Yasushi Sakamoto

The Complete Amino Acid Sequence of Human P2 Protein

Abstract: The complete amino acid sequence of P2 protein from human peripheral nerve myelin was determined from nine staphylococcal protease peptides and four cyanogen bromide peptides. Human P2 protein is composed of 131 amino acids and has a molecular weight of 14,818. Compared to bovine P2 protein, there are replacements at nine positions (human↔bovine): 18(Asp↔Glu), 39(Thr↔Arg), 56(Thr↔Pro), 83(Ile↔Thr), 87(Gln↔Ala), 96(Arg↔Lys), 100(Lys↔Asn), 115 (Ala↔Val), and 121(Gly↔Asp).

PHOSPHORYLATION OF P0 GLYCOPROTEIN IN PERIPHERAL NERVE MYELIN

Abstract: The P0 protein in mammalian PNS myelin is known to undergo several posttranslational modifications, such as glycosylation, acylation, sulfation, and phosphorylation. Phosphorylation of purified P0 protein in vitro was studied comparatively using three enzymes, i.e., calcium/phospholipid‐dependent protein kinase (protein kinase C), calcium/calmodulin‐dependent protein kinase II (CaM kinase II), and the catalytic subunit of cyclic AMP‐dependent protein kinase (A kinase). The phosphorylation of P0 protein by CaM kinase II was the greatest, followed by that by protein kinase C; phosphorylation by A kinase, however, was much lower. In order to identify phosphorylation sites, P0 protein was phosphorylated with [32P]ATP and each kinase and then digested with lysylendopeptidase. The resulting phosphopeptides were isolated by HPLC. Subsequent amino acid sequence analysis and comparison with the known sequence of P0 protein revealed that Ser181 and Ser204 were strongly phosphorylated by both protein kinase C and CaM kinase II. In addition, Ser214 was also phosphorylated by protein kinase C, but not by CaM kinase II. Because all of these sites are located in the cytoplasmic domain of P0 protein, phosphorylation may be important for maintenance of the major dense line of PNS myelin.

Poly(ADP-Ribose) Polymerase-1 Enhances Transcription of the Profibrotic CCN2 Gene

In the fibrotic kidney, tubular epithelial cells express CCN2, formerly known as connective tissue growth factor. Because little is known about the transcriptional regulation of this profibrotic protein, this study investigated the mechanism underlying epithelial cell-selective upregulation of CCN2 in fibrosis. It was found that a previously unidentified cis-regulatory element located in the promoter of the murine CCN2 gene plays an essential role in basal and TGF-beta1-induced gene transcription in tubular epithelial cells; this element acts in conjunction with the Smad-binding element and the basal control element-1. By protein mass fingerprint analysis and de novo sequencing, poly(ADP-ribose) polymerase-1 (PARP-1) was identified as a trans-acting protein factor that binds to this promoter region, which we termed the PARP-1-binding element. In vivo, knockdown of PARP-1 in proximal tubular epithelial cells significantly reduced CCN2 mRNA levels and attenuated interstitial fibrosis in the obstructed kidney. Thus, the PARP-1/PARP-1 binding element complex functions as a nonspecific, fundamental enhancer of both basal and induced CCN2 gene transcription in tubular epithelial cells. This regulatory complex may be a promising target for antifibrotic therapy.

Cytokine-Dependent Modification of IL-12p70 and IL-23 Balance in Dendritic Cells by Ligand Activation of Vα24 Invariant NKT Cells

