Induction of gene expression for immunomodulating cytokines in peripheral blood mononuclear cells in response to orally administered PSK,an immunomodulating protein-bound polysaccharide

The protein-bound polysaccharide extracted from a fungus, PSK, has been used as a biological response modifier in the treatment of cancer patients in Japan for over 16 years. The administration of PSK to tumor-bearing rodents inhibited tumor growth and modulated immune responses. Recently, an in vitro study has revealed that PSK is a strong inducer of cytokine gene expression and production in human peripheral blood mononuclear cells (PBMC). To establish whether PSK has cytokine-inducing activities in vivo, we have orally administered PSK (1 g, the clinical dose) to 12 healthy volunteers and 9 gastric cancer patients who had undergone gastrectomy, and assessed the gene expression for cytokines in PBMC of each subject. As determined by the reverse-transcribed polymerase chain reaction method, the induction of gene expression for both tumor necrosis factor and interleukin-8 (IL-8) was detected in PBMC from 5 of the 12 healthy volunteers (42%) and 4 of the 9 patients (44%). Furthermore, the concentration of serum IL-8 was elevated in 5 healthy volunteers given PSK orally, who had shown induction of IL-8 gene expression, as detected by enzyme-linked immunosorbent assay. These findings indicate that responsiveness of PBMC to PSK, in terms of gene expression and production of cytokines, varies among individuals. Thus, when using PSK to treat cancer patients, it seems advisable to select patients on the basis of their responsiveness to PSK. We speculate that the cytokines induced by PSK might mediate the immunoenhancing action of this agent in vivo.     

Expansion of human autologous cytotoxic T lymphocytes on fixed target tumor cells

Human tumor specific cytotoxic T lymphocytes (CTL) were expanded on formalin-fixed autologous target tumor cells derived from glioblastoma multiforme. Growth response of the CTL restimulated with the fixed target cells was larger than those with live target cells. The results suggest that formalin-fixed tumor cells will be stable sources of tumor antigen for efficient autologous CTL expansion and be useful for adoptive immunotherapy of tumors. This revised version was published online in August 2006 with corrections to the Cover Date.     

Involvement of blood-group-B-active trisaccharides in Ca2+-dependent cell-cell adhesion in the Xenopus blastula

 Despite their wide distribution in various organisms, no physiological roles have been proposed for the human blood-group-ABO (ABH)-active trisaccharides. Here we show that monoclonal antibodies against human blood-group-B-active trisaccharides (B-substance) completely block the Ca²⁺-dependent cell-cell adhesion system of frog (Xenopus laevis) embryonic cells. Synthetic B-substance or B-active glycopeptides also disrupt the Ca²⁺ -dependent cell-cell adhesion. These results suggest that blood-group-B-active substances play a role in cell-cell adhesion. Blood-group-B-active substances were found as glycoproteins and as glycosphingolipids. In order to identify B-active glycoproteins active in cell-cell adhesion, we purified B-active membrane glycoproteins by two-dimensional electrophoresis and found that they are 45- to 58-kDa proteins with pI(s) ranging from 4.0 to 5.3. They are glycosylphosphatidyl inositol (GPI) anchored. Amino acid sequence analysis showed that the purified B-active GPI-anchored proteins are homologues of soluble Xenopus cortical granule lectins (CGL). The results suggest that the B-active membrane glycoproteins are GPI-anchored forms of the lectin and are directly involved in frog Ca ²⁺-dependent cell-cell adhesion. Received: 16 September 1997 / Accepted 19 November 1997     

Rapid and efficient production of L-lactate from xylose using electrodialysis culture-associated product separation

L-Lactate was produced from xylose using electrodialysis culture (ED-C)-associated product separation. In a medium containing 50g xylose/l, the ED-C was completed in only 32h (i.e. less than half the time taken by the control culture, without electrodialysis). At 80g xylose/l, the control culture was unable to consume more than 50g xylose/l, whereas the ED-C showed increased xylose consumption and was completed by 45h. The maximum rate of lactate production in the ED-C was higher than that in the control culture. ED-C was also carried out (at 80g initial xylose/l) with a supply of fresh xylose-free medium. This ED-C was completed within 30h, which represents a reduction in fermentation time of 15h when compared to ED-C without addition of xylose-free medium. Thus, rapid production of L-lactate was achieved by using ED-C which supplied fresh xylose-free medium.     

