Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov.

Based on the 16S rRNA sequences, DNA-DNA homology values, cellular lipid and fatty acid composition, and phenotypic characteristics, a new genus Burkholderia is proposed for the RNA homology group II of genus Pseudomonas. Seven species in this group were transferred to the new genus. Thus seven new combinations, Burkholderia cepacia (Palleroni and Holmes 1981), Burkholderia mallei (Zopf 1885), Burkholderia pseudomallei (Whitmore 1913), Burkholderia caryophylli (Burkholder 1942), Burkholderia gladioli (Severini 1913), Burkholderia pickettii (Ralston et al 1973) and Burkholderia solanacearum (Smith 1896) were proposed.     

Identification of a CA repeat at the TCRA locus using yeast artificial chromosomes: a general method for generating highly polymorphic markers at chosen loci.

The creation of a comprehensive genetic map in human has been limited by the lack of highly polymorphic markers spaced evenly throughout the human genome. We have utilized yeast artificial chromosomes (YAC) containing large human DNA inserts to help identify highly polymorphic (CA)n repeats at a chosen locus. The DNA of a YAC containing the locus was subcloned in M13 vectors, and the recombinants were screened at high stringency to detect preferentially long (CA)n repeats (n greater than 20). These repeats, which are the most likely to be highly polymorphic, were then studied to confirm both the level of polymorphism and their precise genetic location. This strategy has permitted the identification of a new, highly polymorphic CA repeat (77% heterozygosity) at the T cell receptor alpha chain (TCRA) locus on chromosome 14q. It provides a powerful marker for assessing the role of this locus in the susceptibility to autoimmune and infectious diseases. This approach should permit the development of highly polymorphic markers at any targeted locus and rapidly improve the current human genetic map.     

Antitumor effect of RBS (rice bran saccharide) on ENNG-induced carcinogenesis

We examined whether orally administered RBS (rice bran saccharide), prepared from rice bran by hot water extraction, increases immunocompetence, inhibits gastrointestinal carcinogenesis with N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) or shows an antitumor effect. After the administration of RBS, phytohemagglutinin (PHA)- and pokeweed mitogen (PWM)-stimulated blastogenesis of lymphocytes derived from the mesenteric lymph nodes and peripheral blood was enhanced, and the helper/ suppressor T-cell ratio was elevated, and migration activity of peritoneal macrophages was also increased in rats treated continuously with ENNG. ENNG-induced gastrointestinal carcinomas were observed in 43% of those administered RBS (ENNG-RBS) as compared with 88% in the control (ENNG) and 94% in the prednisolone (PRD) group (ENNG-PRD). The 12-month survival rate of rats bearing gastrointestinal cancer was 58% in the ENNG-RBS group as compared with 25% in the ENNG group and 15% in the ENNG-PRD group. RBS prevented the reduction in immunocompetence in the course of carcinogenesis, suppressed carcinogenesis, and prolonged the survival of rats with gastrointestinal cancer. Antitumor activities of RBS are thought to be a kind of host mediated action. The growth inhibition ratio of transplantable ENNG-induced cancer in Wistar rats was 42.1% in the RBS and 51.8% in the 5-FU group. Since little is known about the potent antitumor activity of -glucan, it would be interesting to consider the relationship between the structure and the biological activities of polysaccharides.     

The combined effect of lymphokine activated killer cell and radiation therapy on rat brain tumorin vitro

