Comparative study of inhibitory effects by murine interferon γ and a new bisphosphonate (alendronate) in hypercalcemic,nude mice bearing human tumor (LJC-1-JCK)

The inhibitory effect of murine interferon (muIFN) on humoral hypercalcemia in nude mice bearing lower-jaw cancer (LJC-1-JCK), in which parathyroid-hormone(PTH)-related protein is responsible for causing humoral hypercalcemia by activating bone resorption, was examined in comparison with that of a new bisphosphonate, 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate), muIFN was injected into tumor-bearing nude mice for 5 days before the establishment of hypercalcemia. The increase of plasma calcium concentration was delayed and this effect continued for more than 6 days even after the injection was stopped. Alendronate markedly suppressed hypercalcemia in tumor-bearing nude mice but this inhibitory effect continued for less than 6 days. Neither muIFN nor alendronate affected the tumor volume or serum PTH-related protein concentration. Injection of muIFN into mice for 3 days almost completely abolished the formation of multinucleated osteoclast-like cells from bone marrow cells in vitro, whereas injection of alendronate into mice had no effect. These findings suggested that muIFN suppressed the formation of osteoclasts, resulting in the prolonged decrease of plasma calcium concentration in hypercalcemic tumor-bearing nude mice, whereas alendronate is cytotoxic to functionally mature osteoclasts and inhibited osteoclastic bone resorption, resulting in a marked decrease in the plasma calcium concentration in tumor-bearing hypercalcemic nude mice.     

Construction of Mutant Genes for a Non-Toxic Verotoxin 2 Variant (VT2vp1) of Escherichia coli and Characterization of Purified Mutant Toxins

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed.     

Isolation of Expressed Sequences Encoded by the Human Xq Terminal Portion Using Microclone Probes Generated by Laser Microdissection

The genes that cause a variety of neurologic and neuromuscular disorders have been mapped to the distal region of Xq. In an effort to isolate genes from this area, a regional genomic library of the distal 30% of Xq was constructed from a single metaphase spread by means of laser microdissection and single unique primer-polymerase chain reaction. Using pooled probes of 1000 clones from the genomic library, human brain cDNA libraries were screened for expressed sequences encoded by this region. From the 250,000 cDNA clones screened so far, 10 nonoverlapping sequences that mapped back to the target portion were isolated. The complete nucleotide sequences of these cDNA clones have been determined. Analysis of the sequences indicates that none has significant similarity to previously characterized primate genes. One sequence mapping to Xq27.3-qter contained an open reading frame of 281 amino acids and was expressed in every tissue tested. This gene, as well as others isolated in this manner, may prove to be a candidate gene for heritable disorders mapping to this region.     

Application of fuzzy reasoning to control of glucose and ethanol concentrations in baker's yeast culture

Fuzzy reasoning was applied to control both ethanol and glucose concentrations in fed-batch cultures of baker's yeast. This fuzzy controller consisted of three membership functions (concentrations of dissolved oxygen (DO), ethanol and glucose) and 18 production rules. Fuzzy inference was carried out by IF {A is a and B is b,...#x007D;, THEN {C is c} from the on-line measured concentrations of DO, ethanol and glucose. When medium concentrations of ethanol and glucose in fed-batch culture of baker's yeast were set at 2 g/l and 0.2 g/l, both ethanol and glucose concentrations were controlled at 2.67±0.35 g/l and 0.27±0.25 g/l, respectively, ethanol production was reduced from 26 g/l to 34 g/l, cell yield increased from 0.38 to 0.53 g dry cell/g consumed glucose and ethanol yield decreased from 0.50 to 0.14 g ethanol/g consumed glucose, respectively, as compared with those of the glucose only control at 0.2 g/l.     

