Enhancement of albumin expression in bone tissues with healing rat fractures

The characterization of 66 kDa protein molecule, a major protein component which is produced from femoral-diaphyseal tissues with fracture healing (Igarashi and Yamaguchi [2002] Int. J. Mol. Med. 9:503-508), was investigated. Weaning rats were killed at 7 and 14 days after femoral fracture. When the femoral-diaphyseal tissues with fracture healing were cultured for 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein molecule of approximately 66 kDa was markedly increased in culture medium from bone tissues with fracture healing. N-terminal sequencing of 66 kDa protein indicated that its N-terminus was identical to that of rat albumin. Western blot analysis of medium 66 kDa protein showed expression of albumin. This expression was significantly enhanced by fracture healing. The expression of albumin was seen in the diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues of rat femur. When the femoral-diaphyseal tissues obtained at 7 days after femoral fracture were cultured in a serum-free medium containing either vehicle, parathyroid hormone (1-34) (10(-7) M), insulin-like growth factor-I (10(-8) M) or zinc acexamate (10(-4) M), medium albumin was significantly increased in the presence of those bone-stimulating factors. The addition of albumin (0.5 or 1.0 mg/ml of medium) caused a significant increase in calcium and deoxyribonucleic acid contents in the femoral-diaphyseal and -metaphyseal tissues obtained from normal rats in vitro. The present study demonstrates that fracture healing induces a remarkable production of albumin which is a major protein component produced from femoral-diaphyseal tissues of rats, and that albumin has an anabolic effect on bone components.     

Involvement of histone H1.2 in apoptosis induced by DNA double-strand breaks

It is poorly understood how apoptotic signals arising from DNA damage are transmitted to mitochondria, which release apoptogenic factors into the cytoplasm that activate downstream destruction programs. Here, we identify histone H1.2 as a cytochrome c-releasing factor that appears in the cytoplasm after exposure to X-ray irradiation. While all nuclear histone H1 forms are released into the cytoplasm in a p53-dependent manner after irradiation, only H1.2, but not other H1 forms, induced cytochrome c release from isolated mitochondria in a Bak-dependent manner. Reducing H1.2 expression enhanced cellular resistance to apoptosis induced by X-ray irradiation or etoposide, but not that induced by other stimuli including TNF-alpha and UV irradiation. H1.2-deficient mice exhibited increased cellular resistance in thymocytes and the small intestine to X-ray-induced apoptosis. These results indicate that histone H1.2 plays an important role in transmitting apoptotic signals from the nucleus to the mitochondria following DNA double-strand breaks.     

Origin of cortical microtubules organized at M/G1 interface: recruitment of tubulin from phragmoplast to nascent microtubules

The origin of cortical microtubules (CMTs) was investigated in transgenic BY-2 cells stably expressing a GFP (green fluorescent protein) -tubulin fusion protein (BY-GT16). In a previous study, we found that CMTs were initially organized in the perinuclear regions but then elongated to reach the cell cortex where they formed bright spots, and that the appearance of parallel MTs from the bright spots was followed by the appearance of transverse MTs (Kumagai et al., Plant Cell Physiol. 42, 723-732, 2001). In this study, we investigated the migration of tubulin to the reorganization sites of CMTs at the M/G1 interface. After synchronization of the BY-GT16 cells by aphidicolin, the localization of GFP-tubulin was monitored and analyzed by deconvolution microscopy. GFP-tubulin was found to accumulate on the nuclear surface near the cell plate at the final stage of phragmoplast collapse. Subsequently, GFP-tubulin accumulated again on the nuclear surface opposite the cell plate, where nascent MTs elongated to the cell cortex. The significance of these observations on the mode of CMT organization is discussed.     

Importance of renal mitochondria in the reduction of TEMPOL,a nitroxide radical

Spin probing methods using an electron spin resonance (ESR) spectrometer are used extensively and bring us a lot of information about in vivo redox mechanisms. However, the in vivo reducing mechanisms of exogenous nitroxide radicals, which serve as typical spin probing reagents are not clear. To clarify this, we examined the sequential kinetics of a spin probe, 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) in the in vivo organs, tissue homogenates and subcellular fractions of kidney and liver using an in vivo and X-band ESR spectrometers. As a parameter of reducing activity, we calculated the half-life of TEMPOL from the decay curve of ESR signal intensity. The half-life of TEMPOL in the whole organs and homogenates of the kidney was significantly shorter than that of the liver, this indicates that the kidney has more reducing activity against TEMPOL as compared to the liver. Subcellular fractional studies revealed that this reducing activity of the kidney mainly exists in the mitochondria. Contrarily, in addition to reduction in the mitochondria, TEMPOL in the liver was reduced by the microsome and cytosol.     

Evidence of isozymes for delta6 fatty acid desaturase in rat hepatocytes

The expression of delta6 fatty acid desaturase, previously identified, was suppressed almost completely by hyper expression of the corresponding antisense gene in a transformant of the rat hepatic cell line BRL-3A. Conversion rates of [1-14C] linoleic acid, alpha-linolenic acid, and tetracosapentaenoic acid into the respective delta6 fatty acids were equivalent to those in control cells. This finding suggested that all of these reactions were catalyzed by at least two delta6 desaturase isozymes in rat hepatocytes.     

