The effect of calcium ion concentration on osteoblast viability, proliferation and differentiation in monolayer and 3D culture

Our research group aims to develop an osteochondral composite using type II collagen gel with hydroxyapatite (HAp) deposited on one side. Soaking gels in Ca2+ and phosphate solution is indispensable to HAp deposition, so relationships between cell behavior and Ca2+ concentration were examined in two- and three-dimensional cultures. The present results indicate that 2-4 mM Ca2+ is suitable for proliferation and survival of osteoblasts, whereas slightly higher concentrations (6-8 mM) favor osteoblast differentiation and matrix mineralization in both 2- and 3-dimensional cultures. Higher concentrations (>10 mM) are cytotoxic. Purely from the perspective of calcium deposition, higher concentrations lead to increased accumulation of Ca2+. Culturing cells in phosphate-containing gel in media with Ca2+ also leads to time-dependent formation of HAp in the gel. Considering the viability of embedded cells, culturing scaffolds in media with Ca2+ concentrations around 5mM is useful for both HAp deposition and osteoblast behavior.     

Immunohistochemical localization of glomerular basement membrane antigens in various renal diseases

Summary The immunofluorescent localization of glomerular basement membrane (GBM) antigens was examined in 52 specimens from normal kidneys and in various renal diseases using antisera to human GBM HGBM), IV type collagen (IV Col) and P3 antigen, a rat nephritogen. Anti-HGBM serum normally stained the GBM and the mesangium in a restrictive pattern, anti-IV Col serum stained the GBM and the mesangium in a wider pattern and anti-P3 serum stained only the GBM. In mesangial proliferative glomerulonephritis, including IgA nephropathy pathy and Henoch-Schönlein nephritis, the widened mesangial areas were stained with anti-HGBM and anti-IV Col sera. In membranous nephropathy, the punched-out lesions of thickened GBM were demonstrated with the three antisera in moderate cases and a double linear distribution with fine granulation with anti-HGBM and anti-IV Col sera were revealed in one severe case. In membranoproliferative glomerulonephritis, the expanded mesangium and thickened capillary walls were stained with anti-HGBM and anti-IV Col sera, while the outer line of glomerular capillary walls was only positive with anti-P3 serum. In crescentic glomerulonephritis, the collapsed glomerular tufts were stained normally with anti-HGBM and anti-P3 sera and weakly with anti-IV Col serum. In diabetic nephropathy, anti-HGBM serum stained the GBM in a double linear distribution without reacting with the expanded mesangium; anti-IV Col serum stained the mesangium and the GBM in a less clear double linear fashion while anti-P3 serum stained the GBM as single line. Thin membrane disease and Alport's syndrome had normal reactivity with all antisera. However, in one case of Alport's syndrome anti-HGBM and anti-P3 sera stained the GBM in a focal and segmental pattern, while normal staining with anti-IV Col serum was found. In lesions with adhesions and crescents the staining was positive for HGBM and IV Col and negative for P3; obsolescent glomeruli were stained with anti-HGBM and anti-P3 sera, and had diminished staining with anti-IV Col serum.The identification of the various structural glomerular antigens is useful in the classification of certain types of glomerular diseases. Further insight into the mechanisms underlying these conditions may be obtained in this way.     

Recruitment of katanin p60 by phosphorylated NDEL1, an LIS1 interacting protein, is essential for mitotic cell division and neuronal migration

LIS1 is mutated in the human neuronal migration defect lissencephaly and along with NDEL1 (formerly NUDEL) participates in the regulation of cytoplasmic dynein function during neuronal development. Targeted disruption of Ndel1 suggested that NDEL1 could have other molecular targets that regulate microtubule organization for proper neuronal migration. To further understanding the molecular mechanism of LIS1 and lissencephaly, we identified the katanin p60 microtubule-severing protein as an additional molecular target of NDEL1. We demonstrate that phosphorylation of NDEL1 by Cdk5 facilitates interaction between NDEL1 and p60, suggesting that P-NDEL1 regulates the distribution of katanin p60. Abnormal accumulation of p60 in nucleus of Ndel1 null mutants supports an essential role of NDEL1 in p60 regulation. Complete loss of NDEL1 or expression of dominant negative mutants of p60 in migrating neurons results in defective migration and elongation of nuclear-centrosomal distance. Our results suggest that NDEL1 is essential for mitotic cell division and neuronal migration not only via regulation of cytoplasmic dynein function but also by modulation of katanin p60 localization and function.     