Enzymes related to catecholamine biosynthesis in Tetrahymena pyriformis. Presence of GTP cyclohydrolase I.

We first identified GTP cyclohydrolase I activity (EC 3.5.4.16) in the ciliated protozoa, Tetrahymena pyriformis. The V_max value of the enzyme in the cellular extract of T. pyriformis was 255 pmol mg⁻¹ protein h⁻¹. Michaelis–Menten kinetics indicated a positive cooperative binding of GTP to the enzyme. The GTP concentration producing half-maximal velocity was 0.8 mM. By high-performance liquid chromatography (HPLC) with fluorescence detection, a major peak corresponding to -monapterin (2-amino-4-hydroxy-6-[(1′R,2′R)-1′,2′,3′-trihydroxypropyl]pteridine, -threo-neopterin) and minor peaks of -erythro-neopterin and -erythro-biopterin were found to be present in the cellular extract of Tetrahymena. Thus, it is strongly suggested that Tetrahymena converts GTP into unconjugated pteridine derivatives. In this study, dopamine was detected as the major catecholamine, while neither epinephrine nor norepinephrine was identified. Indeed, this protozoa was shown to possess the activity of a dopamine synthesizing enzyme, aromatic -amino acid decarboxylase. On the other hand, activities of tyrosine hydroxylase or tyrosinase which converts tyrosine into dopa, the substrate of aromatic -amino acid decarboxylase, could not be detected in this protozoa. Furthermore, neither dopamine β-hydroxylase activity nor phenylethanolamine N-methyltransferase activity could be identified by the HPLC methods.     

A 100-kDa Protein Tyrosine Phosphorylation Is Concurrent with β1 Integrin-Mediated Morphological Differentiation in Neuroblastoma and Small Cell Lung Cancer Cells

IMR32, a neuroblastoma cell line, and CADO LC6, a small cell lung cancer (SCLC) cell line, extended neurite-like processes when cultured on fibronectin (FN)-coated surfaces or cultured in a serum-free medium. Monoclonal antibodies against the integrin β1 subunit inhibited this process formation, suggesting that their morphological change is initiated by β1 integrin-dependent signal transduction to the cell interior. Anti-phosphotyrosine immunoblots demonstrated that the phosphorylation level of a 100-kDa protein, but not 125-kDa focal adhesion kindase, correlated well with the morphological change in both cell lines. This 100-kDa protein phosphorylation did not accompany FN-induced morphological changes in NIH 3T3 fibroblasts or A549 adenocarcinoma cells. These findings suggest that neuroblastoma and SCLC may share β1 integrin-mediated signaling events distinct from nonneuronal cells.     

Characterization of species-specifically amplified SINEs in three salmonid species—Chum salmon,pink salmon,and kokanee: The local environment of the genome may be important for the generation of a dominant source gene at a newly retroposed locus

Short interspersed repetitive elements (SINEs), known as theHpaI family, are present in the genomes of all salmonid species (Kido et al.,Proc. Natl. Acad. Sci. USA 1991, 88: 2326–2330). Recently, we showed that the retropositional efficiency of the SINE family in the lineage of chum salmon is extraordinarily high in comparison with that in other salmonid lineages (Takasaki et al.,Proc. Natl. Acad. Sci. USA 1994, 91: 10153–10157). To investigate the reason for this high efficiency, we searched for members of theHpaI SINE family that have been amplified species-specifically in pink salmon. Since the efficiency of the species-specific amplification in pink salmon is not high and since other members of the same subfamily of SINEs were also amplified species-specifically in pink salmon, the actual sequence of this subfamily might not be the cause of the high retropositional efficiency of SINEs in chum salmon. Rather, it appears that a highly dominant source gene for the subfamily may have been newly created by retroposition, and some aspect of the local environment around the site of retroposition may have been responsible for the creation of this dominant source gene in chum salmon. Furthermore, a total of 11 sequences ofHpaI SINEs that have been amplified species-specifically in three salmon lineages was compiled and characterized. Judging from the distribution of members of the same-sequence subfamily of SINEs in different lineages and from the distribution of the different-sequence subfamilies in the same lineage, we have concluded that multiple dispersed loci are responsible for the amplification of SINEs. We also discuss the additional possibility of horizontal transmission of SINEs between species. The availability of the sets of primers used for the detection of the species-specific amplifications of the SINEs provides a convenient and reliable method for identification of these salmonid species.