Kumiko Harada

HLA-A2-restricted CTL epitopes of a novel lung cancer-associated cancer testis antigen, cell division cycle associated 1, can induce tumor-reactive CTL

Toward the development of a novel cancer immunotherapy, we have previously identified several tumor‐associated antigens (TAAs) and the epitopes recognized by human histocompatibility leukocyte (HLA)‐A2/A24‐restricted cytotoxic T lymphocyte (CTL). In this study, we tried to identify a TAA of lung cancer (LC) and its HLA‐A2 restricted CTL epitopes to provide a target antigen useful for cancer immunotherapy of LC. We identified a novel cancer testis antigen, cell division cycle associated gene 1 (CDCA1), overexpressed in nonsmall cell LC using a cDNA microarray analysis. The expression levels of CDCA1 were also increased in the majority of small cell LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers. We used HLA‐A2.1 transgenic mice to identify the HLA‐A2 (A*0201)‐restricted CDCA1 epitopes recognized by mouse CTL, and we investigated whether these peptides could induce CDCA1‐reactive CTLs from the peripheral blood mononuclear cells (PBMCs) of HLA‐A2‐positive donors and a NSCLC patient. Consequently, we found that the CDCA165–73 (YMMPVNSEV) peptide and CDCA1351–359 (KLATAQFKI) peptide could induce peptide‐reactive CTLs in HLA‐A2.1 transgenic mice. In HLA‐A2+ donors, in vitro stimulation of PBMC with these peptides could induce peptide‐reactive CTLs which killed tumor cell lines endogenously expressing both HLA‐A2 and CDCA1. As a result, CDCA1 is a novel cancer‐testis antigen overexpressed in LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers, and CDCA1 may therefore be an ideal TAA useful for the diagnosis and immunotherapy of these cancers.

In vitro and in vivo properties of recombinant human serum albumin from pichia pastoris purified by a method of short processing time

AbstractPurpose. Recombinant human serum albumin (rHSA), secreted by a Pichia pastorisexpression system, was purified by a fast and efficient method, the essential feature of which is strong but reversible binding of the protein to Blue Sepharose. The structural characteristics, stability, and ligand-binding properties of the resulting protein were examined, and pre-clinical studies were performed. Methods. Protein structure was investigated by amino acid sequencing, sodium polyacrylamide gel electrophoresis, CD spectroscopy and chromatography. Stability was examined by denaturation by guanidine hydrochloride and by calorimetry, and ligand binding was studied by ultrafiltration. Rat experiments were performed with 125I-labeled albumin. Results. Far-ultraviolet and near-ultraviolet CD spectra of rHSA were identical to those of human serum albumin isolated from serum (HSA). Mercaptalbumin and non-mercaptalbumin were separated by high-performance liquid chromatography using an N-methylpyridinium polymer-based column. 60% of rHSA existed as mercaptalbumin, a content that is higher than that of a commercial preparation of HSA. Fatty acids, N-acetyl-L-tryptophan and pasteurization had similar effects on the conformational stability of rHSA and HSA. Stereoselective ligand-binding properties (warfarin, phenprocoumon, pranoprofen and ibuprofen) of rHSA were the same as those of HSA. The effect of the neutral to base transition on warfarin (site I-ligand) and dansylsarcosine (site II-ligand) binding to rHSA was also similar to HSA. In vivo studies showed comparable half-lives, excretion and tissue distributions of the two albumin preparations. Conclusion. The present yeast expression system and purification procedure result in rHSA with structural and functional properties very similar to those of HSA.

