Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF

Serum Gc protein (known as vitamin D(3)-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years.     

Identification of an inducible factor secreted by pancreatic cancer cell lines that stimulates the production of fucosylated haptoglobin in hepatoma cells

Fucosylation is one of the most important oligosaccharide modifications and is involved in cancer and inflammation. Recently, fucosylated haptoglobin was identified as a possible tumor marker for pancreatic cancer. The molecular mechanism underlying increases in fucosylated haptoglobin in sera of patients with pancreatic cancer seems to be complicated. Our previous study [N. Okuyama, Y. Ide, M. Nakano, T. Nakagawa, K. Yamanaka, K. Moriwaki, K. Murata, H. Ohigashi, S. Yokoyama, H. Eguchi, O. Ishikawa, T. Ito, M. Kato, A. Kasahara, S. Kawano, J. Gu, N. Taniguchi, E. Miyoshi, Fucosylated haptoglobin is a novel marker for pancreatic cancer: a detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation, Int. J. Cancer 118 (11) (2006) 2803-2808] demonstrated that pancreatic cancer cells secrete a factor, which induces the production of haptoglobin in hepatoma cells. In the present study, we found that interleukin 6 (IL6) expressed in pancreatic cancer is a factor that induces the haptoglobin production, using a neutralizing antibody for IL6. Real-time PCR analyses revealed the up-regulation of fucosylation regulatory genes after IL6 treatment, resulting increases in fucosylated haptoglobin being revealed by a lectin ELISA. This pathway could be one of the possible mechanisms underlying increases in haptoglobin in sera of patients with pancreatic cancer.     

Reduced alpha4beta1 integrin/VCAM-1 interactions lead to impaired pre-B cell repopulation in alpha 1,6-fucosyltransferase deficient mice

Mice with a targeted gene disruption of Fut8 (Fut8(-/-)) showed an abnormality in the transition from pro-B cell to pre-B cell, reduced peripheral B cells, and a decreased immunoglobulin production. Alpha 1,6-fucosyltransferase (FUT8) is responsible for the alpha 1,6 core fucosylation of N-glycans, which could modify the functions of glycoproteins. The loss of a core fucose in both very late antigen 4 (VLA-4, alpha4beta1 integrin) and vascular cell adhesion molecule 1 (VCAM-1) led to a decreased binding between pre-B cells and stromal cells, which impaired pre-B cells generation in Fut8(-/-) mice. Moreover, the B lineage genes, such as CD79a, CD79b, Ebf1, and Tcfe2a, were downregulated in Fut8(-/-) pre-B cells. Indeed, the frequency of preBCR(+)CD79b(low) cells in bone marrow pre-B cells in Fut8(-/-) was much lower than that in Fut8(+/+) cells. These results reveal a new role of core fucosylated N-glycans in mediating early B cell development and functions.     

Potential link between amyloid beta-protein 42 and C-terminal fragment gamma 49-99 of beta-amyloid precursor protein

A novel cleavage of beta-amyloid precursor protein (APP), referred to as epsilon-cleavage, occurs downstream of the gamma-cleavage and generates predominantly a C-terminal fragment (CTFgamma) that begins at Val-50, according to amyloid beta-protein (Abeta) numbering. Whether this cleavage occurs independently of, or is coordinated with, gamma-cleavage is unknown. Using a cell-free system, we show here that, although Abeta40 and CTFgamma 50-99 were the predominant species produced by membranes prepared from cells overexpressing wild-type (wt) APP and wt presenilin (PS) 1 or 2, the production of CTFgamma 49-99, which begins at Leu-49, was remarkably enhanced in membranes from cells overexpressing mutant (mt) APP or mtPS1/2 that increases the production of Abeta42. Furthermore, a gamma-secretase inhibitor, which suppresses Abeta40 production and paradoxically enhances Abeta42 production at low concentrations, caused the proportion of CTFgamma 50-99 to decrease and that of CTFgamma 49-99 to increase significantly. These results strongly suggest a link between the production of Abeta42 and CTFgamma 49-99 and provide an important insight into the mechanisms of altered gamma-cleavage caused by mtAPP and mtPS1/2.     

