Increased expression of protein C-mannosylation in the aortic vessels of diabetic Zucker rats

C-Mannosylation is a novel type of glycosylation in proteins. There are several examples of proteins in which the specific motif Trp-X-X-Trp is mannosylated at the first Trp to produce C-mannosylated Trp (CMW). Although C-mannosylation modifies Trp-X-X-Trp, predicted to be a functional motif of various integral proteins such as cytokine receptors, the physiological or pathological relevance of C-mannosylation in the cell is still not known. In this study, to characterize C-mannosylation in biological samples, we generated specific polyclonal antibodies against CMW by using a chemically synthesized CMW as an antigen. Using the antibody, we investigated the effect of hyperglycemic conditions on protein C-mannosylation in cultured cells and diabetic Zucker fatty rats. We found that protein C-mannosylation was increased in macrophage-like RAW264.7 cells under hyperglycemic conditions compared to low-glucose conditions. Furthermore, C-mannosylation was increased in the aortic vessel wall of Zucker fatty rats. Thrombospondin-1 was identified as a protein modified with C-mannosylation, and its expression was also increased in the aortic tissues of Zucker fatty rats. These results indicate that C-mannosylation is increased in specific tissues or cell types under hyperglycemic conditions, suggesting a pathological role for the increased C-mannosylation in the development of diabetic complications.     

p73-dependent expression of DAN during cisplatin-induced cell death and osteoblast differentiation

We previously reported that DAN, a founding member of the DAN family of secreted proteins, acts as an inhibitor of cell cycle progression and is closely involved in retinoic acid-induced neuroblastoma differentiation. In this study, we found that DAN as well as p73, the recently identified p53 family member, was up-regulated during osteoblast differentiation. Additionally, the expression of DAN was increased in response to cisplatin-induced cell death of neuroblastoma SH-SY5Y cells. Consistent with the previous reports, p73 was accumulated after the treatment with cisplatin. Intriguingly, we found a putative p53/p73-binding site in the 5'-upstream region of the human DAN gene. A luciferase reporter assay and an in vitro DNA-binding experiment revealed that this canonical p53/p73-binding site was a functional responsive element and was specific for p73. Our results suggest that there exists a functional association between DAN and p73 during osteoblast differentiation as well as cisplatin-induced cell death.     

Nepsilon-(Carboxymethyl)lysine induces gamma-glutamylcysteine synthetase in RAW264.7 cells

Advanced glycation end products (AGEs) play an important role in the development of angiopathy in diabetes mellitus and atherosclerosis. Here, we show that adducts of N(epsilon)-(carboxymethyl)lysine (CML), a major AGE, and bovine serum albumin (CML-BSA) stimulated gamma-glutamylcysteine synthetase (gamma-GCS), which is a key enzyme of glutathione (GSH) synthesis, in RAW264.7 mouse macrophage-like cells. CML-BSA stimulated the expression of gamma-GCS heavy subunit (h) time- and dose-dependently and concomitantly increased GSH levels. CML-BSA also stimulated DNA-binding activity of activator protein-1 (AP-1) within 3h, but the stimulatory effect decreased in 5h, and nuclear factor-kappaB (NF-kappaB) with a peak activity at 1h and the stimulatory effect diminished in 3h. Studies of luciferase activity of the gamma-GCSh promoter showed that deletion and mutagenesis of the AP-1-site abolished CML-BSA-induced up-regulation, while that of NF-kappaB-site did not affect CML-BSA-induced activity. CML-BSA also stimulated the activity of protein kinase C, Ras/Raf-1, and MEK/ERK1/2. Inhibition of ERK1/2 abolished CML-BSA-stimulated AP-1 DNA-binding activity and gamma-GCSh mRNA expression. Our results suggest that induction of gamma-GCS by CML adducts seems to increase the defense potential of cells against oxidative stress produced during glycation processes.     

Fatal Pulmonary and Cerebellar Zygomycosis due to <Emphasis Type="Italic">Rhizomucor pusillus</Emphasis> in a Ringed Seal (<Emphasis Type="Italic">Pusa hispida</Emphasis>)