Serum-free culture of rat keratinocytes

Summary Procedures for the serum-free culture of rat keratinocytes have been established. Basal cells prepared from epidermis of newborn rat were stored in liquid nitrogen and used for primary culture. Among the available media, MCDB 153, developed originally for human keratinocyte (HK) culture, was the best for the development of serum-free formulation. To grow rat keratinocytes, bovine serum albumin was arbitrarily substituted for the macromolecule supplements needed for HK culture, i.e. fetal bovine serum protein or bovine pituitary extract. Qualitative and quantitative adjustment of supplements was thereafter made to support rapid cell growth. Satisfactory cell growth was achieved in the optimized medium of MCDB 153 supplemented with growth factors and amino acids: insulin (10 μg/ml), hydrocortisone (0.1 μg/ml), epidermal growth factor (25 ng/ml), calcium chloride (0.2 mM), histidine (0.23 mM), isoleucine (0.05 mM), tryptophane (0.015 mM), threonine (1.25 mM), tyrosine (0.031 mM), alanine (4.08 mM), and albumin (2 mg/ml). This optimized culture system was superior to the original HK culture condition for rapid growth of rat keratinocytes. Under our condition, cells grew as a monolayer, becoming confluent, but without stratification, and were passaged 2 to 3 times without any changes in morphology. The serum-free formulation allows us to control more accurately the concentrations of biomolecules in the medium including lipids and hormones, and therefore will be suitable for the study focusing on lipid metabolism or hormonal regulation of rat keratinocytes.     

The development of emotion regulation ability between the ages of 13 and 18 months in Japanese infants during the strange situation as a function of mothers' styles of emotion expression

The purposes of this study were two-fold. First, it compared Japanese infants' (N=129) abilities to regulate emotions at 13 months and 18 months of age, using the Strange Situation procedure. Second, it examined the relationship between the development of emotion regulation and the mother's emotion expression style as assessed by the Emotion Expression Style Questionnaire (EESQ). The total number of subjects who successfully completed all 8 episodes of the Strange Situation procedure increased significantly during the aging between 13 and 18 months of age, indicating that as a group these infants increased their ability to cope with stressful situations. However, infants who had mothers with negative emotion expression styles did not show greater capacity for emotion regulation at 18 months. These findings suggest that the development of emotion regulation is mediated by the mother's emotion expression style.     

Cytotoxicity Induced by Monoclonal Antibody to α-Fetoprotein Coadministered with Spleen Cells of Nude Mice

This paper describes an attempt to effectively induce antibody-dependent cell-mediated cytotoxicity (ADCC) in nude mice. A monoclonal antibody against α-fetoprotein, 80G, coadministered with spleen cells from other nude mice bearing HuH-7N (xenograft of human hepatoma cell line, HuH-7) significantly suppressed the growth of HuH-7N as compared to treatment with 80G alone. 80G with spleen cells from normal nude mice also had some suppressive effect. In contrast, no effect was observed with each spleen cells alone as well as 80G alone. These results suggest that further supply of effector cells could enhance ADCC activity in nude mice.     

Cloning and Sequencing of Two New Verotoxin 2 Variant Genes of Escherichia coli Isolated from Cases of Human and Bovine Diarrhea

We cloned and sequenced two new Verotoxin 2 (VT2) variant genes: one from an Escherichia coli strain from a case of bovine diarrhea and the other from an E. coli strain from a patient with diarrhea. The nucleotide and amino acid sequences of these two genes were highly homologous with, but distinct from those of the VT2, VT2vha, VT2vhb, SLT-IIv (VT2vp1) and SLT-IIva (VT2vp2) genes. Their nucleotide sequences were much more closely homologous to that of VT2vh than to that of VT2vp. Search for these two new genes in other Verocytotoxin-producing E. coli strains resulted in the isolation of 2 strains carrying one of the new VT2 variant genes, one strain from Tokyo and the other from Canada.     

Genetic variation in the Y chromosome and sex-biased DNA methylation in somatic cells in the mouse

Mammalian Genome - Several lines of evidence suggest that the presence of the Y chromosome influences DNA methylation of autosomal loci. To better understand the impact of the Y chromosome on...