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PubMed2004

[Diversity of invasiveness and matrix metalloproteinases expression profile of human gastric carcinoma xenografted in different tissue environments].

OBJECTIVE: To study the effect of tissue environment on the invasiveness of carcinoma cells and the implication of expression of matrix metalloproteinases. METHODS: Tissue from a human gastric carcinoma was transplanted and passaged subcutaneously in nude mice. After the 3rd passage, the xenografts were also transplanted into the abdominal cavity of nude mice. The invasiveness of xenografts at the two locations were observed morphologically and the expressions of MMP-2, MMP-7, MMP-9, MMP-13, TM1-MMP, TM2-MMP and TM3-MMP were monitored by immunohistochemistry. RESULTS: The subcutaneous xenografts of human gastric carcinoma in nude mice presented as expanding outgrowths with limited invasion. Except for MMP-7, the other 6 MMPs (MMP-2, MMP-9, MMP-13, TM1-MMP, TM2-MMP, TM3-MMP) were not expressed in the neoplastic cells nor in the tumor stroma. In contrast, the intra-peritoneal xenografts displayed an invasive growth pattern accompanied by more fibrous stroma. All MMPs examined were expressed in the tumor cells at the invasive fronts and in the adjacent stroma. CONCLUSIONS: Invasiveness and expression of MMPs were obviously diverse in human gastric carcinoma cells when grafted at different anatomic locations in nude mice, thus indicating: (1) There exists a close interaction between tumor cells and surrounding stromal cells. The tissue environment may play a definitive role in the tumor phenotype. (2) The expression of MMPs is closely related to the growth pattern and the invasiveness of tumor cells. MMPs produced by the stroma cells at the invasion front may be linked to the invasiveness of neoplastic cells.

Animals↗
PubMed1992

Membrane topology of the pBR322 tetracycline resistance protein. TetA-PhoA gene fusions and implications for the mechanism of TetA membrane insertion.

The tetracycline resistance gene of pBR322 encodes a 41-kDa inner membrane protein (TetA) that acts as a tetracycline/H+ antiporter. Based on hydrophobicity profiles, we identified 12 potential transmembrane segments in TetA. We used oligonucleotide deletion mutagenesis to fuse alkaline phosphatase (PhoA) to the C-terminal edge of each of the predicted periplasmic and cytoplasmic segments of TetA. In general, the PhoA activities of the TetA-PhoA fusions support a TetA topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, several TetA-PhoA fusions have unexpected properties. One PhoA fusion to a predicted cytoplasmic segment (C6) has high activity. However, previous protease accessibility studies on the related Tn10 TetA protein indicated that C6 is cytoplasmically localized as predicted (Eckert, B., and Beck, C. F. (1989) J. Biol. Chem. 264, 11663-11670). PhoA fusions to three predicted periplasmic segments (P1, P2, and P5) have low to intermediate activity. In each case, the preceding transmembrane segment (TM1, TM3, and TM9) contains an aspartate (Asp17, Asp86, and Asp287). We show that these aspartates act like signal sequence mutations for PhoA export: (i) Asp----Ala mutations increase the PhoA activity of fusions to P1, P2, and P5. (ii) The signal sequence mutation suppressor prlA402 increases the PhoA activity of these same fusions. We also show that the aspartates in TM1, TM3, and TM9 are critical for wild-type TetA function; they are conserved in related TetA proteins and Asp----Ala mutations reduce or eliminate tetracycline resistance. The properties of the anomalous TetA-PhoA fusions suggest that TetA sequences C-terminal to some cytoplasmic and periplasmic segments are required for the proper localization of those segments, i.e. long range interactions may be more important in determining the membrane topology of TetA than suggested in some general models.

Alkaline Phosphatase↗
PubMed1994

Protein kinase C transiently activated heteromeric N-methyl-D-aspartate receptor channels independent of the phosphorylatable C-terminal splice domain and of consensus phosphorylation sites.

