Pre-clinical evaluation of a new orally-active CCK-2R antagonist, Z-360, in gastrointestinal cancer models.

BACKGROUND: Gastrin has a role in gastrointestinal (GI) malignancy. This study provides pre-clinical evaluation of a novel, orally-active gastrin/cholecystokinin-2 receptor (CCK-2R) antagonist, Z-360. METHODS: (125)I gastrin-17 (G17) displacement and G17-stimulated calcium assays were used in classical CCK-2R-transfected cell lines. Akt phosphorylation was assessed by Western blotting. Z-360 efficacy in vivo was evaluated in three human xenograft models, and microvessel density and apoptosis in these models were investigated by immunohistochemistry. RESULTS: Z-360 inhibited (125)I G17 binding to cells expressing CCK-2R, and G17-stimulated signalling. Reduced Akt phosphorylation in an oesophageal cell-line treated with Z-360 was reversed by co-treatment with G17. Z-360 increased survival in a gastric ascites model (p=0.011) and decreased tumour growth in a hepatic metastasis model (81%, p=0.02). In an orthotopic pancreatic model, Z-360 combined with gemcitabine decreased final tumour weight compared to single agents (84%, p=0.002) and there was increased apoptosis and decreased microvessel density in ex vivo tumour tissue. CONCLUSIONS: These results show that the orally-active CCK-2R antagonist, Z-360 has high sub-nM affinity for classical CCK-2R, is well tolerated in vivo and exerts an anti-tumour effect.

Molecular cloning and functional analysis of three subunits of yeast proteasome.

The genes encoding three subunits of Saccharomyces cerevisiae proteasome were cloned and sequenced. The deduced amino acid sequences were homologous not only to each other (30 to 40% identity) but also to those of rat and Drosophila proteasomes (25 to 65% identity). However, none of these sequences showed any similarity to any other known sequences, including various proteases, suggesting that these proteasome subunits may constitute a unique gene family. Gene disruption analyses revealed that two of the three subunits (subunits Y7 and Y8) are essential for growth, indicating that the proteasome and its individual subunits play an indispensable role in fundamental biological processes. On the other hand, subunit Y13 is not essential; haploid cells with a disrupted Y13 gene can proliferate, although the doubling time is longer than that of cells with nondisrupted genes. In addition, biochemical analysis revealed that proteasome prepared from the Y13 disrupted cells contains tryptic and chymotryptic activities equivalent to those of nondisrupted cells, indicating that the Y13 subunit is not essential for tryptic or chymotryptic activity. However, the chymotryptic activity of the Y13 disrupted cells is not dependent on sodium dodecyl sulfate (SDS), an activator of proteasome, since nearly full activity was observed in the absence of SDS. Thus, the activity in proteasome of the Y13 disrupted cells might result in unregulated intracellular proteolysis, thus leading to the prolonged cell cycle. These results indicate that cloned proteasome subunits having similar sequences to the yeast Y13 subunit are structural, but not catalytic, components of proteasome. It is also suggested that two subunits (Y7 and Y8) might occupy positions essential to proteasome structure or activity, whereas subunit Y13 is in a nonessential but important position.

Taste bud contains both short-lived and long-lived cell populations.

Taste bud cells undergo continual turnover even in adulthood, and their average lifespan has been estimated as approximately 10 days. However, it is not clear whether this figure can be applied to all the different cell types contained in a taste bud. Here, we describe the age and life cycle of taste bud cells in rat circumvallate papillae, and indicate that the lifespan is heterogeneous, ranging from 2 days to over 3 weeks. Taste bud cells were incorporated from the basal proliferative layer in 1-2 days after birth. After incorporation, approximately half of the cells were eliminated within 2-3 days, and the remaining half were maintained with gradual decrease, suggesting that there are at least two types of cells; short-lived cells and long-lived cells. Moreover, above 10% of the incorporated cells were maintained at 3 weeks. In order to gain information about the relationship between the cell functions and the cell age, we carried out double-labeling experiments using 5-bromo-2'-deoxyuridine and each of two markers for in situ hybridization: mammalian achaete-scute homolog 1 (Mash1) and phospholipase C beta 2 (PLCbeta2) as markers of early differentiation and functional taste signaling, respectively. Mash1 expression began immediately after the incorporation and reached a maximum at 5-6 days after birth. Fewer but distinct Mash1-positive cells were still observed after 3 weeks. PLCbeta2 expression was observed from day 5, reached a maximum at day 12, and continued over 3 weeks. Taken together, a taste bud contains both short-lived and long-lived cells: the short-lived cells are eliminated in a time course similar to the surrounding epithelial cells, and the long-lived cells including taste receptor cells have a lifespan longer than the previous estimation.

