An attempt to isolate genes responsible for spontaneous and experimental metastasis in the mouse model

Cancer develops and progresses as genetic alterations occur subsequently. Onset process of cancer has become well understood in some types of cancer, such as colorectal cancers. In this process, responsible alterations were identified in numbers of oncogenes such as k-ras, and tumor suppressor genes such as p53, as Vogelstein proposed earlier in the multistage carcinogenesis theory. In contrast, our understanding remains short to draw such an adequate diagram for the process during which cancer becomes more malignant, i.e., metastatic. To examine the molecular basis for this progression step, mouse metastasis models have been established where tumor cell lines are inoculated into mice and metastasize to specific organs. The model using B16 melanoma cells is one of the most developed. BL6 subline, one of the most metastatic, was obtained from F10 subline simply through six rounds of in vitro selection. Nonetheless, BL6 cells metastasize lungs much more heavily than F10 cells when injected subcutaneously. The difference in gene expression between the two sublines is considered rather small but relevant for spontaneous metastasis. We began our research by elaborating a method for the construction of subtracted cDNA libraries, and made it applicable to BL6 and F10 cells. As a result, we were able to isolate a couple of genes that were expressed differently between the two sublines. As might be expected, each of the genes appeared to play a role more or less in distinct aspects of spontaneous metastasis of B16 melanoma cells. Moreover, similar roles were expected for the genes in the process by which human melanoma cells metastasize.     

Kinetic and structural properties of two isoforms of trypsin isolated from the viscera of Japanese anchovy, Engraulis japonicus

Two isoforms of anchovy trypsin (aT-I and aT-II) were purified from the visceral extracts by (NH₄)₂SO₄ fractionation followed by affinity chromatography, gel filtration, and ion-exchange chromatography. The homogeneity of the purified preparation was evidenced by both native- and SDS-PAGE, and further by gelatin zymography. Identities of aT-I and aT-II as trypsins were established by N-terminal amino acid sequencing, which matched exactly to the corresponding stretches of their respective amino acid sequences obtained by molecular cloning [Ahsan et al. (2000), Marine Biotechnol., in press]. Both isoforms were completely inhibited by serine protease inhibitors as well as by specific trypsin inhibitors. The purified anchovy trypsins showed considerably higher catalytic efficiencies (k_cat/K_m) than bovine trypsin as measured toward benzoyl-arginine p-nitroanilide (BAPA) and benzoyl-arginine ethyl ester (BAEE) at 25°C; in particular, aT-II was 35 times more efficient than its mammalian counterpart against BAPA. This was due mainly to a dramatic decrease of K_m values for anchovy trypsins, which are indicative of an evolutionary response toward increased substrate binding at suboptimal temperatures in the marine environment.     

Bombyx acid cysteine protease (BCP): hormonal regulation of biosynthesis and accumulation in the ovary

We previously reported purification of the cysteine protease from Bombyx eggs (BCP) and the occurrence of the enzyme in various tissues of this insect. In the present paper, we present a detailed analysis of stage-specific changes in activity of BCP between the fourth larval instar and pupal-adult development. A synthetic fluorescent peptide, carbobenzoxy-L-Phenylalanyl-L-Arginine4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA), was used to assay proteolytic activity. When tissue extracts were treated with anti-BCP serum before assay of enzyme activity, most activity towards Z-Phe-Arg-MCA was removed from the extracts. Therefore proteolytic activity in the present experiments is due mainly to BCP. We used Western blot and Northern blot analyses to determine tissue and stage specific expression of the enzyme. In the 5th larval fat body and hemolymph, BCP activity dramatically increased at the time of spinning, returning to the basal level before ecdysis. Northern blot analysis showed that a 1.5 kilobase mRNA which hybridizes to BCPcDNA suddenly appears during this period. Similar results were obtained in 4th instar fat body. In pupal hemolymph and fat body, low basal activity of BCP was detected early (day 0 to day 3 after pupal ecdysis), followed by a pronounced increase to a maximum six days after ecdysis, before returning to the basal level. In ovariectomized female pupae, a significant amount of proteolytic activity accumulated in hemolymph, suggesting that the enzyme is synthesized in the fat body and transferred into the ovary along with vitellogenin. BCP activity increased three days after injection of 20-hydroxyecdysone into ligated pupae. Furthermore, putative BCPmRNA appeared in the fat body within 24 hours after injection. This increase was completely blocked by the administration of cycloheximide. The results suggest that, BCP is synthesized in extraovarian tissues such as fat body and ovarian follicle cells and accumulates in the ovary, thus representing a new class of yolk protein.     

