A novel mutation of the GATA site in the erythroid cell-specific regulatory element of the ABO gene in a blood donor with the Am B phenotype.

The Am and Bm phenotypes are characterized by weak expression of the A or B antigens, respectively, by red blood cells with a normal expression by the saliva of secretors. Deletion of the regulatory element in the first intron of the ABO gene and disruption of the GATA motif in the element were found to be responsible. In this study, we identified a novel mutation within the GATA motif (G>C substitution at position c.28 + 5830) in the regulatory element of the A allele that might diminish transcription activity causing the generation of the Am B phenotype.

Post-transplant lymphoproliferative disorder of the adrenal gland after allogeneic hematopoietic stem cell transplantation: report of two cases and literature review.

Post-transplant lymphoproliferative disorder (PTLD) is one of the life-threatening complications after hematopoietic stem cell transplantation (HSCT) and solid organ transplantation (SOT), and it is associated almost exclusively with Epstein-Barr virus (EBV). We herein report 2 cases of EBV-associated PTLD after allogeneic HSCT localized in the adrenal gland. Both patients developed adrenal tumor within 3 months after HSCT and were successfully treated with rituximab or tapering immunosuppressive agents. Both remained alive without recurrence. A literature review revealed 12 reported cases of PTLD involving the adrenal gland, but the adrenal gland was involved as one of the lesions of advanced-stage PTLD after SOT. To the best of our knowledge, this is the first report to show cases of isolated EBV-associated adrenal PTLD after HSCT. PTLD should be recognized as one of the causes of isolated adrenal tumor after HSCT.

Influence of sugarcane bagasse-derived biochar application on nitrate leaching in calcaric dark red soil.

Application of biochar has been suggested to improve water- and fertilizer-retaining capacity of agricultural soil. The objective of this study was to evaluate the effects of bagasse charcoal (sugarcane [ L.] bagasse-derived biochar) on nitrate (NO) leaching from Shimajiri Maji soil, which has low water- and fertilizer-retaining capacity. The nitrate adsorption properties of bagasse charcoal formed at five pyrolysis temperatures (400-800° C) were investigated to select the most suitable bagasse charcoal for NO adsorption. Nitrate was able to adsorb onto the bagasse charcoal formed at pyrolysis temperatures of 700 to 800° C. Nitrate adsorption by bagasse charcoal (formed at 800° C) that passed through a 2-mm sieve was in a state of nonequilibrium even at 20 h after the addition of 20 mg N L KNO solution. Measurements suggested that the saturated and unsaturated hydraulic conductivity of bagasse charcoal (800° C)-amended soils are affected by changes in soil tortuosity and porosity and the presence of meso- and micropores in the bagasse charcoal, which did not contribute to soil water transfer. In NO leaching studies using bagasse charcoal (800° C)-amended soils with different charcoal contents (0-10% [w/w]), the maximum concentration of NO in effluents from bagasse charcoal-amended soil columns was approximately 5% less than that from a nonamended soil column because of NO adsorption by bagasse charcoal (800° C). We conclude that application of bagasse charcoal (800°C) to the soil will increase the residence time of NO in the root zone of crops and provide greater opportunity for crops to absorb NO.

NMDA induces long-term synaptic depression and dephosphorylation of the GluR1 subunit of AMPA receptors in hippocampus.

Brief bath application of N-methyl-D-aspartate (NMDA) to hippocampal slices produces long-term synaptic depression (LTD) in CA1 that is (1) sensitive to postnatal age, (2) saturable, (3) induced postsynaptically, (4) reversible, and (5) not associated with a change in paired pulse facilitation. Chemically induced LTD (Chem-LTD) and homosynaptic LTD are mutually occluding, suggesting a common expression mechanism. Using phosphorylation site-specific antibodies, we found that induction of chem-LTD produces a persistent dephosphorylation of the GluR1 subunit of AMPA receptors at serine 845, a cAMP-dependent protein kinase (PKA) substrate, but not at serine 831, a substrate of protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase II (CaMKII). These results suggest that dephosphorylation of AMPA receptors is an expression mechanism for LTD and indicate an unexpected role of PKA in the postsynaptic modulation of excitatory synaptic transmission.

Involvement of a postsynaptic protein kinase A substrate in the expression of homosynaptic long-term depression.