Covalent Binding Between Bucillamine Derivatives and Human Serum Albumin

AbstractPurpose. To clarify the mechanism of covalent binding between human serum albumin (HSA) and drugs containing thiol groups, we studied the interactions between HSA and bucillamine (BA) and its derivatives. Methods. To determine the concentration of HSA-drug conjugate, we used columns of N-methylpyridium polymer cross-linked with ethylene glycol dimethacrylate (4VP-Me), and analyzed the reaction between HSA and B A derivatives kinetically. Following pseudo first-order reaction kinetics, the rate constants of reduction of non-mercaptoalbumin (HNA) to mercaptoalbumin (HMA) (ka) and formation of HSA-drug conjugate (kc) were determined. Results. Formation of HSA-drug conjugate was observed only for drugs containing one thiol group. In compound IV, the plots of ka and kc against pH were found to be linear. The HSA-drug conjugate was affected by various factors such as pKa, pH, temparture and the microenviroment of Cys34. The increases in ka and kc. against pH were mainly due to the increase in mercaptide ion concentration. Further, fatty acid affected the microenviroment of Cys34, which increased HSA-drug formation. Conclusions. Cys34 located in a crevice on the surface of the protein plays an important role on the formation of HSA-drug conjugate. These results may be useful for elucidating the reaction mechanisms between various proteins and thiol compounds.

A new 68Ge/68Ga generator system using an organic polymer containing N-methylglucamine groups as adsorbent for 68Ge.

A macroporous styrene-divinylbenzene copolymer containing N-methylglucamine groups was selected for a new 68Ge/68Ga generator system. This resin packed into a column effectively adsorbed the parent nuclide 68Ge. The daughter 68Ga was eluted from the resin with a solution of a low-affinity gallium chelating ligand such as citric or phosphoric acid. The 68Ge leakage was less than 0.0004% of the 68Ge adsorbed on the resin. By simple mixing of transferrin and desferoxamine conjugated HSA and IgG with the eluate from the column, 68Ga-labeling was completed in high yield.

Prognostic and clinical impact of sarcopenia in esophageal squamous cell carcinoma

Recently, depletion of skeletal muscle mass (sarcopenia) has been linked to poor prognosis in several types of cancers, but has not been investigated in esophageal squamous cell carcinoma (ESCC). This retrospective study investigates the relationship between sarcopenia and clinical outcome in ESCC patients treated by surgical resection or definitive chemoradiation therapy (dCRT). The study was retrospectively conducted in a single academic hospital in Kumamoto, Japan, and involved 325 ESCC patients (256 surgical cases and 69 dCRT cases) treated between April 2005 and April 2011. Skeletal muscle mass was quantified by radiologic measures using standard computed tomography scans. The skeletal muscle tissue in the 325 ESCC patients was distributed as follows: mean: 47.10; median: 46.88; standard deviation (SD): 7.39; range: 31.48-71.11; interquartile range, 46.29-47.90. Skeletal muscle tissue was greater in male patients than in female patients (P < 0.0001), but was independent of other clinical and tumor features. Sarcopenia was not significantly associated with overall survival (log rank P = 0.54). Lymph node involvement significantly altered the relationship between sarcopenia and survival rate (P for interaction = 0.026). Sarcopenia significantly reduced the overall survival of patients without lymph node involvement (log rank P = 0.035), but was uncorrelated with overall survival in patients with lymph involvement (log rank, P = 0.31). The anastomosis leakage rate was significantly higher in the sarcopenia group than in the non-sarcopenia group (P = 0.032), but other surgical complications did not significantly differ between the two groups. Sarcopenia in ESCC patients without lymph node involvement is associated with poor prognosis, indicating sarcopenia as a potential biomarker for identifying patients likely to experience an inferior outcome. Moreover, sarcopenia was associated with anastomosis leakage but no other short-term surgical outcome.

Pro- and anti-apoptotic dual functions of the C5a receptor: involvement of regulator of G protein signaling 3 and extracellular signal-regulated kinase.

When apoptosis is initiated by manganese (II) loading, hyperthermia or thapsigargin treatment, human HL-60 and AsPC-1 cells initiate de novo synthesis of the C5a receptor (C5aR) and generation of its ligand, the ribosomal protein S19 (RP S19) homodimer. The ligand–receptor interaction, in an autocrine/paracrine fashion, promotes apoptosis, which can be bypassed by exogenous administration of C5a, another ligand. The proapoptotic function of the RP S19 dimer is reproduced by a C5a/RPS19 chimera that contains the body of C5a and the C-terminal region (Ile134-His145) of RP S19. The RP S19 dimer or C5a/RPS19 and C5a inversely regulate the expression of Regulator of G protein Signaling 3 (RGS3) gene in the apoptosis-initiated cells. Namely, the RP S19-type proteins upregulate RGS3 expression, whereas the C5a reduce it. Transformation of HL-60 cells to overexpress RGS3 promotes apoptosis in association with the downregulation of the Extracellular signal-Regulated Kinase (ERK) signal, and vice versa in the RGS3 knocked-down cells. Consistent with this result, an inhibitor of ERK phosphorylation effectively enhances the apoptotic rate in wild-type HL-60 cells. Moreover, a dominant negative effect on the RP S19 dimer production encourages apoptosis-initiated HL-60 cells with a longer lifespan in mouse than the natural effect. Our data indicate that, in apoptosis-initiated cells, the ligand-dependent C5aR-mediated dual signal affects the fate of cells, either apoptosis execution or survival, through regulation of RGS3 gene expression and subsequent modulation of ERK signal.