A 4-year-old captive ringed seal (Pusa hispida) was treated with subcutaneous antibacterial injections for pus exuding wounds in the skin and associated blubber following a bite attack. Three months after the incident, the animal presented nystagmus and died the following day. At necropsy, there was a 25?×?18?×?25 mm well-delineated, opaque nodular mass in the lung, besides the skin ulcers and localized areas of discoloration in the blubber correlating with the bite wound and injection sites. Histopathology of the pulmonary mass demonstrated severe eosinophilic inflammatory infiltration among numerous intralesional fungal hyphae. The hyphae were irregularly branched, broad and aseptate, consistent of zygomycosis. Magnetic resonance imaging was conducted on the head, which was initially frozen intact, revealing diffuse areas of hyperintensity in the cerebellum. Restricted histopathologic examination of the cerebellum showed severe granulomatous inflammation well spread within the neuroparenchyma, associated with abundant intralesional fungal hyphae similar to those appreciated in the pulmonary mass. Molecular analyses of the fungi in the pulmonary and cerebellar tissue identified the etiologic agent in both sites as Rhizomucor pusillus. The likely route of infection is through inhalation of R. pusillus spores or fragmented hyphae from the environment that developed into an initial pulmonary infection, becoming the source of hematogenous dissemination to the cerebellum. The skin and blubber lesions likely contributed to immunosuppression. Zygomycosis is uncommon in pinnipeds, and the present report emphasizes the importance of considering zygomycete dissemination even when the primary focus is highly confined.     

An Active Efflux System for Heavy Metals in Cisplatin-Resistant Human KB Carcinoma Cells

The mechanism for cisplatin resistance in cisplatin-resistant KCP-4 cells was studied. Although multidrug resistance-associated protein (MRP) was not detected in KCP-4 cells, the cells were more resistant to heavy metals than multidrug-resistant C-A120 cells that overexpressed MRP. KCP-4 cells expressed metallothionein, but it was scarcely involved in cisplatin resistance in these cells. KCP-4 cells did not express canalicular multispecific organic anion transporter (cMOAT). The glutathione(GSH) level was 4.7-fold higher in KCP-4 cells than in KB-3-1 cells. When the GSH level in KCP-4 cells was decreased by treating the cells with buthionine sulfoximine and nitrofurantoin, the accumulation of and sensitivity to cispaltin in the cells were increased. C-A120 cells were only 3.0-fold more resistant to cisplatin than KB-3-1 cells and this resistance was not affected by the increased glutathione level. The accumulation of platinum in C-A120 and KCP-4 cells was 68.5 and 20.4% of that in KB-3-1 cells, respectively, while the intracellular levels of antimony potassium tartrate in C-A120 and KCP-4 cells were 13.2 and 9.9% of that in KB-3-1 cells, respectively. The ATP-dependent efflux of antimony was enhanced in both C-A120 and KCP-4 cells. These results, taken together, suggest an efflux pump for heavy metals different from MRP and cMOAT is involved in cisplatin resistance in KCP-4 cells.     

Structural homogeneity of mitochondrial DNA in the mitochondrial nucleoid of Physarum polycephalum

Mitochondrial DNA (mtDNA) of Physarum polycephalum was isolated gently by CsCl centrifugation. The mtDNA was linear with molecular weights ranging from 25·10⁶ to 45·10⁶ and heterogeneous in size. Nevertheless, thermal transition profiles of the mtDNA suggested that this DNA fraction was more homogeneous than nuclear DNA. Exhaustive digestions of this DNA with restriction endonucleases yielded unique fragments, and then the total of their molecular weights of each digest was around 45·10⁶. This value is equivalent to the maximum molecular weight estimated using electron microscopy and electrophoresis. Moreover, EcoRI digests of the mtDNA fractionated by the sucrose gradient showed unequimolar quantities of large fragments and a high background between bands. These results suggest that the mtDNA of Physarum has a homogeneous base sequence, and that the size heterogeneity of the mtDNA is attributable to degradation of the DNA under isolation procedures. The mtDNA was cleaved by EcoRI and XhoI to yield 16 and 7 fragments, respectively. A physical map of these fragments was constructed using the routine mapping procedures. The physical map showed that the mitochondrial genome of Physarum was linear with a molecular weight of 45·10⁶. We concluded therefore that the mitochondrial nucleoid is a structure in which the homogeneous mtDNA is highly amplified.     

Rhinocerebral Zygomycosis Due to a Lichtheimia ramosa Infection in a Calf: Neural Spread Through the Olfactory Nerves

Mycopathologia - Here, we report a case of rhinocerebral zygomycosis due to a Lichtheimia ramosa infection in a calf. A histopathological examination revealed that a fungus had invaded the brain...     

Generation and characterization of a monoclonal antibody, mH1, raised against mitotic HeLa cells

Hybridoma cell lines were prepared from spleen cells of mouse immunized with mitotic HeLa cells. A monoclonal antibody (mH1), which intensively reacted with cleavage furrows of dividing HeLa cells in immunofluorescence, was obtained. In interphase, this antibody diffusely stained whole HeLa cells. Immunoelectron microscopy showed that mH1 antigens were localized at microvillus projections at the surface of dividing HeLa cells, but definite localization of that antigen was not observed in interphasic cells. Immunoblot analysis showed that mH1 is reactive to 42-kDa and 130-kDa components. Further, the 42-kDa component was identified as a gamma-actin homolog by N-terminal amino acid sequence analysis.