Production of bovine identical twins via transfer of demi-embryos without zonae pellucidae

Embryos were collected nonsurgically from n?turally-cycling or superovulated donors 7 d after estrus. Forty-four morulae, early blastocysts and blastocysts classified as good to excellent were bisected using a fine glass needle to produce forty-four identical demi-embryos. The bisected demi-embryos, without zonae pellucidae, were nonsurgically transferred, either by twin or single transfer. An additional forty-eight embryos were collected from the same donors and transferred as a control. Among the twin transfers, 8 of the 13 recipients became pregnant (61.5%). Seven of them conceived twin fetuses (87%) and one a single fetus. However, only two sets of normal identical twin calves were obtained. Among the single transfers, 72.6% (45/62) of bisected embryos without zonae pellucidae resulted in pregnancy, of which 48.4% (30/62) were identical twins, and 24.2% (15/62) were singletons. Another 27.4% (17/62) of the recipients did not became pregnant. The pregnancy rate for whole embryos with zonae pellucidae was 72.9% (35/48). These data show that there was no significant difference between the pregnancy rates of bisected embryos without zonae pellucidae and whole embryos with zonae pellucidae transferred 7 d after estrus. Bisection of bovine embryos was simplified and even morula stage embryos were transferred without zonae pellucidae.     

Improved embryo yield and condition of donor ovaries in cows after PMSG superovulation with monoclonal anti-PMSG administered shortly after the preovulatory LH peak

Nonlactating Dutch-Friesian cows were selected from a local slaughterhouse and synchronized with Syncro-Mate B. Cows with a normal progesterone pattern were treated with PMSG (3,000 I.U. i.m.) on Day 10 followed by PG (Prosolvin 22.5 mg) 48 h later. Blood samples were collected daily and at hourly intervals from 30 h after PG. Monoclonal anti-PMSG (Neutra-PMSG) was administered i.v. at 5.8 h after the LH peak in 16 cows; controls (n = 16) did not receive Neutra-PMSG. For comparison, 16 additional cows were superovulated with FSH-P in decreasing doses, twice a day (total 32 mg), starting at Day 10. All cows were inseminated at 10 h after the LH peak. Embryos were evaluated on Days 6 and 7 after flushing upon slaughter (recovery 87%). The number of corpora lutea and follicles on the donor ovaries were counted. No significant differences in the concentrations of progesterone and LH were observed between the three superovulation groups. Upon Neutra-PMSG, PMSG in blood was completely neutralized, it was decreased to < 0.5 ug/l at AI from 7.0 ug/l at the LH peak. The number of transferable embryos was significantly higher after Neutra-PMSG (9.1 per cow) than without Neutra-PMSG (5.3). or upon FSH-superovulation (4.6). The number of cysts on the ovaries of Neutra-PMSG-treated cows was reduced similarly to that after FSH-superovulation. Treatment with Neutra-PMSG shortly after the LH peak positively affects final follicular maturation in PMSG-superovulated cows and results in a nearly two-fold increase of transferable embryos.     

Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.     

Rapid repression of quiescence-specific gene expression by epidermal growth factor, insulin, and pp60v-src.

We isolated a cDNA for p20K, a secreted protein preferentially synthesized in nonproliferating cells. p20K mRNA and protein levels declined rapidly following treatment with various mitogens. DNA sequence analysis of the p20K cDNA predicted a novel protein distantly related to alpha 2 mu-globulin and plasma retinol-binding protein.     

Fluorimetric coupled enzyme assay for lysoplasmalogenase activity in liver.

Two new proteins with apparent molecular masses of 53 kDa and 190 kDa have been identified in both sarcoplasmic reticulum and human blood platelets using a monoclonal antibody, FII1b5. The sarcoplasmic reticulum FII1b5 antigens were present in the terminal cisternae fraction, but were absent from light sarcoplasmic reticulum. The platelet and skeletal muscle proteins were not sensitive to digestion with endoglycosidase H under conditions that removed carbohydrate from the 53 kDa glycoprotein in sarcoplasmic reticulum or GPIIIa in platelet microsomes and did not bind 45Ca in a nitrocellulose overlay calcium-binding assay. These results distinguished the FII1b5 antigens from the 53 kDa glycoprotein and calsequestrin of sarcoplasmic reticulum. The 190 kDa platelet and sarcoplasmic reticulum proteins were extracted from membranes with high concentrations of NaCl, indicating that the high molecular mass FII1b5 antigens are peripherally associated with the bilayers. In contrast, the platelet and muscle 53 kDa proteins remained membrane-bound in the presence of high salt concentrations, suggesting that they are integral proteins.     

Effects of antibody to 5 S-RNA-binding protein on protein synthesis in Artemia salina ribosomes.

The effects of an affinity-purified polyclonal antibody to Artemia salina ribosomal protein L5 on protein synthesis in vitro were examined. The antibody interacted with 60 S subunits more strongly than with 80 S ribosomes, and inhibited reassociation of ribosomal subunits to some extent at 5 mM-Mg2+ but not at 10 mM. Polyphenylalanine synthesis in vitro at 10 mM-Mg2+ was significantly inhibited, especially when the antibody was first preincubated with 60 S subunits prior to the assay. The incorporation of amino acid directed by globin mRNA was inhibited only when the preincubation with 60 S subunits was carried out. On the other hand, no effect was detected on elongation factor 2- and 60 S subunit-dependent uncoupled GTPase activity. These results suggest that L5 is probably located at or near the subunit interface and may play an important role in protein synthesis.     

Spontaneous excretion of D-alanine in urine in mutant mice lacking D-amino-acid oxidase.

Urine from mutant mice lacking D-amino-acid oxidase contained a large amount of alanine compared with that from normal mice. Urinary alanine of the mutant mice was sensitive to D-amino-acid oxidase. H.p.l.c. showed that about 94% of the urinary alanine had the D-configuration. These results suggest that D-amino-acid oxidase functions to decompose D-amino acid(s) in normal mice.     

Regulation of mast cell differentiation

Mast cells are a unique class of blood cell. Unlike most blood cells, undifferentiated precursors of mast cells migrate in the bloodstream, invade tissues, proliferate there and then differentiate. Even after differentiation, some mast cells may proliferate extensively. Differentiation of mast cells is regulated by both diffusible growth factors and direct contact with fibroblasts.     

Autoregulation of interleukin 6 and granulocyte-macrophage colony-stimulating factor in the differentiation of myeloid leukemic cells.

Induction of differentiation in one type of clone of mouse myeloid leukemic cells by mouse or human interleukin 6 (IL-6) and in another type of clone by mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to be associated with induction of IL-6 and GM-CSF mRNA and protein. The results indicated that IL-6 and GM-CSF could positively autoregulate their own gene expression during myeloid cell differentiation. It is suggested that this autoregulation may serve to enhance and prolong the signal induced by these proteins in cells transiently exposed to IL-6 or GM-CSF.