Effects of organic modifiers on the chiral recognition by different types of silica-immobilized bovine serum albumin

We prepared three columns containing bovine serum albumin immobilized on silica by different means and the effects of organic modifiers in the eluent on chiral separation were studied using N-substituted amino acids. Adsorption on silica, covalent immobilization to diol-silica with carbonyldiimidazole (CSP-II) and covalent immobilization to amino-silica with glutaraldehyde were studied. CSP-II had the highest stereoselectivity and was the most affected by organic modifiers in the eluent. The hydrophobicity of amino acid moiety affected the chiral recognition of N-benzoylamino acids and the aromaticity of the N-substituted group was important.

Phosphorylation of SET Protein at Ser171 by Protein Kinase D2 Diminishes Its Inhibitory Effect on Protein Phosphatase 2A

We previously reported that protein kinase D2 (PKD2) in T cells is promptly activated after T-cell receptor (TCR) stimulation and involved in the activation of interleukin-2 promoter and T cell death, and that one of its candidate substrate is SET protein, a natural inhibitor for protein phosphatase 2A (PP2A). In this study, we investigated the target amino acid residues of SET phosphorylated by PKD2 and the effects of phosphorylation of SET on PP2A phosphatase activity. In vitro kinase assay using various recombinant SET mutants having Ser/Thr to Ala substitutions revealed that Ser171 of SET is one of the sites phosphorylated by PKD2. Recombinant SET with phosphorylation-mimic Ser171 to Glu substitution reduced its inhibitory effects on PP2A phosphatase activity compared with Ser171 to Ala substituted or wild-type SET. In addition, knockdown of PKD2 in Jurkat cells by RNAi or treatment of human CD4+ T cell clone with the PKD2 inhibitor Gö6976 resulted in reduced PP2A activity after TCR-stimulation judged from phosphorylation status of Tyr307 of the catalytic subunit of PP2A. These results suggest that PKD2 is involved in the regulation of PP2A activity in activated T cells through phosphorylation of Ser171 of SET.

Hydroxamamide as a chelating moiety for the preparation of 99mTc-radiopharmaceuticals III. Characterization of various 99mTc-hydroxamamides

Abstract Five hydroxamamide (Ham) derivatives with various nonpolar substituents and different lipophilicities have been selected as ligands for preparation of 99m Tc-complexes. Analysis via high performance liquid chromatography (HPLC) resolved the 99m Tc-complexes of each ligand into two radioactive components, which are considered as isomers. The stability of both components was studied at pH 5 and 7, showing good stability at pH 7, but some transition between the components at pH 5. Use of a mixture of two ligands for complexation showed not only the complexes of the separate ligands, but also complexes containing both ligands. These results indicate that hydroxamamides very likely form complexes composed of Ham and 99m Tc in a 2:1 molar ratio. This may also explain the stability of the 99m Tc-Ham complexes in spite of the bidentate character of a Ham ligand.

High-performance liquid chromatography of proteins on n-methylpyridinium polymer columns

Two types of 4-methylpyridinium polymers (4VP-DVB-Me and 4VP-EG-Me, cross-linked with divinylbenzene and ethylene glycol dimethacrylate, respectively) were employed for the analysis of proteins in ion-exchange high-performance liquid chromatography. These polymers had different physical properties in the dry state, but showed similar retentions in size-exclusion chromatography using carbohydrate standards. Generally, the 4VP-EG-Me column was superior to the 4VP-DVB-Me column with regard to separation and recovery of proteins.