Thein vitro effect of a combined treatment with lymphokine activated killer (LAK) cell and radiation therapy on rat brain tumor was examined using⁵¹Cr release assay. The tumor cell-line used in this experiment was 9L rat brain tumor derived from a Fischer 344 rat. LAK cells were obtained by culturing rat lymphocytes with recombinant human interleukin 2 for at least 3 days. The cytotoxic activity of the LAK cells was examined by⁵¹Cr release assay. Irradiation was done by exposing the microtiter plate in which the¹⁵Cr labeled 9L cells and LAK cells were cultured to a¹³⁷Cs gamma cell unit. Without irradiation, there was 18% cytotoxicity in the 1:100 tumor-to-LAK cell ratio specimen after 24 hrs cocultivation. However, if 5 Gy of irradiation was given, followed by 12 hrs incubation, the cytotoxicity was enhanced significantly at the same cell ratio (30%). This enhancement effect was the most prominent when the cell ratio was 1:100 and the irradiation dose was 5 Gy. To generate the enhancement effect, an incubation time of over 8 hrs both before and after irradiation was required. The supernatant of the LAK cells showed 19.8% and 11.4% cytotoxicity with and without irradiation, respectively. This result indicates the participation of a cytotoxic factor released from LAK cells.This work is supported in part by grant from Univeristy of Tsukuba Project Research.     

Increase in serotonin 5-HT1A receptors in prefrontal and temporal cortices of brains from patients with chronic schizophrenia.

Binding studies with [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT), a specific serotonin1A (5-HT1A) receptor agonist, were done on the autopsied brains from control subjects and from patients with chronic schizophrenia. All the patients and controls were of the Japanese race. In the controls, representative Scatchard plots for the specific [3H]8-OH-DPAT bindings in the prefrontal cortex and hippocampus revealed a single component of high affinity binding site (Kd value = 5.7 and 5.9 nM, Bmax value = 80.1 and 101.0 fmol/mg protein, respectively). The [3H]8-OH-DPAT bindings to the prefrontal cortex and hippocampus were potently inhibited by serotonin (IC50 = 6.3 x 10(-9) M) and 5-HT1A agonists (IC50 = 5.0 x 10(-9) - 2.3 x 10(-7) M), while other neurotransmitters, 5-HT2 and 5-HT3 related compounds did not inhibit the binding (IC50 greater than 10(-5) M). The bindings were decreased in the presence of 0.1mM GTP and 0.1mM GppNHp but not in the presence of 0.1mM GMP. In the prefrontal and temporal cortices of schizophrenics, there was a significant increase in the specific [3H]8-OH-DPAT binding, by 40% and 60%, respectively, with no change in the hippocampus, amygdala, cingulum, motor cortex, parietal or occipital cortex, as compared to findings in the controls. Scatchard analysis showed that this increased binding reflects changes in the number of sites but not in the affinity. The effect of 0.1mM GppNHp on the binding to prefrontal cortex was observed in both controls and schizophrenic patients. The bindings were significantly greater in the schizophrenic patients than in controls, in the presence of 0.1mM GppNHp. Our findings suggest that there are GTP-sensitive 5-HT1A sites in the human brain and that selective increases in GTP-sensitive 5-HT1A sites in the prefrontal and temporal cortices of schizophrenics relate to the pathophysiology of schizophrenia.     

TEI-9063, a stable and highly specific prostacyclin analogue for the prostacyclin receptor in mastocytoma P-815 cells.

The prostacyclin (PGI2) analogues, TEI-9063 and its methyl ester, TEI-1324, have been compared with another stable analogue, iloprost, with respect to binding to the PGI2 receptor, stimulation of adenylate cyclase activity and inhibition of thrombin-induced Ca2+ mobilization in mastocytoma P-815 cells. TEI-9063 displaced the [3H]iloprost binding to the membrane fraction, the IC50 value being 3 nM, but showed very low affinity for the PGE receptor. TEI-9063 dose dependently stimulated cAMP formation in the cells and GTP-dependent adenylate cyclase activity in the membrane fraction, the EC50 value being 50 and 10 nM, respectively. Furthermore, TEI-9063 prevented the thrombin-induced increase in the intracellular Ca2+ concentration, the IC50 value being 50 nM. These IC50 and EC50 values are lower than those obtained for iloprost. On the other hand, those of TEI-1324 were about two-orders higher. Although PGI2 lost its ability to stimulate cAMP formation by preincubation for 20 min at 37 degrees C, TEI-9063 completely retained its ability after 60-min preincubation. These results demonstrate that TEI-9063 is a stable and stronger agonist for the PGI2 receptor than iloprost, and that it prevents thrombin-induced Ca2+ mobilization through stimulation of the adenylate cyclase system in mastocytoma cells.     

