DNA polymerase activity in preimplantation mouse embryos.

DNA polymerase activity was measured in mouse embryos at stages before implantation to determine whether it increases in proportion to the amount of DNA synthesis, as it does in populations of differentiated mammalian cells, or remains constant, as it does in early sea urchin embryos. Total enzyme activity was found to be relatively unchanged following fertilization and in the first few cleavage stages. However, between the 12- and 120-cell (blastocyst) stage, the amount of activity increased by several-fold. These results indicate that the relationship between amount of DNA polymerase activity and DNA synthesis in mouse embryos exhibits two phases: in the early cleavage phase it is similar to that in sea urchin embryos, whereas, in the blastocyst phase, it is similar to that in differentiated mammalian cells.     

Protein kinase activity in mouse mammary carcinoma.

C3H mouse mammary carcinoma contains cyclic AMP-independent (C) and dependent (RC) protein kinases and a specific cyclic AMP-binding protein (R). The specific activities of C, RC and R are markedly lower in carcinoma than the normal mammary cells. Protein kinase preparation from neoplastic cells showed markedly higher ration of $\text{C}\text{RC}$ and lower responsiveness to cyclic AMP for the activation of the enzyme than the normal cells.     

Decreased lysyl oxidase activity in the aneurysm-prone, mottled mouse.

Inbred mice bearing certain alleles at the Mottled locus have defects in connective tissue which result in weakness of skin and of blood vessels. Previous studies have established that cross-links in collagen and elastin are decreased in these animals due to impaired formation of lysine-derived aldehydes. Lysyl oxidase activity in extracts of skin is markedly lower in those prepared from affected animals than control mice. An inhibitor of lysyl oxidase is present in equal amounts in affected and control skins and does not account for diminished activity found in affected animals.     

Metastasis-stimulating activity in the mouse uterus

Mouse uterine luminal proteins are thought to play important roles in inducing diapausing blastocysts to implant into the uterine wall. Employing a syngeneic teratocarcinoma cell line (402AX), we demonstrate that neoplastic cells are better able to invade and metastasize if they are coinjected with uterine fluid from pregnant or estrogen-primed mice. This metastasizing activity of uterine fluid was partially purified by using disc polyacrylamide electrophoresis and gel filtration chromatography. Preliminary experiments indicate that the post-albumin and albumin bands contain most of the bioactivity. Furthermore, these bands contain smaller molecular weight proteins (less than 14,000) than can be separated by detergent and mild acetic acid (0.1 N) treatment.     

Decrease in DNA methylase activity during preimplantation development in the mouse.

During early mouse development, there are large-scale changes in DNA methylation. These changes may be due to the availability or stability of the enzyme, DNA methyltransferase (methylase), which is responsible for maintenance of DNA methylation. A microassay for methylase activity in preimplantation embryos shows that the level of maternally inherited enzyme is extremely high in the egg and that this activity is stable for the first three cleavage divisions. However, from the 8-cell to the blastocyst stage, there is a marked and absolute decrease in enzyme activity.     

A single-base-pair substitution abolishes D-amino-acid oxidase activity in the mouse.

Mutant ddY/DAO- mice lacking D-amino-acid oxidase (DAO) activity were examined for the cause of their lack of enzyme activity. Total RNA was extracted from the kidney of the ddY/DAO- mice and cDNA was synthesized. After cDNA encoding DAO was amplified by the polymerase chain reaction it was cloned into a plasmid and sequenced. Comparison of the DAO cDNA sequence with that of normal BALB/c mice revealed the presence of a single-base substitution (G----A) which causes a Gly-181----Arg substitution in the middle of the enzyme molecule. The mutant DAO cDNA was inserted into an expression vector and was expressed in transfected COS-1 cells. The transfected cells synthesized the DAO mutant protein, but they did not show DAO activity. In contrast, when cells were transfected with an expression vector carrying wild-type DAO cDNA, where the substituted base-pair was replaced by a normal base-pair, they showed DAO activity. These results indicate that the single base-pair substitution is the cause of the loss of DAO activity in the ddY/DAO- mice.     

Proliferation-dependency of gamma-glutamyl hydrolase activity in various mouse cells.

The activity of gamma-glutamyl hydrolase, the enzyme which deglutamylates folyl and antifolyl polyglutamates, changed significantly in mouse cells during different phases of growth, being about two times lower in actively proliferating mice splenocytes and fibroblasts than in nondividing cells. In EAC cells growing in vivo the lowest activity was observed in cells in the logarythmic phase. Methotrexate treatment of mice in a dose of 500 mg/kg body weight increased the activity of the enzyme in EAC cells about 1.5 times. We suggest that gamma-glutamyl hydrolase is a proliferating dependent enzyme which together with folypolyglutamate synthetase ensures in cells an appropriate amount of folates in the form of polyglutamates necessary for optimizing folate-dependent biosynthetic activities.     

Ultrastructural demonstration of NAD-pyrophosphorylase activity in mouse liver nuclei.

Ultrastructural demonstration of NAD-pyrophosphorylase activity (E.C.2.7.7.1) in isolated mouse liver nuclei was investigated with the use of an electronhistochemical procedure based on the precipitation of pyrophosphate ions with lead ions under conditions permitting simultaneous ATPase inhibition by formaldehyde/ethanol prefixation. In isolated mouse liver nuclei activity of NAD-pyrophosphorylase was found in nucleoli, in interchromatin granules, coiled bodies and strand-like structures in nucleoplasm.     