Polyethylene/phospholipid polymer alloy as an alternative to poly(vinylchloride)-based materials

To develop new biomaterials for making medical devices, polymer alloys composed of a phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), and polyethylene (PE) were prepared. The PE/PMPC alloy membrane could be obtained by a combination of solution mixing and solvent evaporation methods using xylene and n-butanol mixture as a solvent. Moreover, thermal treatment was applied to improve the mechanical properties of the PE/PMPC alloy membrane. In the PE/PMPC alloy membrane, the PMPC domains were located not only inside the membrane but also at the surface. Surface analysis of the PE/PMPC alloy membrane with X-ray photoelectron spectroscopy, wettability evaluation, and dynamic contact angle measurements revealed that the phospholipid polar groups in the PMPC covered the surface even after thermal treatment. Blood compatibility tests with attention to platelet adhesion and change in morphology of adhered platelets showed that the PE/PMPC alloy membrane had excellent platelet adhesion resistance. We finally concluded that the PE/PMPC alloy could be used as biomaterials instead of poly(vinyl chloride)-based materials.     

Effects of non-MHC background genes on the induction of CD4+ T cells that prevent rejection of a highly immunogenic tumor, FBL-3

This study showed that non-MHC genes common to (DBA/2 H-2^d)and (DBA/1 H-2^q) gave rise to suppressor T (T_a) cells in thehybrid F₁ mice between C57BL/6 (B6) strain in the antl-FBL-3tumor responses. FBL-3, a Friend virus-induced tumor cell lineof B6 mouse origin, is highly immunogenic as shown by findingsthat syngenelc and hybrid F₁ mice with several other inbredstrains rejected up to 3 x 10⁷ tumor cells inoculated s.c. andgenerated potent CTL responses after mixed lymphocyte tumorcell culture. In contrast to these mice, (B6 x DBA/2) and (B6x DBA/1)F1 mice did not reject the tumor as the tumor dosesincreased. Progressive tumor growth in these F₁ mice was blockedby an I.p. Injection of cyclophosphamlde (250 mg/kg) on day10, but not on day 5, after tumor cell inoculation. Antl-CD4(GK1.5) mAb exerted similar therapeutic effects against tumorwhen given twice, between day 0 and 10, whereas the additionalinjection of antl-CD8 mAb enhanced the tumor growth in micethat otherwise rejected the tumor. Thus, In the response of(B6 x DBA/2)F, mice to FBL-3 tumor cells, CD4⁺ T₈ seemed todown-regulate the immunologically mediated regression of thetumor produced by CD8⁺ CTL. This was evidenced by limiting dilutionculture analyses, which showed that the frequency of an FBL-3-speclflcCTL precursor in the (B6 x DBA/2)F1 mice that rejected the tumorwith antl-CD4 mAb was 7- to 9-fold higher than that in micein which the tumor regressed spontaneously. That more than onegene was involved in suppressor T cell induction was shown bythe tumor growth pattern in (B6 x DBA/2)F₁ x B6 backcross andB6D2F2 mice.     

