Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells

Cells were isolated from stage X embryos of a line of Barred Plymouth Rock chickens (that have black pigment in their feathers due to the recessive allele at the I locus) and injected into the subgerminal cavity of embryos from an inbred line of Dwarf White Leghorns (that have white feathers due to the dominant allele at the I locus). Of 53 Dwarf White Leghorn embryos that were injected with Barred Plymouth Rock blastodermal cells, 6 (11.3%) were phenotypically chimeric with respect to feather colour and one (a male) survived to hatching. The distribution of black feathers in the recipients was variable and not limited to a particular region although, in all but one case, the donor cell lineage was evident in the head. The male somatic chimera was mated to several Barred Plymouth Rock hens to determine the extent to which donor cells had been incorporated into his testes. Of 719 chicks hatched from these matings, 2 were phenotypically Barred Plymouth Rocks demonstrating that cells capable of incorporation into the germline had been transferred. Fingerprints of the blood and sperm DNA from the germline chimera indicated that both of these tissues were different from those of the inbred line of Dwarf White Leghorns. Bands that were present in fingerprints of blood DNA from the chimera and not present in those of the Dwarf White Leghorns were observed in those of the Barred Plymouth Rocks.(ABSTRACT TRUNCATED AT 250 WORDS)     

The fate of female donor blastodermal cells in male chimeric chickens.

In previous experiments in our laboratories, chickens that are chimeric in their gamete, melanocyte, and blood cell populations have been produced by injection of dispersed stage X blastodermal donor cells into the subgerminal cavity of stage X recipient embryos. In some experiments, donor cells were transfected with reporter gene constructs prior to injection as a preliminary step in the production of transgenic birds. Chimerism was assessed by test mating, observation of plumage, and DNA fingerprinting. Methods were sought that would provide a relatively rapid analysis of the spatial distribution of descendants of donor cells in chimeras to assess the efficacy of various methods of chimera construction. To date, the sex of donor and recipient embryos was not known and, therefore, numerous mixed sex chimeras must have been constructed by chance, since donor cells were usually collected from several embryos rather than from individual embryos. The presence of female-derived cells was determined by in situ hybridization using a W-chromosome-specific DNA probe, using smears of washed erythrocytes from 16 phenotypically male chimeric chickens ranging in age from 4 days to 42 months posthatching. The proportion of female cells detected in the erythrocyte samples was zero (eight samples) or very low (0.020-0.083%), although 1% of the erythrocytes from a phenotypically male chick that was killed 4 days after hatch were female-derived. The low proportions of female-derived cells were surprising, considering that most of these chimeras had been produced by the injection of cells pooled from several donor embryos and most recipients had been exposed to gamma irradiation prior to injection, thus dramatically enhancing the level of incorporation of donor cells into the resulting chimeras. By contrast, 0-100% of the erythrocytes were female-derived in blood samples taken at 10 days of incubation from the chorioallantois of seven phenotypically normal male embryos that resulted from the injection of blastodermal cells pooled from five embryos into irradiated recipient embryos. Approximately 70% of the erythrocytes in a blood sample from a phenotypically normal female chimeric embryo were female-derived, and 100% of the erythrocytes examined from an intersex embryo bearing a right testis and a left ovary were female-derived. These results indicate that female-derived cells can contribute to the formation of erythropoietic tissue during the early development of what will become a phenotypically male chimeric embryo. It would appear, therefore, that female-derived cells are blocked in development or destroyed, or certain male-female combinations of cells may be lethal prior to hatching.(ABSTRACT TRUNCATED AT 400 WORDS)     

Absence of oxytocin-neurophysin messenger RNA in the day-18 bovine conceptus

Total cellular RNA was isolated from conceptus tissue obtained from 22 superovulated cows 18 days after artificial insemination. Total RNA was also isolated from luteal tissue from 3 cyclic cows 7 and 8 days after oestrus. Luteal and conceptus RNA were simultaneously subjected to formaldehyde-agarose gel electrophoresis and transferred to nitrocellulose by bidirectional diffusion blotting. Northern blots were probed using cDNAs specific for bovine oxytocin and bovine beta-actin gene sequences. Hybridization of the oxytocin cDNA to RNA was consistently observed on autoradiographs as a 0.6 kilobase (kb) band in lanes containing corpus luteum RNA, but was not detected in lanes containing conceptus RNA. The presence of conceptus RNA on the blots was confirmed by hybridization of the actin cDNA to conceptus RNA, which resulted in a 2.0 kb band on autoradiographs. These results suggest that oxytocin is not synthesized by the bovine conceptus on Day 18 of gestation.     