Rat calpastatin has diverged primary sequence from other mammalian calpastatins but retains functionally important sequences.

Rat calpastatin was cloned for cDNA and sequenced. It comprises 603 amino acid residues and contains four repeats of approx. 140 amino acid residues, each of which has TIPPxYr sequence responsible for calpain-inhibitory activity. However, rat calpastatin has three deletions of 30-40 amino acid residues in the nonessential regions for the inhibitory activity, and consequently, the deduced molecular size is significantly smaller than those of other mammalian calpastatins.

Molecular cloning of cDNAs for two Xenopus proteasome subunits and their expression in adult tissues.

Proteasome, a large protein complex with ATP-dependent protease activities, is composed of non-identical but closely related multi-subunits. Using cDNAs for rat proteasome subunits as probes, we obtained three cDNA clones for two Xenopus proteasome subunits from ovary cDNA library. The primary structures of the three cDNAs showed high homology to the corresponding proteasome subunits of other mammalian species (above 90%) and also considerable homology to those of Drosophila and yeast. These results indicate that the sequences of proteasome subunits are well conserved during evolution. Northern blot hybridization revealed that RNAs for the newly isolated subunits (XC8 and XC9) and the previously isolated subunit (XC3) occur at very high levels in testis and ovary, at moderately high levels in lung, skin kidney and spleen, and at low levels in liver, stomach and muscle. It was also shown that relative amounts of the mRNAs for the three subunits are similar in all the adult tissues examined. From these results, we concluded that the expression of the genes for the three subunits (XC3 XC8 and XC9-1) takes place in a roughly coordinated manner in different adult tissues.

Molecular cloning of a fish gene encoding a novel seven-transmembrane receptor related distantly to catecholamine, histamine, and serotonin receptors.

A genomic DNA fragment encoding a G protein-coupled seven-transmembrane receptor was isolated from Medaka fish, Oryzias latipes. The encoded protein is similar in sequence to other receptors including catecholamine, histamine and serotonin receptors. However, the similarity is much lower than those among members of these receptor subfamilies, thus suggesting this seven-transmembrane receptor to be an orphan receptor whose ligand has not yet been identified. Genomic Southern blot analysis suggested that the fish genome contains additional receptor genes related to the isolated gene, indicating that this novel receptor, possibly with its related receptors, might constitute a novel subfamily of the seven-transmembrane receptor superfamily.

Identification of two alpha-subunit species of GTP-binding proteins, Galpha15 and Galphaq, expressed in rat taste buds.

We cloned cDNAs for two G protein alpha-subunits belonging to the Galphaq family, each capable of activating PLCbeta, from rat tongue. One is a Galphaq in the narrow sense, and the other, termed rat Galpha15, is a rat counterpart of mouse Galpha15, sharing an amino acid sequence similarity of 94%. RT-PCR and Northern blot analysis demonstrated that rat Galpha15 and Galphaq were distinctly expressed in tongue epithelia containing taste buds. Immunostaining also showed that rat Galpha15, together with the Galphaq, was localized mainly in taste buds. These studies suggest the possibility that these two Galpha proteins function for taste signal transduction in sensory cells.

Organization and primary sequence of multiple genes coding for the apopolysialoglycoproteins of rainbow trout.

We have shown that the mRNAs for apopolysialoglycoproteins (apoPSGP) of rainbow trout contain various numbers of a repetitive sequence of 39 base-pairs encoding mature apoPSGP, and that this sequence is bordered by highly homologous 5' and 3' regions encoding pre-, pro- and telopeptides. These mRNAs are thought to be transcribed from different genes that constitute a large multiple gene family (more than 100 members). Here, we have determined the structures of several members of the apoPSGP gene family. The results show that two of three genomic DNA fragments contain two independent apoPSGP genes in the same orientation with unrelated sequences intervening. Five characterized genes have essentially the same organization and sequence. Each gene has four exons, and CAAT and TATA sequences were found in the 5'-flanking regions. However, two noteworthy differences were observed among the five genes; a diversity in the number of the 39 base-pair repeats, also observed among the cDNA clones, and a one-base polymorphism in the 39 base-pair repeat, which causes an amino acid change. This polymorphism was not detected among the cDNA clones obtained. The boundary positions of the genes are various and contain no transposon-like structures. The variation in the number of repeats and the absence of a rule for bordering positions of the genes suggest that apoPSGP genes may have been amplified by gene duplications, unequal recombination, and selection of chromosomes having larger numbers of apoPSGP genes.

