Reduced DNase I sensitivity of the rearranged c-myc gene in somatic cell hybrids between murine plasmacytoma cells and fibroblasts

In mouse plasmacytoma (MPC) S194, the rearranged c-myc gene was much more sensitive to DNase I digestion than the nonrearranged gene. The sensitivity of the rearranged c-myc was markedly reduced to the same extent as that of the nonrearranged one in hybrids between the MPC cells and the fibroblasts, but not in a hybrid between the MPC and the spleen cells. These results suggest that trans-acting factors in fibroblasts alter the DNase I-sensitive structure of the rearranged c-myc gene.     

Specific induction of metallothionein synthesis by mitochondrial oxidative stress.

Metallothionein (MT), a sulfhydryl-rich protein, may be increased by administration of a variety of agents, including metals, cytokines and oxidative stress agents. Mitochondria are a major source of reactive oxygen species, but antioxidant systems against mitochondrial free radicals are not fully understood. In this study, we examined the induction of MT synthesis by administration of mitochondrial-specific reactive oxygen generators such as antimycin A (AA), an electron transfer inhibitor, and 2,4-dinitrophenol (DNP), an uncoupling agent. Subcutaneous administration of AA to mice significantly increased the hepatic MT concentration in a dose- and time-dependent manner. AA slightly elevated glutathione peroxidase (GSHPx) activity, but the rate of increase in GSHPx (1.3-fold) was smaller than that in MT (11.8-fold). Other antioxidants such as catalase, manganese-superoxide dismutase (Mn-SOD), copper/zinc-superoxide dismutase (Cu/Zn-SOD) and GSHPx were not activated by AA treatment. Moreover, administration of DNP induced the synthesis of MT in the liver. Although DNP slightly elevated Mn-SOD activity, the rate of increase in Mn-SOD (1.3-fold) was smaller than that in MT (3.7-fold). Other antioxidants such as catalase, Cu/Zn-SOD and GSHPx were not activated by DNP treatment. These data suggest that MT plays a major role in protection against oxidative stress induced in mitochondria.     

Requirement of N-myc protein down-regulation for neuronal differentiation in the spinal cord

Previous work has indicated that N-myc expression occurs widely in the developing central nervous system, but its level changes dynamically with region- and stage-specificities. We show in the present report that in the developing spinal cord of the mouse, N-myc protein expression takes place in the ventricular zone and reaches its maximum at the outermost layer, but is extinct in the intermediate zone, indicating that N-myc protein is not expressed in mature neurons. We examined the effect of forced, persistent N-myc expression in development of the spinal cord in order to understand the functional significance of N-myc down-regulation. We made embryonic stem (ES) cell lines that constitutively expressed N-myc at a high level, then produced mouse embryo chimeras with a high contribution of the ES cells. The majority of the chimeras developed to day 12 with normal gross morphology, but in these chimeras neuronal differentiation in the spinal cord was perturbed at the histological level. Intermediate zones and ventral horns were formed, but the expression of N-CAM and neurofilaments was diminished. Chimeras using β-galactosidase-expressing recipient embryos indicated that inhibition of the neuronal differentiation was a cell-autonomous effect of persistent N-myc expression. These observations indicate that N-myc down-regulation in individual cells is required for full differentiation of neurons.     

Hemolytic C-type lectin CEL-III from sea cucumber expressed in transgenic mosquitoes impairs malaria parasite development

The midgut environment of anopheline mosquitoes plays an important role in the development of the malaria parasite. Using genetic manipulation of anopheline mosquitoes to change the environment in the mosquito midgut may inhibit development of the malaria parasite, thus blocking malaria transmission. Here we generate transgenic Anopheles stephensi mosquitoes that express the C-type lectin CEL-III from the sea cucumber, Cucumaria echinata, in a midgut-specific manner. CEL-III has strong and rapid hemolytic activity toward human and rat erythrocytes in the presence of serum. Importantly, CEL-III binds to ookinetes, leading to strong inhibition of ookinete formation in vitro with an IC(50) of 15 nM. Thus, CEL-III exhibits not only hemolytic activity but also cytotoxicity toward ookinetes. In these transgenic mosquitoes, sporogonic development of Plasmodium berghei is severely impaired. Moderate, but significant inhibition was found against Plasmodium falciparum. To our knowledge, this is the first demonstration of stably engineered anophelines that affect the Plasmodium transmission dynamics of human malaria. Although our laboratory-based research does not have immediate applications to block natural malaria transmission, these findings have significant implications for the generation of refractory mosquitoes to all species of human Plasmodium and elucidation of mosquito-parasite interactions.     

