM or COL showed none of these differentiative, biochemical or morphogenetic changes. Hence, differentiation of blastomeres and morphogenesis is apparently coupled with nuclear divisions and the information does not reside within the blastomeres at the 2-cell or 8-cell stage. The trophoblastic vesicles produced after cleavage arrest subsequently gave rise to only trophoblast giant cells and no embryonic derivatives were detected.     

Ultrastructure and ATPase activity of the rectal gland of the chondrichthyean fish Hydrolagus colliei (Holocephali)

Summary The anatomy, histology, ultrastructure and ATPase activity of the intramural rectal gland of the chondrichthyean Hydrolagus colliei, are described. The cells of the rectal gland of Hydrolagus demonstrate the same well developed lateral and basal cisternae, elongate mitochondria and luminal border as those of their elasmobranch counterparts. ATPase activity within the rectal gland of Hydrolagus is as intense as that in a number of elasmobranchs examined in the course of the study. Despite its primitive intramural location the rectal gland of Hydrolagus respresents a homolog of the more specialized and better known elasmobranch gland and appears as well suited for cation excretion.     

Mortality in Grey seal pups: incidence and causes

Pup production and mortality of beach breeding Grey seals was studied at two contrasting types of rookery, the cliff-bound island of Ramsey, Dyfed, and the low, grassy island of Auskerry, Orkney. Thirty-five per cent of pups died on Ramsey compared with 14 % on Auskerry. Almost half of the Ramsey mortality was accounted for by animals being lost from the beaches so that they were not available for analysis. The main proximate causes of death revealed by post-mortem examination were starvation and infections. The other conditions which accounted for a small proportion of deaths were drowning, trauma, non-viability, stillbirths, atelectasis, dystokia and heart abnormality. Certain pathogens associated with disease were common to both sites, while others were specific to Ramsey or Auskerry.
The most important ultimate cause of pup deaths appeared to be failure of the mother/pup bond. The differences in levels of mortality between the sites is thought to be due to differences in the topography of the beaches. Pup survival on the narrow cliff-bound beaches, or in caves, on Ramsey was reduced either directly through pups being washed off, or indirectly by overcrowding of animals at high tide.     

Synergistic effect of ribose and 2-deoxy-ribose with nutrition and auxin in rooting hypocotyl cuttings of Phaseolus mungo

Exogenously supplied ribose and deoxyribose in a medium containingsucrose+ IAA considerably enhanced the formation of roots onhypocotyl cuttings of Phaseolus mungo with intact apex andleaves.The effect increased with increasing concentration of pentosesugars and was more pronounced with deoxyribose than with ribosesugar. (Received October 25, 1975; )     

Hypoxanthine-guanine phosphoribosyltransferase: Mosaicism in the peripheral erythrocytes of a heterozygote for a normal and a mutant enzyme

A mutant form of erythrocyte hypoxanthine-guanine phosphoribosyltransferase with an abnormal isoenzyme pattern was found in a patient with a partial enzyme deficiency and X-linked gout. This abnormal pattern was a marker for the mutant enzyme in hemolysate from the heterozygote for the enzyme deficiency.These studies were generously supported by the Medical Research Council of Canada (MRC # MA4758) and the Canadian Arthritis and Rheumatism Society.     

Prostaglandin biosynthesis and stimulation of cyclic AMP in primary monolayer cultures of epithelial cells from mouse mammary gland

Cyclic AMP levels in primary monolayer cultures of epithelial cells prepared from mid-pregnant mice are stimulated by prostaglandin E₁ and E₂. Prostaglandin F_1α and F_2α have only a slight effect upon cyclic AMP levels. In the absence of phosphodiesterase inhibitors the rise in cyclic AMP produced by PGE₁ is only transient and the levels return to normal within 30 minutes. High concentrations (16 mM) of theophylline are needed to prevent this decline, suggesting that the phosphodiesterase activity of epithelial cells in culture is high. However, theophylline alone produced only a small increase in basal cyclic AMP levels even over a 2-hour period indicating that basal cyclic AMP is turned over more slowly than cyclic AMP produced in response to stimulation with PGE₁.Both PGE and PGF synthesis were monitored using radioimmunoassay procedures previously reported. The observed levels were found to decrease as cell density increased and were sensitive to the addition of agents such as collagen and naproxen.     

Determination of plasma and tissue levels of tyramine by radioimmunoassay.