The subcellular distribution of poly-A-degrading activity in mouse kidney.

Most (95%) of the poly-A-degrading activity of the mouse kidney was found in the cytoplasmic fraction and only 5% was found in the nuclear fraction; 43% of the poly-A-degrading activity of the cytoplasm was found in the mitochondria, 22% in the microsomes, and 30% in the soluble fraction. Differences in activity and specificity indicate that poly A is degraded in the nucleus by enzymes that are separate and distinct from the enzymes in the cytoplasm that degrade poly A. The nuclear poly-A-degrading activity can be separated into an endonuclease with a general specificity and exonuclease, similar to one found in Ehrlich ascites tumor cells, which shows some specificity for poly A.     

No clastogenic activity of a senna extract in the mouse micronucleus assay.

In previous studies, an analytically well-defined senna extract, commonly used as a laxative, gave positive responses in vitro in the Ames test and in the CHO assay. Therefore, the objective of this study was to investigate the genotoxic activity of the same senna extract in an in vivo genotoxicity assay by means of the generally acknowledged MNT. After administration of an oral dose of 2000 mg senna extract/kg to NMRI mice of both genders, which is equivalent to 119 mg potential rhein/kg, 5.74 mg potential aloeemodin/kg and 0. 28 mg potential emodin/kg, there were no elevated levels of micronuclei in bone marrow cells. Kinetic studies were performed in parallel to demonstrate target organ availability. Highest concentrations in the plasma were reached after 1 h with 3.4 microg rhein/ml and 0.065 microg aloeemodin/ml. In all cases, emodin was below the limit of quantification. From the results, the in vitro clastogenic activity of the senna extract could not be confirmed in the mouse micronucleus assay. Together with further negative in vivo genotoxicity studies with anthranoids, the conclusion can be drawn that there is no indication so far demonstrating a genotoxic risk for patients taking senna laxatives.     

Control of microtubule nucleating activity in the cytoplasm of maturing mouse oocytes.

Taxol, a drug which promotes microtubule assembly, was used to assess the microtubule nucleating activity of pericentriolar material (PCM) in mouse oocytes prevented from undergoing germinal vesicle breakdown (GVBD), compared with oocytes allowed to proceed normally through GVBD and also in nucleate and anucleate oocyte fragments. Both immunofluorescence staining and ultrastructural analysis reveal that taxol induces aster formation in the cortex of oocytes undergoing GVBD, while formation of a continuous sheet of microtubule bundles parallel to the membrane is induced in metabolically GV-arrested oocytes. Since taxol also induces the formation of asters in anucleate as well as in nucleate oocyte fragments, provided they are not treated with activators of protein kinases A or C, it is concluded that microtubule nucleating activity is related to the acquisition of Maturation Promoting Factor (MPF) and does not require mixing between the nucleoplasm and cytoplasm.     

Phosphofructokinase activity in the preimplantation mouse embryo

Summary Phosphofructokinase activity remains relatively constant during the preimplantation period in the mouse, with a low point at day 4 (approximately 3.0×1^–11 moles of substrate converted per embryo per hour).     

Mutagenic activity of 2-amino-N6-hydroxyadenine in the mouse spot test.

The base analogue 2-amino-N6-hydroxyadenine (AHA) was mutagenic in the spot test in (T x HT)F1 mouse embryos. Females were injected with single doses of 20 or 40 mg AHA per kg body weight on the 9th day of pregnancy. To rank the mutagenic potency of different compounds, the frequencies of genetically relevant spots induced by 1 mg/kg body weight were calculated. The observed somatic mutation frequency for 1 mg/kg AHA was lower (1.95 x 10(-3)) spots of genetic relevance) than that of mitomycin C (16 x 10(-3)), ethylnitrosourea (6.8 x 10(-3)) and cyclophosphamide (6.4 x 10(-3)) and therefore AHA was not classified as a very potent mutagen in this test system. The doubling dose to induce genetically relevant spots was calculated to be 20 mg/kg b.w. Based on these data, AHA is suggested to be a candidate to induce recessive specific-locus mutations in germ cells of mice.     

Prolonged antioestrogenic activity of ICI 46, 474 in the ovariectomized mouse.

Subcutaneous administration of ICI 46,474 (3 mg) to ovariectomized mice was found to produce vaginal refractoriness to subcutaneously administered oestradiol for up to 6 weeks. Tritiated oestradiol-17 beta accumulated in the uterus and vagina of the ovariectomized mouse, with maximum accumulation at 3 to 4 hr. This property of the target tissues was used to investigate the binding of tritiated oestradiol after the administration of 3 mg ICI 46,474 to ovariectomized mice. Radioactivity in the vagina was found to be comparable to control values 6 weeks after ICI 46,474 administration; uterine levels of radioactivity returned to control values by 10 weeks. Administration of ICI 46,474 had an oestrogenic effect upon the mouse uterus whereas the vagina appeared to be initially stimulated and was then unable to respond to or to bind oestradiol.     

Alkaline phosphatase activity of the mouse

Plasma alkaline phosphatase (PAP) activity of young adult CD mice is low (e.g. 1/10th of that of rat) and is lower in males than in females. The variability of PAP was assessed in groups of mice kept under different husbandry conditions (e.g. caging, diet, SPF). PAP activities of fed and fasted mice are similar. After comparing their acrylamide electrophoresis and the action of several inhibitors upon the PAP and the alkaline phosphatase of various organ extracts, it is suggested that the two isoenzymes of PAP (indicated by heat inhibition) probably arise from bone and liver.