Establishment of germline-competent embryonic stem cell lines from the MSM/Ms strain

MSM/Ms is an inbred mouse strain established from the Japanese wild mouse, Mus musculus molossinus, which has been phylogenetically distinct from common laboratory mouse strains for about 1 million years. The nucleotide substitution rate between MSM/Ms and C57BL/6 is estimated to be 0.96%. MSM/Ms mice display unique characteristics not observed in the commonly used laboratory strains, including an extremely low incidence of tumor development, high locomotor activity, and resistance to high-fat-diet-induced diabetes. Thus, functional genomic analyses using MSM/Ms should provide a powerful tool for the identification of novel phenotypes and gene functions. We report here the derivation of germline-competent embryonic stem (ES) cell lines from MSM/Ms blastocysts, allowing genetic manipulation of the M. m. molossinus genome. Fifteen blastocysts were cultured in ES cell medium and three ES lines, Mol/MSM-1, -2, and -3, were established. They were tested for germline competency by aggregation with ICR morulae and germline chimeras were obtained from all three lines. We also injected Mol/MSM-1 ES cells into blastocysts of ICR or C57BL/6 × BDF1 mice and found that blastocyst injection resulted in a higher production rate of chimeric mice than did aggregation. Furthermore, Mol/MSM-1 subclones electroporated with a gene trap vector were also highly efficient at producing germline chimeras using C57BL/6 × BDF1 blastocyst injection. This Mol/MSM-1 ES line should provide an excellent new tool allowing the genetic manipulation of the MSM/Ms genome.     

Thalidomide inhibits epidermal growth factor-induced cell growth in mouse and human monocytic leukemia cells via Ras inactivation

The effect of thalidomide on epidermal growth factor (EGF)-induced cell growth was examined. Thalidomide inhibited EGF-induced cell growth in mouse and human monocytic leukemia cells, RAW 264.7, U937 and THP-1. Thalidomide inhibited EGF-induced phosphorylation of extracellular signal regulated kinase (ERK) 1/2, but not p38 and stress-activated protein kinase (SAPK)/JNK. The phosphorylation of MEK1/2 and Raf at Ser 338 as the upstream molecules of ERK 1/2 was also prevented by thalidomide. Further, it inhibited EGF-induced Ras activation through preventing the transition to GTP-bound active Ras. Thalidomide inhibited the Ras activation induced by lipopolysaccharide (LPS) and vascular endothelial growth factor (VEGF) as well as EGF. There was no significant difference in the expression and function of EGF receptor between thalidomide-treated and non-treated cells. Therefore, thalidomide was suggested to inhibit EGF-induced cell growth via inactivation of Ras.     

Sugar Compositions, Intrinsic Viscosities and Molecular Weights of Hemicellulosic Polysaccharides of the Coleoptile Cell Walls in a Semi-Brachytic and a Normal Type Barley

The sugar compositions and intrinsic viscosities of the hemicellulosicpolysaccharides of the coleoptile cell wall were determinedin a normal type barley and a semi-brachytic type which producedless IAA than the normal type. The major sugar components ofhemicelluloses for both strains were arabinose (Ara), xylose(Xyl) and glucose (Glc). The Ara and Xyl content per unit lengthin the normal type did not change during growth, while thosein the semi-brachytic type decreased during growth. The Glccontents per unit length and per coleoptile decreased duringgrowth in both types of barley. The intrinsic viscosity of hemicellulosesfrom the coleoptile of the normal type was lower than that ofthe semi-brachytic type. These results suggested that the synthesis of arabinoxylan keptpace with the growth of the coleoptile in the normal type butnot in the semi-brachytic type, and that the average mol wtof the hemicelluloses in the normal type was lower than thatin the semibrachytic type. These chemical and physical changesin the hemicellulosic polysaccharides may account for the stuntedcoleoptile of the semi-brachytic barley with its less amountof endogenous IAA. (Received March 14, 1984; Accepted June 8, 1984)     

Response of hepatic function to hepatic copper deposition in rats fed a diet containing copper

Fischer rats were a fed diet supplied with copper chloride (150–600 ppm) for 60 d from weaning. Serum (glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) activities were increased with the increase of Cu concentration in the diet. Biliary excretion of Cu was related to the dietary Cu level. Depositions of hepatic and renal Cu were also related to the dietary Cu level in a dose-dependent manner. In particular, hepatic (155.2±13.3 μg/g) and renal (44.9±4.4 μg/g) Cu concentrations increased abruptly in the Cu-600 ppm group. In the liver, about 60% of Cu was distributed in the soluble fraction (100,000 g supernatant). In the Cu-600 ppm group, 25% of cystosolic Cu was bound to metallothionein (MT). Our results suggest that chronic exposure to Cu appears to have a deleterious effect on the hepatic function, and further, that even in rats with normal biliary Cu excretion, clearance of Cu from the liver may be marginal when dietary Cu is near the 600-ppm level. Although Cu is an essential nutrient, an overload of Cu should be avoided.     

Ester Formation by Alcohol Acetyltransferase from Brewers’ Yeast

Alcohol acetyltransferase responsible for the formation of acetate esters during beer fermentation was found to be localized at the cell membrane of brewers’ yeast. This cell membrane-bound enzyme was purified 120-fold by solubilization with Triton X-100, gel filtration on a Sepharose 6B column and chromatography on a DEAE-Sephadex A-50 column. The enzyme was most active at 30°C at pH 7 ? 8. It was least active against C₃ alcohol among C₁ ? C₆ alcohols, and slightly more active against straight-chain alcohols than against branched-chain alcohols with the same carbon number. The enzyme was strongly inhibited by unsaturated fatty acids, heavy metal ions and sulfhydryl reagents.     

Immunocytochemical localization of secretory acetylcholinesterase of the parasitic nematode Nippostrongylus brasiliensis

Various parasitic nematodes secrete acetylcholinesterase (AChE). In this study, the localization of AChE in the nematode Nippostrongylus brasiliensis and the secretory forms of AChE in culture fluid were examined. A thiocholine method revealed that AChE activity was localized in the subventral glands, which have a secretory and excretory function via a duct connected to the excretory pore. By electron microscopy, AChE activity was found mainly in the matrix of secretory granules, and sometimes in the Golgi apparatus in the subventral gland cells. These results show that nematode AChE is produced and stored in the subventral glands. Monoclonal antibodies against AChE of human erythrocytes or electric rays also bound to the nematode subventral gland, suggesting immuno-cross-reactivity of AChE among these species. When AChE activity in the nematode excretory-secretory product was examined by SDS polyacrylamide gel electrophoresis combined with the thiocholine method, intense activity was demonstrated as a single band at 74kDa. Immunoblot analysis showed specific recognition of this molecule by IgE and IgG1 antibodies, but not by IgG2a antibody, in nematode-infected rat sera. These results indicate that the nematode AChE molecule produced in and secreted from the subventral glands is antigenic for the production of IgE/IgG1 in host animals.