Antibodies were prepared against tyramine. The antigen was prepared as follows: p-Aminohippuric acid was coupled to mBSA using a carbodiimide reagent. The amino group was diazotized an attached to the aromatif ring of TYR. The immunogen in Freund's complete adjuvant was injected into rabbits. The specificity of the resulting antibody was determined by radioimmunoassay. Using random-labeled TYR-3H, TYR, its metabolites, phenethylamine analogs, catecholamines, and certain amino acids were evaluated by a competitive binding assay method. With this technique 4 ng of TYR inhibited the binding of TYR-3H by 50%. The radioimmunoassay of TYR was used to measure the plasma, urine, and tissue levels of TYR in rabbits. The plasma disappearance curve of TYR revealed a biphasic pattern with t1/2 of 2 min and 54 min. The highest concentration of TYR was found in adrenals and spleen. The factthat the major metabolites of TYR and a series of pharmacologically important sympathomimetics and catecholamines did not interfere, makes the radioimmunoassay of TYR a useful, simple, sensitive, and spedific method for the direct analysis of TYR in biological meterials.     

Optimization of conditions for the enzymatic hydrolysis of phytoestrogen conjugates in urine and plasma

Optimal pH, temperature, and concentration of enzyme conditions for the rate of hydrolysis of five isoflavone conjugates (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone) from two biological matrices (urine and plasma) were studied using beta-glucuronidase from Helix pomatia. In addition, the use of mixtures of beta-glucuronidase and sulfatase enzymes from different sources was investigated to find enzyme preparations that contained lower amounts of naturally present phytoestrogens. Quantification of aglycones spiked with (13)C(3)-labeled internal standards was carried out by LC-MS/MS. In urine, all of the phytoestrogen conjugates hydrolyzed within 2h under standard hydrolysis conditions (24mul H. pomatia, pH 5, 37 degrees C). Hydrolysis rates were improved at 45 degrees C and by doubling the enzyme concentration and may be used to further reduce hydrolysis times down to 100min. In plasma, a 16-h hydrolysis was required to ensure complete hydrolysis of all conjugates. As with urine, the use of increased temperature or increased enzyme concentration reduced hydrolysis times for most analytes. However, the rate of hydrolysis in plasma was significantly slower than that in urine for all analytes except enterodiol, for which the reverse was true. Neither increased temperature nor increased enzyme concentration increased the rate of hydrolysis of enterolactone. Hydrolysis at pH 6 proved to be detrimental to hydrolysis of phytoestrogen conjugates, especially those in plasma. Other enzyme preparations from different sources, such as beta-glucuronidase from Escherichia coli, were found to contain lower amounts of contaminating phytoestrogens and showed increased enzyme activity for isoflavones, but lower activity for lignans, when used with other sulfatase enzymes. In addition, this involved complicating the analytical procedure through using mixtures of enzymes. Therefore, the use of beta-glucuronidase from H. pomatia combined with an enzyme "blank" to correct for phytoestrogen contamination was shown to be a suitable method for hydrolysis of phytoestrogens.     

The 30-Base-Pair Deletion in Chinese Variants of the Epstein-Barr Virus LMP1 Gene Is Not the Major Effector of Functional Differences between Variant LMP1 Genes in Human Lymphocytes

One group of sequence variants of Epstein-Barr virus is characterized by a 10-amino-acid deletion within the CTAR-2 functional domain of the latent membrane protein, LMP1. A role for this deletion in enhancing the tumorigenicity of the viral oncogene in rodent fibroblasts was recently demonstrated. We examined the effect of this deletion upon LMP1 function in four human lymphoid cell lines by using three natural variants of LMP1: the prototype B95.8 gene and the CAO and AG876 genes, both of which have codons 343 to 352 of the B95.8-LMP1 deleted. These experiments revealed that LMP1-mediated upregulation of CD40 and CD54 was markedly impaired (by 60 to 90%) with CAO-LMP1 compared with B95.8-LMP1. In contrast, the function of AG876-LMP1 was indistinguishable from that of B95.8-LMP1 in two lines and was only slightly impaired in the other two lines. Activation of NF-κB by CAO-LMP1 was not impaired in any of the lines; rather, activation of an NF-κB reporter by CAO-LMP1 was consistently about twofold greater than the activation with B95.8- or AG876-LMP1. Therefore, while the CAO-LMP1 is functionally distinct from the prototype B95.8-LMP1 in human lymphocytes, the 10-amino-acid deletion appears not to be directly responsible. This conclusion was confirmed by using a B95.8-LMP1 mutant with codons 343 to 352 deleted and chimerae of CAO- and B95.8-LMP1 in which the CTAR-2 domains of these genes were exchanged. Sequences outside the CTAR-2 domain were implicated in the distinct functional characteristics of CAO-LMP1 in human lymphoid cells.