A Drosophila homolog of human proto-oncogene ret transiently expressed in embryonic neuronal precursor cells including neuroblasts and CNS cells.

We have identified a Drosophila gene encoding a putative receptor tyrosine kinase by screening a genomic DNA library with a DNA probe for a Drosophila homolog of fibroblast growth factor receptors. The newly isolated gene codes for a transmembrane protein most similar in sequence to a mammalian proto-oncogene ret; thus, the gene was termed Dret. Dret mRNA is transcribed in very small amounts in the embryonic, larval, and pupal stages. Whole mount in situ hybridization experiments revealed that the mRNA is transiently expressed in neuroblasts in early embryos. In late embryos, Dret mRNA was detected in subpopulations of differentiating CNS and PNS cells. In addition, Dret expression was affected in neurogenic mutants. These results suggest that Dret can be considered as a functional homolog of mammalian ret and should play important roles in neurogenesis.

Cloning and sequence analysis of the genomic DNA fragment encoding oryzacystatin.

A genomic DNA clone encoding oryzacystatin (Oc), a cysteine proteinase inhibitor (cystatin) of rice, was isolated from a lambda EMBL3 phage library constructed with Sau3AI partial digests of rice chromosomal DNA, by screening with an oc cDNA as a probe. The restriction map of the isolated DNA fragment was consistent with the pattern of the genomic Southern-blot analysis using a cDNA probe, and consequently, the gene is considered to be a single-copy gene. The oc gene is about 1.4 kb long and composed of three exons and two introns. The first intron (336 bp) intervenes between Ala-38 and Asp-39. The second intron (372 bp) exists in the 3'-noncoding region at the G residue next to the stop codon. S1 nuclease mapping showed the major transcription start point (tsp) at A, 104 bp upstream from the start codon (ATG). Typical CAT and TATA box sequences were found in the 5'-upstream region of the tsp. The nucleotide sequences around the TATA box, the tsp, the start codon, and the stop codon essentially matched the consensus sequences of other higher plant genes. The intron boundaries of the oc gene were quite different from those of the human kininogen-encoding gene and the human salivary cystatin (cystatin S)-encoding gene.

Cloning of cDNA and genomic DNA encoding fibroblast growth factor receptor-4 of Xenopus laevis.

We have isolated and characterized the cDNA and genomic DNA encoding fibroblast growth factor receptor-4 of Xenopus laevis (XFGFR-4). The gene encompassing the total coding sequence spans about 10 kb, consists of 17 exons, and has an organization very similar to those of mammalian genes encoding FGFR-1 and -2, except that the XFGFR-4 gene does not contain an alternative exon for the third immunoglobulin-like domain nor an internal poly(A)-addition site. Thus, XFGFR-4 appears not to generate multiple forms of mRNA, as are identified for the mammalian FGFR-1, -2 and -3 genes. The amino-acid sequence of XFGFR-4 shows high homology to other vertebrate FGFR-4 species, but the similarity was significantly lower than in the cases of FGFR-1 and -2. Northern blot analysis showed the XFGFR-4 mRNA to occur throughout X. laevis early embryogenesis in a profile different from those of X. laevis FGFR-1 and -2.

Distinct expression of two Drosophila homologs of fibroblast growth factor receptors in imaginal discs.

The expression of two Drosophila homologs of FGF receptors (DRF1 and DRF2) in imaginal discs was studied. DFR1 mRNA was observed in several imaginal discs, whereas DFR2 mRNA was not detected. DFR1 expression in the wing and leg discs took place in probable myoblasts in a pattern similar to that of twist, a mesodermal gene. The mRNA was also detected in the morphogenetic furrow and its posterior region of the eye disc and around the proliferation center of the brain. These results suggest that DFR1 is involved in the development of mesodermal and neuronal cells constituting the adult body.

Identification of seven novel protein-tyrosine kinase genes of Drosophila by the polymerase chain reaction.

We used the polymerase chain reaction to identify 7 novel tyrosine-kinase genes (dtk1 to -7) in Drosophila melanogaster, dtk4 coded for a part of the kinase catalytic domain nearly identical in sequence to that of the human receptor for insulin-like growth factor 1, whereas sequences encoded by dtk1 and dtk2 were highly homologous to that of the chicken fibroblast growth factor receptor.

Identification of four FGF receptor genes in Medaka fish (Oryzias latipes).