Localization of the locus responsible for Chediak-Higashi syndrome in cattle to bovine chromosome 28

Chediak-Higashi syndrome in Japanese black cattle is a hereditary disease with prolonged bleeding time and partial albinism. In the present study, we mapped the locus responsible for the disease (CHS) by linkage analysis using microsatellite genotypes of paternal half-sib pedigrees obtained from commercial herds. Analysis revealed significant linkage between the CHS locus and marker loci on the proximal end of bovine chromosome 28. The CHS locus was mapped on the region incorporating the microsatellite markers BMC6020, BM2892, and RM016 with recombination fraction 0 and lod score 4.9-11.2. We also assigned the bovine CHS1/LYST, the homologue of the gene responsible for human Chediak-Higashi syndrome, to bovine chromosome 28 using a bovine/murine somatic cell hybrid panel. These findings suggest that a mutation in the CHS1/LYST gene is likely to be responsible for Chediak-Higashi syndrome in Japanese black cattle.     

The changes in glycosylation after partial hepatectomy enhance collagen binding of vitronectin in plasma

Vitronectin is a multifunctional glycoprotein present in the extracellular matrix and plasma. Changes in rat vitronectin were studied during liver regeneration after partial hepatectomy. Carbohydrate concentrations of vitronectin decreased to 2/3 of sham-operated rats at 24 h after partial hepatectomy. Carbohydrate composition and lectin reactivity indicated that N-glycosylation and sialylation of vitronectin changed markedly after partial hepatectomy, while amino acid composition did not change significantly. We previously showed that deN-glycosylation of vitronectin in vitro affects collagen binding among various ligands (Yoneda et al., Biochemistry (1998) 37, 6351-6360). Vitronectins from partially hepatectomized rats at 24 h were found to exhibit markedly enhanced binding to type I collagen. The effect of sialylation on collagen binding was further examined using enzymatically deglycosylated vitronectin of nonoperated rats. Collagen binding increased by 1.2 times after deN-glycosylation of vitronectin, while it increased more than 2.9 times after desialylation. Various glycosyltransferases in liver are known to change after partial hepatectomy, including the attenuation of N-oligosaccharide transferase. The findings therefore suggest that the collagen binding of vitronectin is modulated by the alteration of peptide glycosylation caused by postoperative physiological changes of glycosyltransferases and that the change may contribute to tissue remodeling processes.     

Molecular characterization of a novel intracellular hyaluronan-binding protein

Hyaluronan has well defined functions in extracellular matrices and at the surface of cells. However, several studies have now shown that significant pools of hyaluronan are also present intracellularly, but its function therein is unknown. One avenue of investigation that may assist in defining the function of intracellular hyaluronan is to identify intracellular hyaluronan-binding proteins. In previous studies we identified CDC37, a cell cycle regulatory protein, using a monoclonal antibody that recognizes a novel group of hyaluronan-binding proteins. In this study, we have identified a second hyaluronan-binding protein with this antibody and characterized its properties. This protein, which we have termed IHABP4, was also found to be an intracellular and a specific hyaluronan-binding protein, containing several hyaluronan-binding motifs: (R/K)[X(7)](R/K) (where R/K denotes arginine or lysine and X denotes non-acidic amino acids). Furthermore, we have determined the gene organization of IHABP4 and cloned cDNAs for the chick, mouse, and human homologs. Comparison of the deduced chick, mouse, and human protein sequences showed that the hyaluronan-binding motifs, (R/K)[X(7)](R/K), in these sequences are conserved; both chick and mouse IHABP4 were shown directly to bind hyaluronan. Biochemical fractionation and immunofluorescent localization of epitope-tagged IHABP4 indicated that it is mainly present in the cytoplasm. These data support the possibility that intracellular hyaluronan and its binding proteins may play important roles in cell behavior.     

The insect salivary protein, prolixin-S, inhibits factor IXa generation and Xase complex formation in the blood coagulation pathway

Prolixin-S is a salivary anticoagulant of the blood-sucking insect, Rhodnius prolixus, and known as an inhibitor of the intrinsic Xase. We report here its inhibitory mechanisms with additional important anticoagulation activities. We found prolixin-S specifically bound to factor IX/IXa in the presence of Ca(2+) ions. Light scattering and surface plasmon resonance studies showed that prolixin-S interfered with factor IX/IXa binding to the phospholipid membrane, indicating that prolixin-S inhibit Xase activity of factor IXa by interference with its Xase complex formation. Furthermore, reconstitution experiments showed that prolixin-S binding to factor IX strongly inhibited factor IXa generation by factor XIa. We also found that prolixin-S inhibited factor IXa generation by factor VIIa-tissue factor complex and factor IXalpha generation by factor Xa. These results suggest that prolixin-S inhibits both intrinsic and extrinsic coagulations by sequential inhibition of all coagulation pathways in which factor IX participates. It was also suggested that prolixin-S may bind to factor IX/IXa by recognizing conformational change of the Gla domain induced by Ca(2+) binding.