Caenorhabditis elegans Rab escort protein (REP‐1) differently regulates each Rab protein function and localization in a tissue‐dependent manner

Rab proteins play a critical role in intracellular vesicle trafficking and require post‐translational modification by adding lipids at the C‐terminus for proper functions. This modification is preceded by the formation of a trimeric protein complex with the Rab escort protein (REP) and the Rab geranylgeranyltransferase (RabGGTase). However, the genetic hierarchy among these proteins and the tissue‐specificity of each protein function are not yet clearly understood. Here we identified the Caenorhabditis elegans rep‐1 gene and found that a rep‐1 mutant showed a mild defect in synaptic transmission and defecation behaviors. Genetic analyses using the exocytic Rab mutants rab‐3 or rab‐27 suggested that rep‐1 functions only in the RAB‐27 pathway, and not in the RAB‐3 pathway, for synaptic transmission at neuromuscular junctions. However, the disruption of REP‐1 did not cause defecation defects compared to severe defects in either RAB‐27 or RabGGTase disruption, suggesting that REP‐1 is not essential for RAB‐27 signaling in defection. Some Rab proteins did not physically interact with REP‐1, and localization of these Rab proteins was not severely affected by REP‐1 disruption. These findings suggest that REP‐1 functions are required in specific Rab pathways and in specific tissues, and that some Rab proteins are functionally prenylated without REP‐1.     

Identification of cembratriene-4,6-diol as antitumor-promoting agent from cigarette smoke condensate

Cigarette smoke condensate (CSC) was separated into severalfractions and each was tested for an inhibitory effect on theearly antigen (EA) of Epstein-Barr virus (EBV) which can beinduced by 12-0-tetradecanoylphorbol-13-acetate (TPA) in Rajicells. Two diastereoisomers of 2,7,11-cembratriene-4,6-diol(- and ß-CBT) were isolated from the neutral fractionsof CSC and these showed potent inhibitory effects on the inductionof EBV-EA by TPA. The doses of - and ß-CBT requiredfor 50% inhibition of EBV-EA induction by TPA were 7.7 and 6.7µg/ml, respectively, which are comparable with those ofretinoic acid, a potent inhibitor of induction of epideral ornithinedecarboxylase (ODC) activity and tumor promotion by TPA in mice.Application of - and ß-CBT to mouse skin prior totreatment with TPA inhibited TPA-induced ODC activity. The degreeof inhibition was dependent on the dose and application of 16.5µmol/mouse of - and ß-CBT resulted in a 50 and40% reduction, respectively, of the maximum of the ODC activityinduced as a result of treatment with TPA. In initiation-promotionexperiments, -CBT markedly inhibited the promoting effect ofTPA on skin tumor formation in mice which were initiated with7,12-dimethylbenz[a]anthracene, but ß-CBT was foundto be less effective. Application of 3.3 µmol of -CBT40 min prior to treatment with TPA (1 µg) resulted ina 53% reduction in the number of papillomas per mouse. Our presentdata suggest that EBV-EA inhibition assay using Raji cells iseffective for the first screening of inhibitors of tumor promotion,and provide evidence that CSC contains antitumor-promoting agentsin addition to carcinogenic and tumor-promoting agents alreadyreported.     

Cyclosporine and partial liver allotransplants in a simplified rat model

We designed a simplified rat model to assess the effects of cyclosporine (CsA) on liver transplants. Auxiliary liver grafts consisting of 20 per cent of the whole liver were supplied with portal inflow and the bile was drained into the stomach. The cuff technique was used routinely for both end-to-end and end-to-side venous anastomoses. The host liver was totally deprived of portal inflow. Two inbred strains of rats, LEW and F344, were used for isograft (LEW----LEW) and allograft (F344----LEW). All survivors were killed at the end of the fourth week after transplantation. During four weeks, the isografts increased by 108 per cent in wet liver weight. Animals with allograft were treated with CsA given i.m., in a daily dose of 20 and 40 mg/kg for the first ten days post transplantation. The CsA-treated rats were free from parenchymal destruction of the allograft, as was characteristic of the allograft controls. The rats given CsA in the dose of 40 mg/kg showed a 173 per cent increase in the wet liver weight of the transplant, such being greater than allografts in rats on 20 mg/kg of CsA (100 per cent) and even than isografts. These results indicate that auxiliary partial liver grafts benefit from the use of CsA not only through suppression of allorejection but through a potential "hepatotrophic effect" of the agent.