Amperometric detection of the bacterial metabolic regulation with a microbial array chip

A microbial array chip with collagen gel spots entrapping living bacterial cells has been applied to investigate the metabolic regulation in Paracoccus denitrificans. Scanning electrochemical microscopy (SECM) was used to monitor the ferrocyanide production that reflects the electron flow in the respiratory chain located within the internal membrane of P. denitrificans. The ferrocyanide production from P. denitrificans largely depends on the types of the carbon source (glucose or lactate), suggesting that the electron flow rate in the respiratory chain depends on the activity of the metabolic pathway located up-stream of the respiratory chain. More importantly, it was found that the enzymes affecting glucose catabolic reactions were significantly up-regulated in cultures with a nutrient agar medium containing D-(+)-glucose as a sole carbon source. Enzyme assays using crude extracts of P. denitrificans were carried out to identify the enzymes expressed at a higher level in cultures supplemented with D-(+)-glucose. It was confirmed that the pyruvate kinase and enzymes of the overall Entner-Doudoroff pathway were highly induced in cultures containing D-(+)-glucose.     

Phenotypic error threshold; additivity and epistasis in RNA evolution

Background  

The error threshold puts a limit on the amount of information maintainable in Darwinian evolution. The error threshold was first formulated in terms of genotypes. However, if a genotype-phenotype map involves redundancy ("mutational neutrality"), the error threshold should be formulated in terms of phenotypes since there is no unique fittest genotype. A previous study formulated the error threshold in terms of phenotypes, and their results showed that a rather low degree of mutational neutrality can increase the error threshold unlimitedly.     

Activation of macrophages by ether analogues of lysophospholipids

Summary Inflammation processes cause activation of phospholipase A in plasma membranes resulting in the production of various lysophospholipids. Treatment of mice with L--lysophosphatidyl-DL-glycerol (lyso-Pg) resulted in an enhanced ingestion activity of peritoneal macrophages as did other lysophospholipids. However, lyso-Pg is rather toxic as indicated by a rapid decrease in macrophage activity 3 days after treatment while macrophage activity of lysophosphatidylcholine-treated mice continued to increase at least up to the 6th day after treatment. Alkyl-lysophospholipid derivatives, racemic 1-0-octadecyl-2-methylglycero-3-phosphocholine and -phosphoethanolamine stimulated mouse macrophages for Fc-mediated ingestion. Decomposed products of alkyl-lysophospholipids, alkylglycerols, were also found to be excellent activators of macrophages not only for ingestion of IgG-coated target cells but also antibody-mediated tumoricidal activity. Macrophages from mice treated with alkylglycerols developed superoxide generating capacity. Furthermore, alkylglycerols were found to be tumoricidal by direct contact with retinoblastoma cells. Therefore, the advantage of the potential application of alkylglycerols as chemotherapeutic agents is that they have dual beneficial effects: potentiation of macrophage activity and cytotoxicity to malignant cells.     

Effects of inflammation products on immune systems

Summary Microbial infection causes inflammation which stimulates macrophage functions. One of the inflammatory products, lysophosphatidylcholine (lyso-Pc), can stimulate macrophage activities. Treatment of mice with lyso-Pc enhanced spreading and ingestion activities of peritoneal macrophages. In vitro treatment of macrophages with lyso-Pc greatly enhanced spreading but not ingestion activities. However, incubation of a mixture of adherent and nonadherent cells with lyso-Pc produced a markedly enhanced ingestion activity of macrophages, implying the contribution of nonadherent cells to the stimulation of macrophages. Time course studies of the stimulation of these macrophages showed that spreading activity is stimulated immediately, even 30 min, after their contact with lyso-Pc while induction of ingestion activity requires a latent period of about 5 h. When the specificity of the macrophage receptors for ingestion was analyzed using defined immunoglobulins (i.e., IgG and IgM) with or without complement, lyso-Pc-activated macrophages efficiently ingested IgG-coated sheep erythrocytes independent of complement. However, macrophages of the same lyso-Pc-treated mice did not ingest erythrocytes coated with IgM and complement. These observations suggest that lyso-Pc-stimulated macrophages ingest the targets via Fc-receptors but not C3b receptors.     

Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12: identification of a new mutation (recQ1) that blocks the RecF recombination pathway

Summary An Escherichia coli K12 mutant resistant to thymineless death (TLD) was isolated, and its genetic analysis led us to identify a new mutation (recQ1) located between corA and metE on the standard linkage map. The mutation was found to result in increased sensitivity to ultraviolet light and deficiency in conjugational recombination when placed in the recBC sbcB background, indicating that it blocked the RecF pathway of recobbination. It seemed likely that this mutation is also capable of causing partial resistance to TLD, but we reserve the possibility of a separate mutation closely linked to recQ1 giving rise to this phenotype. The original mutant was shown to carry an additional mutation probably in the vicinity of the uhp locus, which was also required for the full TLD resistance of the mutant to be expressed.Abbreviations UV ultraviolet light - NG N-methyl-N'-nitro-N-nitrosoguanidine - Km Kanamycin - Sm streptomycin - TLD thymineless death - ¹ resistant - ^s sensitive