CD1d-restricted invariant NKT (iNKT) cells play crucial roles in various types of immune responses, including autoimmune diseases, infectious diseases and tumor surveillance. The mechanisms underlying their adjuvant functions are well understood. Nevertheless, although IL-4 and IL-10 production characterize iNKT cells able to prevent or ameliorate some autoimmune diseases and inflammatory conditions, the precise mechanisms by which iNKT cells exert immune regulatory function remain elusive. This study demonstrates that the activation of human iNKT cells by their specific ligand α-galactosylceramide enhances IL-12p70 while inhibiting the IL-23 production by monocyte-derived dendritic cells, and in turn down-regulating the IL-17 production by memory CD4+ Th cells. The ability of the iNKT cells to regulate the differential production of IL-12p70/IL-23 is mainly mediated by a remarkable hallmark of their function to produce both Th1 and Th2 cytokines. In particular, the down-regulation of IL-23 is markedly associated with a production of IL-4 and IL-10 from iNKT cells. Moreover, Th2 cytokines, such as IL-4 and IL-13 play a crucial role in defining the biased production of IL-12p70/IL-23 by enhancement of IL-12p70 in synergy with IFN-γ, whereas inhibition of the IFN-γ-promoted IL-23 production. Collectively, the results suggest that iNKT cells modify the IL-12p70/IL-23 balance to enhance the IL-12p70-induced cell-mediated immunity and suppress the IL-23-dependent inflammatory pathologies. These results may account for the long-appreciated contrasting beneficial and adverse consequence of ligand activation of iNKT cells.

Monoclonal antibodies specific to the integral membrane protein P0 of bovine peripheral nerve myelin

Two monoclonal antibodies (mAbs), 58A and 46E, were generated against the major protein P0 of bovine peripheral nervous system myelin (PNSM). The reactivities of the mAbs were assessed by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry. Both mAbs, 58A and 46E, reacted to PNSM of bovine, human, rat and rabbit, but not to chicken PNSM or the brains of rat and rabbit. In the Western blot, these mAbs showed specific binding to bovine P0 as well as deglycosylated P0, but not to myelin-associated glycoprotein (MAG) of bovine spinal cord. The analyses of the lysylendopeptidase-digested peptides of bovine P0 revealed that the epitopes for the mAbs 58A and 46E were located on the amino acid residues 68-79 and 210-216, respectively. Since the mAbs 58A and 46E recognize the extracellular domain and the cytoplasmic domain of P0, respectively, they could be useful for studies on P0s role in myelin formation, its adhesive properties, and functions of the N-terminal extracellular and C-terminal cytoplasmic domains of the protein.

Structural study of the glycosylated and unglycosylated forms of a genetic variant of human serum albumin (63 Asp → Asn)

A genetic variant of human serum albumin (alloalbumin) exhibited atypical electrophoretic mobility and chromatographic behavior apparently because of the effect of a point substitution on the molecular conformation. Three forms of albumin were isolated by DEAE HPLC chromatography: normal albumin, and two variant forms V1 and V2. The point substitution (Asp-63-->Asn) generated a canonical tripeptide acceptor sequence for glycosylation with an N-linked oligosaccharide (Asn-Lys-Ser). Neuraminidase digestion followed by electrophoresis showed that the V2 variant form was glycosylated and the V1 form was not. Time-of-flight mass spectrometry yielded a molecular weight of about 2000 for the carbohydrate. Structural analysis of the carbohydrate was done by chromatographic comparison of the pyridylaminated derivatives with standards and was confirmed by proton NMR of the three pronase glycopeptides and of the pyridylaminated oligosaccharide. The oligosaccharide had a complex biantennary structure with two sialic acid residues. In normal albumin Asp-63 is exposed and is adjacent to the first disulfide bond, Cys-62-->Cys-53. The apparent effect on molecular conformation resulting in incomplete glycosylation and atypical electrophoretic mobility suggests that glycosylation may interfere with disulfide bond formation at this site.