Regulation of nuclear translocation of extracellular signal-regulated kinase 5 by active nuclear import and export mechanisms

Extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinase family, plays an important role in growth factor signaling to the nucleus. However, molecular mechanisms regulating subcellular localization of ERK5 have remained unclear. Here, we show that nucleocytoplasmic shuttling of ERK5 is regulated by a bipartite nuclear localization signal-dependent nuclear import mechanism and a CRM1-dependent nuclear export mechanism. Our results show that the N-terminal half of ERK5 binds to the C-terminal half and that this binding is necessary for nuclear export of ERK5. They further show that the activating phosphorylation of ERK5 by MEK5 results in the dissociation of the binding between the N- and C-terminal halves and thus inhibits nuclear export of ERK5, causing its nuclear import. These results reveal the mechanism by which the activating phosphorylation of ERK5 induces its nuclear import and suggest a novel example of a phosphorylation-dependent control mechanism for nucleocytoplasmic shuttling of proteins.     

Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells

Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, β-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.     

Testicular Angiotensin-converting enzyme with different glycan modification: characterization on glycosylphosphatidylinositol-anchored protein releasing and dipeptidase activities

We have previously found that the angiotensin-converting enzyme (ACE) carries GPI-anchored protein releasing activity (GPIase) as well as dipeptidase activity. Testicular ACE (tACE), the male germinal specific isozyme, plays a crucial role in male fertilization. The amino-terminal region of this isozyme is different from that of somatic isozyme (sACE) and contains potential O-linked glycosylation sites. By multiple mutagenesis after an in silico prediction, amino acid residues acquiring O-glycans were assigned. Both GPIase and dipeptidase activities were compared between O-glycan null mutant and wild-type molecules, but no differences were found. Furthermore, the wild-type tACE was produced in two different cells (COS7 and CHO) and its activities compared. The GPIase activity, but not dipeptidase, was apparently higher for CHO-derived molecule than COS7. Sensitivity to neuraminidase and O-glycosidase digestions and the profile of glycosylation were quite different between these two molecules. Moreover, serial digestions with neuraminidase and O-glycosidase have no influence on GPIase activity of both molecules, suggesting that the sialylation and the presence of O-glycan has no influence on tACE enzyme activities, while the set of glycans modulate GPIase activity.     

Glycan-binding profile of a D-galactose binding lectin purified from the annelid, Perinereis nuntia ver. vallata

A lectin recognizing D-galactose was purified from the pacific annelid Perinereis nuntia ver. vallata (Polychaeta) by affinity chromatography. Hemagglutinating activity, with a very low titer suggesting the presence of lectin appeared in the supernatant from the homogenization of body with Tris-buffered saline. However, dialyzed supernatant from the precipitate homogenized by galactose in the buffer revealed strong hemagglutinating activity against human erythrocytes. The crude supernatant was applied onto lactosyl–agarose column, and only the supernatant eluted from precipitate with galactose was obtained a galactose-binding lectin with 32 kDa polypeptide was obtained from the supernatant of the precipitate, extracted in presence of galactose. It suggests that the lectin tightly binds with glycoconjugate as endogenous ligand(s) in the tissue. Hemagglutinating activity against trypsinized and glutaraldehyde-fixed human erythrocytes was specifically inhibited by D-galactose, N-acetyl-D-galactosamine, lactose, melibiose, and asialofetuin. Glycan-binding profile of the lectin analyzed by frontal affinity chromatography shows that the lectin recognizes branched complex type N-linked oligosaccharides and both type 1 (Galβ1-3GlcNAc) and type 2 (Galβ1-4GlcNAc) lactosamine. The surface plasmon resonance study of the lectin against asialofetuin showed the k_ass and k_diss values are 5.14 × 10⁴ M^− 1 s^− 1 and 2.9 × 10⁻³ s^− 1, respectively. The partial primary structure of the lectin reveals 182 amino acids with novel sequence.