A clinical trial of live attenuated varicella vaccine (Biken) in children with malignant diseases

The live attenuated varicella vaccine (Biken) derived from the Oka strain was used for immunization of childhood cancer patients in remission but on chemotherapy. Thirty nine patients were immunized without any severe adverse effects but 6 recipients had a small number of vesicles. Seroresponses were observed in 90% of recipients examined. During the follow-up period 6 leukemia recipients were suffered from natural varicella in family or community contacts. No vaccinee developed herpes-zoster during this study period.     

Degradation of glycerol using hydrothermal process

Sub-critical or supercritical water was utilized for the degradation of glycerol in an environmentally benign reaction. The reaction was carried out in a batch reactor in the temperature range of 473-673 K, pressure of 30 MPa, and reaction time of 20-60 min. The effects of temperature and reaction time were observed. The degradation of glycerol produced acetaldehyde, acrolein, allyl alcohol and un-identified products. The highest yield of acrolein, acetaldehyde and allyl alcohol were 0.20, 7.17, 96.69 mol%, respectively. Glycerol conversion was 99.92 mol%. While acetaldehyde was formed only in sub-critical water and allyl alcohol only in supercritical water, acrolein was formed in both. The kinetics of the global reaction displayed a pseudo-first-order. The activation energy at subcritical water was 39.6 kJ/mol. Based on the results, this method could be an efficient method for glycerol degradation because the high conversion of glycerol was obtained.     

Creation of a lysine-deficient LIGHT mutant with the capacity for site-specific PEGylation and low affinity for a decoy receptor

The cytokine LIGHT is a promising candidate for cancer therapy. However, the therapeutic effect of LIGHT as a systemic anticancer agent is currently insufficient because of its instability and its binding to nonfunctional soluble decoy receptor 3 (DcR3), which is overexpressed in various tumors. Modification of proteins with polyethylene glycol (PEGylation) can improve their in vivo stability, but PEGylation may occur randomly at all lysine residues and the NH₂-terminus; therefore, PEGylated proteins are generally heterogeneous and have decreased bioactivity. In this study, we attempted to create a lysine-deficient LIGHT mutant that could be PEGylated site-specifically and would have lower affinity for DcR3. We prepared phage libraries expressing LIGHT mutants in which all the lysine residues were replaced with other amino acids. A lysine-deficient LIGHT mutant [mLIGHT-Lys(−)] was isolated by panning against lymphotoxin β receptor (LTβR). mLIGHT-Lys(−) could be site-specifically PEGylated at its NH₂-terminus, yielding molecular uniformity and in vitro bioactivity equal to that of non-PEGylated, wild-type LIGHT. Furthermore, mLIGHT-Lys(−) was not trapped by the nonfunctional DcR3, despite binding to its functional receptors. These results suggest that mLIGHT-Lys(−) might be a useful candidate for cancer therapy.     

Evolutionary Origin of GnIH and NPFF in Chordates: Insights from Novel Amphioxus RFamide Peptides

Gonadotropin-inhibitory hormone (GnIH) is a newly identified hypothalamic neuropeptide that inhibits pituitary hormone secretion in vertebrates. GnIH has an LPXRFamide (X = L or Q) motif at the C-terminal in representative species of gnathostomes. On the other hand, neuropeptide FF (NPFF), a neuropeptide characterized as a pain-modulatory neuropeptide, in vertebrates has a PQRFamide motif similar to the C-terminal of GnIH, suggesting that GnIH and NPFF have diverged from a common ancestor. Because GnIH and NPFF belong to the RFamide peptide family in vertebrates, protochordate RFamide peptides may provide important insights into the evolutionary origin of GnIH and NPFF. In this study, we identified a novel gene encoding RFamide peptides and two genes of their putative receptors in the amphioxus Branchiostoma japonicum. Molecular phylogenetic analysis and synteny analysis indicated that these genes are closely related to the genes of GnIH and NPFF and their receptors of vertebrates. We further identified mature RFamide peptides and their receptors in protochordates. The identified amphioxus RFamide peptides inhibited forskolin induced cAMP signaling in the COS-7 cells with one of the identified amphioxus RFamide peptide receptors expressed. These results indicate that the identified protochordate RFamide peptide gene is a common ancestral form of GnIH and NPFF genes, suggesting that the origin of GnIH and NPFF may date back to the time of the emergence of early chordates. GnIH gene and NPFF gene may have diverged by whole-genome duplication in the course of vertebrate evolution.     

Predicted multiple selected reaction monitoring to screen activated drug-mediated modifications on human serum albumin

Metabolic activation of drugs frequently generates electrophilic products that may undergo covalent binding to biological macromolecules, such as proteins and DNA. The resulting covalent adducts are of considerable concern in drug discovery and development. Several strategies for assessing the potential risks of candidate drugs have been reported. Of these, glutathione trapping is the most commonly used method together with mass spectrometry. Furthermore, drug-mediated protein modifications have been studied using serum albumin and CYP enzymes to clarify target amino acids and mechanism-based inhibition, respectively. In this article, we introduce a practical way to screen drug-mediated protein modifications. The method, referred to as “predicted multiple selected reaction monitoring,” is based on the selected reaction monitoring (SRM) strategy, but targets all possible chemically modified tryptic peptides. The creation of SRM lists may require patience; however, this strategy could facilitate more sensitive screening compared with the common strategy of data-dependent product ion scanning. Ketoprofen-N-hydroxysuccinimidyl ester (equivalent to glucuronide) and N-acetyl-p-benzoquinone imine (NAPQI) were allowed to react with human serum albumin as a model experiment. Using this strategy, 11 ketoprofen-adduction sites (at Lys^{137, 195, 199, 212, 351, 402, 432, 436, 525, 536, and 541}) and 1 NAPQI-adduction site (at Cys³⁴) were easily identified.     