Four types of cDNA clones encoding tyrosine kinases highly homologous to mammalian fibroblast growth factor receptors (FGF-R) were isolated from Medaka fish (Oryzias latipes) by the reverse transcription-polymerase chain reaction. Comparison of the four deduced amino acid sequences with four known mammalian FGF-Rs indicated that four FGF-R species corresponding to mammalian FGF-Rs exist universally in vertebrates including fishes, although FGF-R4 might have diverged sequences between fishes and mammals. Each of four FGF-R genes is transcribed to various extents as multiple mRNAs possibly by alternative splicing in adult fishes.

Virion-associated RNA polymerase of bacteriophage phi 6 synthesizes three complete transcripts of double-stranded RNA genome in vitro.

Three single-stranded RNA transcripts synthesized in vitro by a virion-associated RNA polymerase of bacteriophage phi 6 were sequenced at their 5'- and 3'-termini. The sequences agreed with those of the + strands of the 3 double-stranded RNA segments [FEBS Lett. (1982) 141,111-115]. The results show that the transcription by phi 6 RNA polymerase initiates exactly at the 3'-ends of the template RNAs (-strands of the genomic RNA) and terminates exactly at the 5'-ends.

Gene organization of oryzacystatin-II, a new cystatin superfamily member of plant origin, is closely related to that of oryzacystatin-I but different from those of animal cystatins.

The gene structure of oryzacystatin-II, a new cystatin superfamily member of rice seed origin, was determined. It spans approximately 2.5 kbp and comprises 3 exons. The number of exons and the intron-breakpoints coincide with those of oryzacystatin-I, the first well-defined plant cystatin. However, no similar sequences were observed between the two oryzacystatin genes in 5'-upstream regulatory regions, even though both are expressed specifically during the ripening stage of rice seeds. The gene organization of these two plant cystatins is generally different from that of animal cystatins.

Gene structure of calcium-dependent protease retains the ancestral organization of the calcium-binding protein gene.

The gene structure of calcium-dependent protease (Ca2+-protease) was determined. It comprises at least 21 exons, and these were assigned to the 4 functional domains of the protease. The protease domain does not show clear correlation between exons and functional units, but the calmodulin-like calcium-binding domain shows strong correlation. Each of the 4 consecutive calcium-binding regions in the C-terminal part of Ca2+-protease is encoded by one exon. This gene structure supports the idea that the 4 calcium-binding regions of calcium-binding proteins such as calmodulin arose by 2 steps of gene duplication.

Multiple genes for G protein-coupled receptors and their expression in lingual epithelia.

Using the polymerase chain reaction (PCR), we identified a gene family including more than 60 members which encoded similar G protein-coupled seven-transmembrane receptors. Sequence analyses of six representatives out of the 60 PCR clones showed that they had significant structural similarity to olfactory and optic receptors. Their expression is restricted in the surface of lingual epithelia.

An ice nucleation active gene of Erwinia ananas. Sequence similarity to those of Pseudomonas species and regions required for ice nucleation activity.

The ice nucleation active gene, inaA, of Erwinia ananas IN-10 has been sequenced. This gene encodes a protein composed of 1322 amino acid residues. The inaA protein contains a 1120-residue segment consisting of 70 repeats of closely related 16 amino acid motifs (R-domain), which is flanked by N- and C-terminal sequences (N- and C-domains, respectively). Its primary structure is similar to, but not identical with, those of Pseudomonas inaW and inaZ gene products. By truncating the inaA gene to various extents, it was found that deletion of the C-domain resulted in complete loss of the ice nucleation activity, whereas removal of the N-domain led to a moderate decrease in the activity. Complete loss of the activity was also observed when the N-domain plus a large part of the P-domain were deleted. It is suggested that the C-domain is required for the assembly of inaA protein to form a functional ice nucleus.

Complete amino acid sequence of the large subunit of the low-Ca2+-requiring form of human Ca2+-activated neutral protease (muCANP) deduced from its cDNA sequence.

The complete amino acid sequence of the large subunit (catalytic subunit) of human low-Ca2+-requiring-calcium-activated neutral protease (muCANP) was deduced from its cDNA base sequence. It is composed of 714 amino acid residues and its sequence is highly homologous to the chicken CANP sequence determined previously. Human muCANP, like chicken CANP, has a clear 4-domain structure, and their fundamental structures are essentially the same, although their Ca2+ sensitivities are significantly different. The role of each domain in the Ca2+ sensitivity and protease activity of CANP is discussed on the basis of sequence comparison.