In vitro evaluation of antioxidant defense mechanism and hemocompatibility of mauran

Mauran (MR), a highly polyanionic sulfated exopolysaccharide was extracted from moderately halophilic bacterium; Halomonas maura and characterized using X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. Purified MR was evaluated for antioxidant defense mechanisms under in vitro conditions using L929, mouse fibroblast cell line and mice liver homogenate. It was demonstrated that MR could impart protective effect against oxidative stress in both cells and tissue up to a concentration of 500 μg, which is found to be safe under laboratory conditions. Various enzymatic and non-enzymatic parameters of antioxidant mechanisms were evaluated and concluded that MR has the tendency to maintain a balance of antioxidative enzymes with in the test systems studied. Also, hemocompatibility assay performed revealed that MR has a lesser hemolytic index and exhibited a prolonged clotting time, which shows both antihemolytic, and antithrombogenic nature respectively. Furthermore, absorption studies performed using fluorescent-labeled MR confirmed that MR accumulated within the cell cytoplasm neither induced cellular lysis nor affected the cell integrity.

Multifunctional Cu 2'x Te Nanocubes Mediated Combination Therapy for Multi-Drug Resistant MDA MB 453

Hypermethylated cancer populations are hard to treat due to their enhanced chemo-resistance, characterized by aberrant methylated DNA subunits. Herein, we report on invoking response from such a cancer lineage to chemotherapy utilizing multifunctional copper telluride (Cu2−XTe) nanocubes (NCs) as photothermal and photodynamic agents, leading to significant anticancer activity. The NCs additionally possessed photoacoustic and X-ray contrast imaging abilities that could serve in image-guided therapeutic studies.

Applications of Fourier transform infrared spectroscopy, Fourier transform infrared microscopy and near-infrared spectroscopy to cancer research

Glycogen levels in human lung and colorectal cancerous tissues were measured by the Fourier transform (FT-IR) spectroscopic method. Reliability of this method was confirmed by chemical analyses of the same tissues used for the FT-IR spectroscopic measurements, suggesting that this spectroscopic method has a high specificity and sensitivity in discriminating human cancerous tissues from noncancerous tissues. The glycogen levels in the tissues were compared with the clinical, histological and histopathological factors of the cancer, demonstrating that glycogen is a critical factor in understanding the biological nature of neoplastic diseases. Furthermore, direct measurement of a very small amount of tissue by a FT-IR microscope suggested that it could be used as a diagnostic instrument for various tissue samples obtained via a fine needle biopsy procedure. The progressive alterations in rat mammary gland tumors were investigated by a near-infrared (NIR) spectrometer with a fiber optic probe. A lipid band due to the first overtone of n-alkane was used to quantitatively evaluate malignant changes in the tumors. NIR spectroscopy may offer the potential for non‒invasive, in vivo diagnosis of human cancers.

Structure of a Major Oligosaccharide of PASII/PMP22 Glycoprotein in Bovine Peripheral Nerve Myelin

The amino acid sequence of the glycopeptide obtained from bovine PASII/PMP22 protein in the PNS myelin was determined to be Gln‐Asn‐Cys‐Ser‐Thr, where the asparagine was glycosylated. To eliminate all the contaminated Po glycopeptides from the PASII/PMP22 glycopeptide preparation, we used a fluorescent probe, N‐[2‐(2‐pyridylamino)ethyl]maleimide, which reacts with the cysteine of the PASII/PMP22 glycopeptides. The labeled PASII/PMP22 glycopeptides were isolated by HPLC and were digested further with glycopeptidase A. The resultant oligosaccharides were conjugated with 2‐aminopyridine (PA) as a fluorescent tag. One major PA‐oligosaccharide, OPPE1, was purified by HPLC. The structure of OPPE1 was elucidated by fast atom bombardment mass spectrometry and 1H‐NMR studies and comparing the derivatives of PA‐OPPE1 and PA‐oligosaccharides of γ‐globulin on HPLC. The structure, SO4‐3GlcAβ1‐3Galβ1‐4GlcNAcβ1‐2Manα1‐6(GlcNAcβ1‐4) (GlcNAcβ1‐2Manα1‐3)Manβ1‐4GlcNAcβ1‐4(Fucα1‐6)GlcNAc‐PA, was identical to the pyridylaminated form of the major oligosaccharide D8 of bovine Po previously reported.