Hydrolysis of galactosylceramide is catalyzed by two genetically distinct acid beta-galactosidases

Two genetically distinct acid beta-galactosidases are apparently involved in the hydrolysis of galactosylceramide in fibroblasts. These beta-galactosidases were activated by different bile salts. The classical galactosylceramidase (galactosylceramidase I, EC 3.2.1.46) was activated by sodium taurocholate, while the other galactosylceramidase (galactosylceramidase II) was activated by sodium cholate. The former was genetically lacking in globoid cell leukodystrophy (GLD) and the latter in GM1 gangliosidosis. Galactosylceramidase II cross-reacted with antibody raised against purified GM1 ganglioside beta-galactosidase (EC 3.2.1.23) from the human placenta. The purified beta-galactosidase had galactosylceramidase II activity, which was competitively inhibited by GM1 ganglioside. Thus, galactosylceramidase II seems to be identical to GM1 ganglioside beta-galactosidase and lactosylceramidase II. Galactosylceramidase II had a very low affinity for galactosylsphingosine. In the galactosylceramide-loading tests using fibroblasts from patients with GLD and GM1 gangliosidosis, both cell lines hydrolyzed the incorporated galactosylceramide, with lower rates than control fibroblasts but higher than the fibroblasts from patients with I-cell disease, in which both galactosylceramidase I and II were deficient. These results indicate that galactosylceramide is hydrolyzed by two genetically distinct beta-galactosidases and explain well that galactosylsphingosine but not galactosylceramide accumulates in the brain of patients with GLD.     

Mechanism of action of a streptococcal preparation (OK-432) in prevention of autoimmune diabetes in NOD mice. Suppression of generation of effector cells for pancreatic B cell destruction

We have previously reported that treatment with a streptococcal preparation (OK-432), one of the biologic response modifiers, inhibits insulitis and development of autoimmune diabetes in nonobese diabetic (NOD) mice and Bio-Breeding rats used as animal models of insulin-dependent diabetes mellitus (IDDM). We studied the mechanism by which OK-432 inhibited development of IDDM in NOD mice. In female NOD mice diabetes spontaneously developed from 10 to 15 wk of age and the cumulative incidence of diabetes amounted to 74.7% at 30 wk of age. NOD mice, however, never developed diabetes over the observation period of up to 45 wk of age when they were i.p. injected with 0.1 mg of OK-432 weekly from 4 to 15 wk of age. OK-432 treatment in younger age had a stronger inhibitory effect on the development of diabetes. Diabetes was transferred to young, irradiated mice by spleen cells of nontreated, adult mice, but barely transferred by those of OK-432-treated mice. Furthermore, these spleen cells of OK-432-treated mice did not suppress transfer of diabetes by diabetic mice spleen cells. Treatment with cyclophosphamide promoted development of diabetes in nontreated NOD mice due to removal of suppressor cells. However, cyclophosphamide did not show the promotive effect in OK-432-treated mice. Taken together, these results indicate that OK-432 treatment prevented development of diabetes mainly by suppression in generation of the effector cells for pancreatic B cell destruction. The same OK-432 treatment did not suppress the immune response to exogenous AG such as xenogeneic SRBC and allogeneic tumor cells. The study suggests that BRM in the natural environment such as streptococci may suppress induction and progression of autoimmunity and be useful for the immunotherapy of human IDDM.     

Galactosylceramide- and lactosylceramide-loading studies in cultured fibroblasts from normal individuals and patients with globoid cell leukodystrophy (Krabbe's disease) and GM1-gangliosidosis

The metabolism of galactosylceramide and lactosylceramide in cultured fibroblasts was studied using the lipid-loading test. These compounds were incorporated into the fibroblasts yet only small amounts of the incorporated lipids were hydrolyzed unless additional phospholipid was mixed with the glycolipid before loading. Among phospholipids, phosphatidylserine was the most effective for incorporation and hydrolysis of the glycolipids, while phosphatidylcholine inhibited the incorporation of the glycolipids. Using filtration techniques, light scattering analyses and subcellular fractionation, the particle size of glycolipid in the culture medium was found to be critically important for the incorporation of the lipids into the cells and their transportation to the lysosomes. The particle sizes of the glycolipids were decreased by mixing with phosphatidylserine. Furthermore, the negative charge in phosphatidylserine may be necessary for the glycolipid transportation into the lysosomes. In fibroblasts from patients with globoid cell leukodystrophy, 40-50% of galactosylceramide was hydrolyzed on the 4th day of culture, a time when the control fibroblasts had hydrolyzed it about 80%. This finding is in contrast with observations made on fibroblasts with other sphingolipidoses which showed near-zero degradation in corresponding substrate-loading tests. In fibroblasts from patients with either globoid cell leukodystrophy of GM1-gangliosidosis, hydrolysis of lactosylceramide was fairly normal yet somewhat lower than control values on any day of culture, thereby indicating that, in the loading tests, lactosylceramide seems to be hydrolyzed with similar levels of enzyme activities by two distinct beta-galactosidases, galactosylceramidase and GM1-ganglioside beta-galactosidase.     

Benzoxazinones from Coix lachryma-jobi var. ma-yuen

The absolute configurations of 2-O-β-D-glucopyranosyl-7-methoxy-1,4(2H)-benzoxazin-3-one and three congeners isolated from Coix lachryma-jobi var. ma-yuen were determined by X-ray analysis and chemical correlation as the 2R type. ¹³C NMR spectra of all congeners and coixol were fully assigned.