Changes of carp myosin subfragment-1 induced by temperature acclimation

Summary Heavy meromyosin subfragment-1 (S1) was prepared by -chymotrypsin from myosin of carp acclimated to either 10°C or 30°C for a minimum of 5 weeks. The objective of these studies was to document thermally-induced changes in the myosin molecule and to extend previous observations. Ca²⁺- and K⁺ (EDTA)-ATPase activities of cold-acclimated carp S1 were 1.1 and 0.8 mol P_i·min^-1·mg^-1, respectively, and these values did not differ significantly from those of warm-acclimated carp. The inactivation rate constant (K_D) of S1 from cold-acclimated carp was 32.1x10^-4· s^-1, compared to 13.2x10^-4·s^-1 for warm-acclimated carp. The maximum initial velocity of acto-S1 Mg²⁺-ATPase activity at pH 7.0 in 0.05 M KCl was 9.3 s^-1 with cold-acclimated carp, about 3.7 times higher than that for warm-acclimated carp. However, no significant difference was observed in the apparent affinity of S1 to actin. Peptides maps of the heavy chain of S1 were different and suggested distinct isoforms for the myosins from warm- and cold-acclimated muscle.Abbreviations ATPase adenosine 5-triphosphatase - DTNB 5,5-dithiobis (2-nitrobenzoic acid) - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol bis (-aminoethylether)-N,N,N,N-tetraacetic acid - K _D inactivation rate constant - K _m apparent dissociation constant - P _i inorganic -phosphate - PMSF phenylmethane-sulfonyl fluoride - S ₁ heavy meromyosin subfragment-1 - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis - TPCK N-tosyl-l-phenylalanyl chloromethyl ketone - V _max maximum initial velocity     

Two monoclonal antibodies against small-cell lung cancer show existence of synergism in binding

Summary Murine IgG1 monoclonal antibodies (mAbs), ITK-2 and ITK-3, were generated against a small-cell lung cancer (SCLC) cell line. Enzyme-linked immunosorbent assay using a variety of established cell lines as substrates, immunoperoxidase staining of freshly frozen tissue sections, and fluorescence-activated cell sorter analysis of peripheral blood leukocytes showed that these mAbs recognize a part of the SCLC-associated cluster 1 antigen. In immunoprecipitation studies, both ITK-2 and ITK-3 bound to a 145-kDa glycoprotein of SCLC cell membrane extracts, as did MOC-1 and NKH-1, which both recognize the cluster 1 antigen. However, because the binding of¹²⁵I-labeled ITK-2 to SCLC cells was not inhibited by MOC-1 or NKH-1, the binding site of ITK-2 on SCLC cells appeared to be different from that of either MOC-1 or NKH-1. Unexpectedly, binding of¹²⁵I-labeled ITK-2 to SCLC cells increased in the presence of ITK-3. This ITK-3-induced increase in ITK-2 binding was due partly to an increase in the number of binding sites for ITK-2 on SCLC cells. Addition of ITK-3 may, therefore, improve the effectiveness of ITK-2-based tumor detection or therapy.     

Detection of tetrodotoxin by thin-layer chromatography/fast atom bombardment mass spectrometry

A new method for detection of tetrodotoxin (TTX) by thin-layer chromatography/fast atom bombardment (FAB) mass spectrometry was developed. TTX and/or related substances were separated by TLC on LHP-K high-performance precoated plates, with a solvent system of pyridine:ethyl acetate:acetic acid:water (15:5:3:4). The plates were subjected to positive FAB mass spectrometry, under scanning within a mass range from m/z 100 to 500. TTX was identified by selected ion-monitored chromatograms at m/z 320 (M + H)+ and 302 (M + H - H2O)+, along with full scan positive ion FAB mass spectrometry. The limit of detection for TTX was about 0.1 micrograms. TTX was also detected by cellulose acetate membrane electrophoresis/FAB mass spectrometry.