Comprehensive DNA microarray expression profiles of tumors in tenascin-C-knockout mice

Tenascin-C (TNC), an extracellular matrix glycoprotein, plays a pivotal role in tumor growth. However, the mechanism whereby TNC affects tumor biology remains unclear. To investigate the exact role of TNC in primary tumor growth, a mouse mammary tumor cell line, GLMT1, was first developed. Subsequently, global gene expression in GLMT1-derived tumors was compared between wild-type (WT) and TNC-knockout (TNKO) mice. Tumors in WT mice were significantly larger than those in TNKO mice. DNA microarray analysis revealed 447 up and 667 downregulated in the tumors inoculated into TNKO mice as compared to tumors in WT mice. Validation by quantitative gene expression analysis showed that Tnc, Cxcl1, Cxcl2, and Cxcr2 were significantly upregulated in WT mice. We hypothesize that TNC stimulates the CXCL1/2-CXCR2 pathway involved in cancer cell proliferation.     

Early development and neurogenesis of Temnopleurus reevesii

Sea urchins are model non‐chordate deuterostomes, and studying the nervous system of their embryos can aid in the understanding of the universal mechanisms of neurogenesis. However, despite the long history of sea urchin embryology research, the molecular mechanisms of their neurogenesis have not been well investigated, in part because neurons appear relatively late during embryogenesis. In this study, we used the species Temnopleurus reevesii as a new sea urchin model and investigated the detail of its development and neurogenesis during early embryogenesis. We found that the embryos of T. reevesii were tolerant of high temperatures and could be cultured successfully at 15–30°C during early embryogenesis. At 30°C, the embryos developed rapidly enough that the neurons appeared at just after 24 h. This is faster than the development of other model urchins, such as Hemicentrotus pulcherrimus or Strongylocentrotus purpuratus. In addition, the body of the embryo was highly transparent, allowing the details of the neural network to be easily captured by ordinary epifluorescent and confocal microscopy without any additional treatments. Because of its rapid development and high transparency during embryogenesis, T. reevesii may be a suitable sea urchin model for studying neurogenesis. Moreover, the males and females are easily distinguishable, and the style of early cleavages is intriguingly unusual, suggesting that this sea urchin might be a good candidate for addressing not only neurology but also cell and developmental biology.     

KIF13B enhances the endocytosis of LRP1 by recruiting LRP1 to caveolae

Multifunctional low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) recognizes and internalizes a large number of diverse ligands, including LDL and factor VIII. However, little is known about the regulation of LRP1 endocytosis. Here, we show that a microtubule-based motor protein, KIF13B, in an unexpected and unconventional function, enhances caveolin-dependent endocytosis of LRP1. KIF13B was highly expressed in the liver and was localized on the sinusoidal plasma membrane of hepatocytes. KIF13B knockout (KO) mice showed elevated levels of serum cholesterol and factor VIII, and KO MEFs showed decreased uptake of LDL. Exogenous KIF13B, initially localized on the plasma membrane with caveolae, was translocated to the vesicles in the cytoplasm with LRP1 and caveolin-1. KIF13B bound to hDLG1 and utrophin, which, in turn, bound to LRP1 and caveolae, respectively. These linkages were required for the KIF13B-enhanced endocytosis of LRP1. Thus, we propose that KIF13B, working as a scaffold, recruits LRP1 to caveolae via LRP1–hDLG1–KIF13B–utrophin–caveolae linkage and enhances the endocytosis of LRP1.     

Glycoprotein Ib distribution on the surface of platelets in resting and activation states: An electron microscope study

Summary Using a monoclonal antibody (TM60) against glycoprotein (GP) Ib, we determined immunocytochemically how GPIb is distributed on the platelet surface. When glutaraldehyde-fixed platelets were incubated with TM60, a uniform distribution of ferritin particles which represent the localization of GPIb was observed on the surface membrane of platelets. The particles were distributed at intervals of about 100 nm. The number of ferritin particles on the surface of one side were 2070–4150 (2940 ± 790; mean ±s.d.,n = 10) under the scanning electron microscope. The distribution of ferritin particles was somewhat disarranged on the surface of unfixed platelets incubated with TM60 compared to that in the fixed platelets. Cluster-like structures of ferritin particles were observed in several places. When platelets were activated with ristocetin or thrombin, the distribution of ferritin particles was disturbed and cluster formation was observed in several places on the surface. These findings suggest that GPIb is uniformly distributed on the surface of platelets in the resting state, and that cluster formation occurs during activation of platelets.     

Effect of carbon sources on the rates of cyclic AMP synthesis, excretion, and degradation, and the ability to produce beta-galactosidase in Escherichia coli.

We have determined the rates of adenosine 3',5'-cyclic monophosphate (cAMP) synthesis, excretion, and degradation, and the cAMP pool size in Escherichia coli grown on various carbon sources. We have found that the cAMP pool size increases in approximate proportion to increases in the cAMP synthetic rate. Although the combined rate of excretion and degradation of cAMP is in approximate proportion to the cAMP pool size, no such regular relationship is seen between the cAMP pool size and either the excretion rate or the degradation rate. Using a method which we have developed for determining the cellular efficiency of enzyme production (termed 'cellular' rate), we have reexamined the relationship between cAMP pool size and the rate of beta-galactosidase production. Although there exists an overall trend of increasing rate of beta-galactosidase production with increasing cAMP pool size, large variations in the rates of beta-galactosidase production are seen even under culture conditions which yield similar cAMP pool sizes. This suggests that the intracellular level of cAMP cannot be the unique regulator of beta-galactosidase production.     

In vivo preparation of [32P]cAMP and thin-layer chromatography of cAMP-related compounds.

A simple and rapid method for preparing [32P]adenosine 3'5'-cyclic monophosphate (cAMP) is described. A culture of an Escherichia coli mutant which excretes cAMP about 150 times faster than does a wild-type strain was incubated overnight with [32P]orthophosphate of high specific activity (e.g., 4000 Ci/mol (1 Ci = 37 GBq). The [32P]cAMP which accumulated extracellularly was then purified to 99.9% radiochemical purity in less than 4 h by adsorption to charcoal and alumina column chromatography. A two-dimensional chromatography system using a PEI-cellulose plate is also described which should prove useful for studying cAMP metabolism with 32P- or 3H-labeled cAMP or ATP.     

Meal feeding schedule and ventromedial hypothalamic lesions do not affect rhythm of pineal serotonin N-acetyltransferase activity in rats

Effects of meal feeding schedule and bilateral lesions of the ventromedial hypothalamus (VMH) on the circadian rhythm of pineal serotonin N-acetyltransferase (SNAT) activity were examined in rats, under LD (12:12) condition. Neither meal feeding nor VMH lesions affected the phase of the circadian rhythm of pineal SNAT activity, but the VMH lesions reduced the level. Meal feeding caused a shift of the phases of the daily rhythms of phosphoenolpyruvate carboxykinase and tyrosine aminotransferase activities in the liver. These findings suggest that the circadian rhythm of pineal SNAT activity is not entrained by the food intake, and that the VMH does not function as a master oscillator of the rhythm.