Cellulolytic Activity of Clostridium acetobutylicum

Clostridium acetobutylicum NRRL B527 and ATCC 824 exhibited extracellular and cell-bound endoglucanase and cellobiase activities during growth in a chemically defined medium with cellobiose as the sole source of carbohydrate. For both strains, the endoglucanase was found to be mainly extracellular (70 to 90%) during growth in continuous or batch cultures with the pH maintained at 5.2, whereas the cellobiase was mainly cell associated (60 to 90%). During continuous cultivation of strain B527 with cellobiose as the limiting nutrient, maximum production of the endoglucanase and cellobiase occurred at pH values of 5.2 and 4.8, respectively. In the carbon-limited continuous cultures, strain 824 produced similar levels of endoglucanase, cellobiosidase, and cellobiase activities regardless of the carbon source used. However, in ammonium- or phosphate-limited cultures, with an excess of glucose, only 1/10 of the endoglucanase was produced, and neither cellobiosidase nor cellobiase activities were detectable. A crude extracellular enzyme preparation from strain B527 hydrolyzed carboxymethylcellulose and phosphoric acid-swollen cellulose readily and microcrystalline cellulose (A vicel) to a lesser extent. Glucose accounted for more than 90% of the reducing sugar produced by the hydrolysis of acid-swollen cellulose and Avicel. Strain B527 did not grow in medium with acid-swollen cellulose as the sole source of carbohydrate, although it grew readily on the products obtained by hydrolyzing the cellulose in vitro with a preparation of extracellular cellulase derived from the same organism.     

Evidence that noradrenaline increases pyruvate dehydrogenase activity and decreases acetyl-CoA carboxylase activity in rat interscapular brown adipose tissue in vivo.

The rate of fatty acid synthesis in interscapular brown adipose tissue of female cold-adapted rats, as measured by the incorporation of 3H from 3H2O into tissue lipid, was decreased by about 70% after injection of noradrenaline. There was a similar decrease in the activity of acetyl-CoA carboxylase. In contrast, the proportion of pyruvate dehydrogenase in its active non-phosphorylated form was greatly increased after injection of noradrenaline. This finding suggests that the oxidation of glucose may be important in noradrenaline-induced thermogenesis in rat brown adipose tissue.     

The distribution and ultrastructure of sensory elements in the baroreceptor region of the truncus arteriosus of the lizard Trachydosaums rugosus

Summary The proximal truncus arteriosus of the lizard Trachydosaurus rugosus was studied with light-, fluorescence and electron-microscopical techniques. Three vessels comprised the truncus: the pulmonary, left aortic, and caroticoaortic arteries. Right and left truncal nerves, each derived from the ipsilateral vagus nerve, innervated the truncus, particularly its proximal 3 mm.Ultrastructurally, the nerves had a variety of appearances: some were clearly adrenergic, c-type or p-type. A number of profiles contained large numbers of mitochondria and were classified as sensory. Some profiles defied exact classification, having characteristics common to two different types of profile.Within the outer medial layers, profiles up to 7 m in diameter were found. These contained large numbers of mitochondria, myelin bodies and structures intermediate between the two. In addition, the profiles contained large amounts of glycogen and small numbers of vesicles. These nerve fibres were classified as baroreceptors, since they closely resemble carotid sinus and aortic arch baroreceptors in mammals.Large numbers of chromaffin cells were found, particularly in the common wall of the pulmonary and left aortic arteries. Many of these cells emitted a long tapering process, which sometimes entered a nearby nerve bundle. Sensory, p-type and c-type profiles, but not adrenergic profiles, made extensive close contacts with chromaffin cells.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Partial purification and properties of a beta-glucosidase from Erwinia herbicola Y46.

A constitutive beta-glucosidase of Erwinia herbicola Y46 was studied as a prerequisite to an assessment of its significance in the release of bacteriotoxic aglycones from plant beta-glucosides, and the possible effects of the aglycones on the course of such plant diseases as "fire-blight". The enzyme was purified 86.5-fold from crude extracts of cells grown on yeast beef broth. Ammonium sulfate precipitation, DEAE-cellulose fractionation, and gel filtration through Sephadex G-100 resulted in a preparation having one peak of activity on isoelectrofocussing, on gel filtration through Sephadex G-200, and on polyacrylamide gel electrophoresis. The latter techniques demonstrated, in addition to the major protein band associated with activity, a single minor impurity. The enzyme was active against p-nitrophenyl-beta-glucoside (p-NPG) and phloridzin, but showed only very slight activity against salicin and arbutin, and no detectable activity against beta-methyl-D-glucoside, cellobiose, lactose, and esculin. The production of beta-glucosidase was maximum at the late log phase of growth on yeast beef broth medium and declined somewhat thereafter. The incorporation of inducers (carbohydrates) in defined basal medium resulted in only small variations in specific activity in the resulting cells; The activity (p-NPG substrate) was not inhibited by D-glucose, phloretin, esculin, salicin, arbutin, lactose, or cellobiose, but was slightly inhibited by 1.0 mM phloridzin. Slight inhibition was observed in the presence of sulfhydryl reagents (iodoacetamide, p-chloromercuribenzoate), but sodium azide, ethylene-diaminetetraacetic acid, Cu2+, and Zn2+ ions produced no effect. The activity was stable, in both crude and purified preparations, over the pH ranges 6.0-7.5 (100% activity) and 4.5-greater than 8.5 (50% activity). The enzyme retained 80% activity after 30 min at 50 degrees C, but only 25% after 30 min at 60 degrees C. The enzyme had a mean K-m value (phloridzin) of 1.35 times 10-4 M, an isoelectric point of 4.75, a molecular weight, determined by Sephadex G-200 gel filtration, of about 122 000, and an optimum pH for activity of 6.5-7.0.