Hippocampal N-methyl-D-aspartate (NMDA) receptor-dependent long-term synaptic depression (LTD) is associated with a persistent dephosphorylation of the GluR1 subunit of AMPA receptors at a site (Ser-845) phosphorylated by cAMP-dependent protein kinase (PKA). In the present study, we show that dephosphorylation of a postsynaptic PKA substrate may be crucial for LTD expression. PKA activators inhibited both AMPA receptor dephosphorylation and LTD. Injection of a cAMP analog into postsynaptic neurons prevented LTD induction and reversed previously established homosynaptic LTD without affecting baseline synaptic transmission. Moreover, infusing a PKA inhibitor into postsynaptic cells produced synaptic depression that occluded homosynaptic LTD. These findings suggest that dephosphorylation of a PKA site on AMPA receptors may be one mechanism for NMDA receptor-dependent homosynaptic LTD expression.

Functioning adrenocortical oncocytoma: the first documented case producing interleukin-6 and review of the literature.

Adrenocortical oncocytoma is an extremely rare and predominantly non-functioning tumor. We herein report the first case of an adrenocortical oncocytoma that produces interleukin (IL)-6. A 38-yr-old woman was referred for treatment of a 4-cm adrenal mass. Laboratory test results showed elevated inflammatory parameters. Intriguingly, IL-6 serum level was also high at 30 pg/ml (normal 0-4 pg/ml). The patient underwent laparoscopic right adrenalectomy. Microscopic examination showed that the tumor was an adrenocortical oncocytoma with a unique peripheral lymphoid cuff with germinal centers. Electron microscopy demonstrated that the cytoplasm of the neoplastic cells was packed with numerous abnormal mitochondria. Three observations lead us to consider that this tumor was the primary source of serum IL-6. First, the IL-6 level in blood collected from the right adrenal vein was highest (527 pg/ml) among intra-operative blood samples. Second, neoplastic cells stained positively for IL-6. Third, the serum IL-6 returned to normal levels immediately after surgery.

Recurrent Spindle Cell Carcinoma Shows Features of Mesenchymal Stem Cells.

This study investigated a case of spindle cell carcinoma (SpCC) in tongue pathological lesions. The patient experienced a local recurrence and distant metastasis after surgical intervention. Although standard chemotherapy was administered, a granulomatous mass continued to develop. This aggressive growth led to survival of the tumor. Secondary debulking surgery was performed to improve the patient's quality of life at the request of the patient. Using a tissue sample derived from the secondary debulking surgery, we performed an analysis of the tumor's cell surface antigens, differentiation potential, metastatic ability, and inhibition potential by anticancer reagents. In vitro analysis revealed that the cell population grown under adherent culture conditions expressed the mesenchymal stem cell (MSC) markers CD73, CD90, and CD105. The cell line established from this SpCC contained colony-forming unit fibroblasts (CFU-Fs) and exhibited multipotent differentiation into several mesenchymal lineages, including bone, cartilage, and fat. The SpCC cells also displayed vigorous mobilization. These characteristics suggested that they had the differentiation potential of mesenchymal cells, especially MSCs, rather than that of epithelial cells. The surgical specimen analyzed in this study resisted the molecular target reagent cetuximab, which is an epidermal growth factor receptor inhibitor. This clinical insight revealed that chemotherapy-resistant SpCC cells have different characteristics compared to most other cancer cells, which are sensitive to cetuximab. Our cell death assay revealed that SpCC cell death was induced by the anticancer drug imatinib, which is known to inhibit protein tyrosine kinase activity of ABL, platelet-derived growth factor receptor α (PDGFRα), and KIT. Here, we report recurrent SpCC with characteristics of MSCs and potential for treatment with imatinib.

Interferons modulate the expression of hormone receptors on the surface of murine melanoma cells.

The effects of IFN-alpha, IFN-beta, and IFN-gamma on the differentiation of murine melanoma cells has been studied, in the presence and absence of melanocyte-stimulating hormone (MSH); the cells were highly responsive to treatment with MSH, which increased the rate of melanin production 25-fold and tyrosinase activity 6-fold within 4 d. Treatment of melanoma cells with IFN-alpha, IFN-beta, or IFN-gamma alone had no stimulatory effect on melanin production, but when the cells were cultured with IFN in the presence of MSH, pigment production was significantly and synergistically increased relative to cells cultured with MSH only. Flow cytometric analysis revealed that levels of tyrosinase in the cells were not affected by MSH or by IFN, which suggests that stimulation of melanogenic activity occurred by activation of a preexisting cellular enzyme. Scatchard analyses showed that the number of MSH receptors on IFN-treated cells was significantly increased (approximately 2.5-fold) relative to untreated cells (approximately 61,000/cell). These findings demonstrate that IFN stimulate differentiation (that is, pigmentation) of melanocytes by increasing the expression of surface MSH receptors; this in turn suggests that such a mechanism may in part be responsible for postinflammatory skin pigmentation, and provides an additional basis for action in the clinical responses of melanoma to IFN treatment.