Interaction of Cytotoxic Bicyclic Peptides, Theonellamides A and F, with Glutamate Dehydrogenase and 17β-Hydroxysteroid Dehydrogenase IV

Theonellamide A, a bicyclic peptide isolated from a Theonella sponge, was fixed on hydrazide-containing gel beads and screened for its binding proteins from rabbit liver tissues. Analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed that two major proteins of 80 kDa and 55 kDa interacted with theonellamide A. The interaction between theonellamide A and two proteins was confirmed by competition experiments in which these two proteins failed to bind to theonellamide A–conjugated gel beads in the presence of theonellamide A or F. Amino-terminal amino acid sequence analysis of peptide fragments derived from the binding proteins by lysylendopeptidase digestion demonstrated that the 80-kDa and 55-kDa proteins were 17β-hydroxysteroid dehydrogenase IV and glutamate dehydrogenase, respectively. In an in vitro assay system, amination of α-ketoglutarate by glutamate dehydrogenase was activated with theonellamide F, although this effect was weaker than that with adenosine diphosphate, a well-known activator. Received October 15, 1999; accepted January 4, 2000.     

The yabG gene of Bacillus subtilis encodes a sporulation specific protease which is involved in the processing of several spore coat proteins

The synthesis and proteolysis of the spore coat proteins, SpoIVA and YrbA, of Bacillus subtilis were analyzed using antisera. Almost no intact full-length proteins of either type were extracted from wild-type spores, while yabG mutant spores contained intact SpoIVA and YrbA proteins. We purified recombinant YrbA and YabG proteins from Escherichia coli transformants and found that YrbA was cleaved to the smaller moiety in the presence of YabG in vitro. These observations indicate that YabG is a protease involved in the proteolysis and maturation of SpoIVA and YrbA proteins, conserved with the cortex and/or coat assembly by B. subtilis.     

C-ABI3, the Carrot Homologue of the Arabidopsis ABI3, is Expressed during Both Zygotic and Somatic Embryogenesis and Functions in the Regulation of Embryo-Specific ABA-Inducible Genes

A carrot gene homologous to the ABI3 gene of Arabidopsis wasisolated from a carrot somatic embryo cDNA library and designatedC-ABI3. The sequence of C-ABI3 was very similar to those ofABI3 of Arabidopsis and VP1 of maize in certain conserved regions.The expression of C-ABI3 was detected specifically in embryogeniccells, somatic embryos and developing seeds. Thus, expressionof C-ABI3 was limited to tissues that acquired desiccation tolerancein response to endogenous or exogenous abscisic acid (ABA).Endogenous levels of ABA in seeds increased transiently andthen desiccation of seeds started. The expression of C-ABI3in developing seeds was observed prior to the increase in levelsof endogenous ABA that was followed by desiccation of seeds.In transgenic mature leaves in which C-ABI3 was ectopicallyexpressed, expression of ECP31, ECP63 and ECP40 was inducedby treatment with ABA, which indicates that the expression ofECP genes was controlled by the pathway(s) that involved C-ABI3and ABA. This suggests that C-ABI3 has the same function asVP1/ABI3 factor in carrot somatic embryos. (Received March 4, 1998; Accepted September 4, 1998)     

Gas transport in serpentine microporous tubes under steady and pulsatile blood flow conditions

A serpentine gas exchange unit was built with cylindrical tubular microporous membranes featuring periodic arcs with a fixed curvature ratio (ratio of tube radius to radius of curvature) of 1/14 and circular angles between 30 and 360 deg. Oxygen transfer was measured under steady and pulsatile blood flow conditions in vitro and ex vivo to assess the design features which most effectively augment gas transfer. Under steady blood flow conditions, oxygen transfer increased with circular angles beyond 70 deg. Under pulsatile conditions, a wide range of geometrical and fluid mechanical parameters could be combined to enhance gas transfer performance, which eventually depended upon the secondary Reynolds number and the Womersley parameter.     

Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling

To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10^-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10^-18 moles of the p24/assay corresponds to ca. 10³ copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (10² copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.