Genetic recombination at different points in the Npro-coding region of bovine viral diarrhea viruses and the potentials to change their antigenicities and pathogenicities.

Cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain KS86-1 cp was isolated from a cow persistently infected with non-cytopathogenic (ncp) BVDV strain KS86-ncp after development of mucosal disease by superinfection with cp BVDV strain Nose. cp BVDV strains 799cp and 839cp were also isolated from independent cattle that developed mucosal disease by superinfection with cp BVDV KS86-1cp. In the present study, genetic analysis revealed that the genes of cp BVDV strains 799cp and 839cp were chimeras between the genes of the persisting ncp BVDVs and that of superinfecting KS86-1cp. The genetic recombination that generates 799cp occurred between the identical points in the N(pro) gene region, whereas genetic recombination that generates 839cp occurred between different points in the N(pro) gene region. Both 799cp and 839cp were inherited Jiv gene of KS86-1cp strain and envelope protein genes of the persisting viruses. In addition, neutralization test disclosed that antigenicities of 799cp, 839cp, and KS86-1cp were also similar to each persisting virus. These findings indicate that exogenous cp BVDV containing insertion of Jiv gene in the 5 terminal region can induce genetic recombination with the original ncp BVDV at different points in the N(pro) gene region, and those viruses have high potential to change those antigenicities and pathogenicities by RNA recombination.

Development of an immunochromatographic test kit for rapid detection of bovine viral diarrhea virus antigen.

An immunochromatographic test was developed for rapid diagnosis of bovine viral diarrhea virus (BVDV) infections using monoclonal antibodies against the nonstructural protein, NS3, of the virus. The kit detected specifically the NS3 of various BVDV strains. Using the kit, leukocyte extracts of cattle infected persistently with BVDV were found positive while those of healthy cattle were negative. The sensitivity and specificity of this kit in compared with virus isolation were 100% and 97.2%, respectively. Furthermore, the test also gave positive results for calves infected acutely with BVDV in experimental infection. The BVDV antigen was detected in 1 ml of blood using a relatively simple procedure. This test kit should be useful for rapid diagnosis of BVD.

A unique tumor rejection antigen from the S91 murine malignant melanoma.

We have identified and described the characteristics of a unique tumor rejection antigen (tumor-specific transplantation antigen) obtained from the murine malignant melanoma S91. This antigen is highly restricted to the autologous melanoma and provides striking inhibition of its growth. Previously, we described common or shared tumor-specific transplantation antigens on the murine malignant melanomas B16 F10, K1735, JB/RH, and JB/MS. No cross-reactivity was obtained in this study between S91 and those four other malignant melanomas. The common tumor-specific transplantation antigen resides on a glycoprotein molecule with a molecular weight of 65,000, termed B700, that shares homology with serum albumin as determined by NH2-terminal amino acid sequencing. B700, however, purified from S91 proved to be ineffective as an immunogen.

Mouse-human chimeric antibody against the multidrug transporter P-glycoprotein.

In an effort to devise an effective treatment for human drug-resistant cancers, we have generated a monoclonal antibody, MRK16, reactive to the multidrug transporter P-glycoprotein. The monoclonal antibody inhibited the growth of human drug-resistant tumor cells in a xenograft model, suggesting its potential usefulness in the immunotherapy of drug-resistant cancers. In this study, we have developed a recombinant chimeric antibody in which the antigen-recognizing variable regions of MRK16 are joined with the constant regions of human antibodies. When human effector cells were used, the chimeric antibody, MH162, was more effective in killing drug-resistant tumor cells than the all-mouse monoclonal MRK16. The chimeric antibody against the multidrug transporter P-glycoprotein will be a useful agent in immunotherapy of human drug-resistant cancers.

Characterization of phosphorylation sites on the glutamate receptor 4 subunit of the AMPA receptors.

Recent studies have suggested that protein phosphorylation of glutamate receptors may play an important role in synaptic transmission. Specifically, the phosphorylation of AMPA receptors has been implicated in cellular models of synaptic plasticity. The phosphorylation of the glutamate receptor 1 (GluR1) subunit of AMPA receptors by protein kinase A (PKA), protein kinase C (PKC), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been characterized extensively. Phosphorylation of this subunit occurs exclusively on the intracellular C-terminal domain. However, the GluR1 subunit C terminus shows low homology to the other AMPA receptor subunits. In this paper we characterized the phosphorylation of AMPA receptor subunit GluR4, using site-specific mutagenesis and biochemical techniques. We found that GluR4 is phosphorylated on serine 842 within the C-terminal domain in vitro and in vivo. Serine 842 is phosphorylated by PKA, PKC, and CaMKII in vitro and is phosphorylated in transfected cells by PKA. Two-dimensional phosphopeptide analysis indicates that serine 842 is the major phosphorylation site on GluR4. In addition, we identified threonine 830 as a potential PKC phosphorylation site. These results suggest that GluR4, which is the most rapidly desensitizing AMPA receptor subunit, may be modulated by phosphorylation.

Escherichia coli OmpA retains a folded structure in the presence of sodium dodecyl sulfate due to a high kinetic barrier to unfolding.

Escherichia coli OmpA can be solubilized by sodium dodecyl sulfate (SDS) in its folded structure, and it unfolds upon heating. Although the heat-denatured OmpA remains unfolded after lowering the temperature, the addition of a non-ionic surfactant, octyl glucoside results in refolding of unfolded OmpA. In the present study, we investigated the refolding kinetics of OmpA in a mixed surfactant system of SDS and octyl glucoside using far- and near-UV circular dichroism and fluorescence spectroscopies. We found four kinetic phases in the refolding reaction, which logarithmically depended on the weight fraction of octyl glucoside. We also examined the unfolding kinetics of OmpA upon heating in the presence of SDS by temperature jump experiments. A comparison of the rate constants for the refolding and the unfolding reactions in SDS-only solution at 30 degrees C revealed that the folded form of OmpA in SDS solution is less stable than the unfolding form, and that the unfolding is virtually unobservable near room temperature due to a high kinetic barrier.

Effect of salt concentration of buffer on the binding of sodium dodecyl sulfate and on the viscosity behavior of the protein polypeptide derived from bovine serum albumin in the presence of the surfactant.

The complex between SDS and a protein polypeptide derived from bovine serum albumin was characterized with respect to binding of SDS and viscosity behavior. The amount of bound SDS increased from 1.0 to 2.2 g/g with increase of the buffer concentration from 10 to 220 mM. A logarithmic plot of the amount of bound SDS against the buffer concentration gave a linear relation like in the plot where the number of SDS molecules constituting a spherical micelle of SDS is plotted similarly. The increase in the buffer concentration up to 25 mM, from 25 to 100 mM and beyond 100 mM, was accompanied by a sharp rise, monotonic decrease and levelling-off of the intrinsic viscosity in the respective region. In the region 45-175 mM, a linear relation was found between the intrinsic viscosity and reciprocal square root of the buffer concentration. The observed changes can be interpreted as follows: (1), the electrostatic repulsion between charges introduced by the bound SDS caused the initial increase; (2), shielding of the charges as the result of ion condensation with further increase in ionic strength caused the viscosity drop and subsequent levelling-off. The characteristics of the plots are consistent with the necklace model proposed previously for such complexes in which SDS is bound to a protein polypeptide forming micelle-like clusters and which behave like a flexible polyelectrolyte (Shirahama, K., Tsujii, K. and Takagi, T. (1974) J. Biochem. 75, 309-319).

Estimation of molecular weights of membrane proteins in the presence of SDS by low-angle laser light scattering combined with high-performance porous silica gel chromatography. Confirmation of the trimer structure of porin of the E. coli outer membrane.

An assessment study was carried out to evaluate the performance of the low-angle laser light-scattering technique combined with high-performance porous silica gel chromatography in the presence of sodium dodecyl sulfate and precision differential refractometry. It was found that the combined technique is highly promising as a reliable method for determining the molecular weight of a membrane protein solubilized by the surfactant. As a test, molecular weights of porin forming the permeability channel of the outer membrane of E. coli B in an oligomeric form were measured before and after heat treatment, which is known to cause dissociation. The results obtained indicate that the porin oligomer is a trimer with stoichiometric composition.