We have expressed dual subunit combinations of isoforms of the N-methyl-D-aspartate receptor, NR1A-NR2A and NR1C-NR2A, in Xenopus oocytes. We show that both forms of the receptor are stereospecifically activated by low concentrations (10 nM) of the phorbol ester 4-beta-phorbol 12-myristate 13-acetate, known to activate protein kinase C (PKC). The activation is transient, and, after reaching a maximum in about 10 min, it decreases rapidly in spite of the continuous presence of phorbol ester. The addition of 2 microM oleoylacetylglycerol had similar consequences. NR1C differs from NR1A by a deletion of 37 amino acids that include four consensus phosphorylation sites for PKC in the C-terminal region. The corresponding peptide has been shown to become phosphorylated upon activation of PKC in neurons (Tingley, W. G., Roche, K. W., Thompson, A. K., and Huganir, R. L. (1993) Nature 364, 70-73). However, the activity of NR1C-NR2A receptors was stimulated 7-fold, twice the potentiation observed for NR1A-NR2A. By site-specific mutagenesis of NR1C and NR2A, we removed additional consensus PKC phosphorylation sites located between TM3 and TM4. Coexpression of these mutant subunits showed a similar response to phorbol esters as wild type receptors. Our results indicate that neither the predicted consensus phosphorylation sites between transmembrane sequences TM3 and TM4 nor the phosphorylatable C-terminal splice domain is essential for the modulation of N-methyl-D-aspartate receptors by PKC.

Amino Acid Sequence↗
PubMed1993

Growth factor dependency and gene expression in preneoplastic mouse mammary epithelial cells.

The TM preneoplastic mammary outgrowth lines were established in vivo from mammary epithelial cell lines and have been characterized with respect to their tumorigenic, morphological, and functional properties. The TM outgrowth lines were then established as in vitro cell lines. A comparison of the growth factor dependencies of the TM preneoplastic lines and their progenitor cell lines grown in monolayer cell culture indicated that the TM preneoplastic cell lines exhibited a decreased dependence on epidermal growth factor for growth in vitro. The exception to this result was the TM3 cell line which still exhibited a marked dependence on epidermal growth factor for growth. An examination of several genes for mRNA levels indicated that the expression of c-neu, c-H-ras, c-myc, and retinoblastoma was not elevated in those TM preneoplasias which exhibited a decreased dependence on epidermal growth factor. Additionally, there was no evidence that c-H-ras was activated in the preneoplastic outgrowths or tumors. In contrast, mouse mammary tumor virus long terminal repeat mRNA was increased in preneoplasias and tumors, whereas gelsolin mRNA was decreased in tumors but not in preneoplasias. The down-regulation of gelsolin mRNA is consistent with recent reports in human breast cancers. These results together with those reported in another paper (D. Medina et al., Cancer Res., 53: 663-667, 1993) indicate that the TM3 outgrowth line is a minimally deviated preneoplasia which does not share many of the molecular alterations exhibited in tumorigenic TM preneoplastic outgrowth lines. These data, along with other recent data, are interpreted in a hypothesis which views the three essential characteristics of the mammary preneoplastic state as independent and dissociable genetic alterations. Importantly, each of the three characteristics is independently isolated in one or more of the in vivo outgrowth populations. These outgrowth lines should allow identification of the nature and function of the molecular alterations associated with each stage of mammary preneoplasia.

Animals↗
PubMed1996

The alpha and omega of G-protein coupled receptors: a novel method for classification. Part 1.

In this paper, a novel method is presented for classification of 113 neurotransmitter and opioid receptors that is based on the counting of amino acid residues. With the use of sequence alignment, ten conserved key residues were identified: alpha (the first Met residue of the sequence), Asn in the tentative first transmembrane domain (TM1), Asp (TM2), Cys (top of TM3), Arg (bottom of TM3), Trp (TM4), Cys (in the loop between TM4 and TM5), Pro (TM5), Pro (TM6), Pro (TM7) and omega (the last residue of the sequence). The number of residues between these key residues is unique for each receptor or receptor subtype and is used for classification. The number of residues between two key residues defines a segment. The sum of the segments before or after a key residue is defined as a partition. In total, 73 possible classification schemes were found using two or three segments, partitions or a combination of segments and partitions. The surprising and striking results is that each of the sequences examined can be characterized by a code consisting of two or three figures. Each figure represents a number of amino acid residues.

Amino Acid Sequence↗
PubMed1996

Hydrophilic side chains in the third and seventh transmembrane helical domains of human A2A adenosine receptors are required for ligand recognition.

Hydrophilic residues of the G protein-coupled human A2A adenosine receptor that are potentially involved in the binding of the ribose moiety of adenosine were targeted for mutagenesis. Residues in a T88QSS91 sequence in the third transmembrane helical domain (TM3) were individually replaced with alanine and other amino acids. Two additional serine residues in TM7 that were previously shown to be involved in ligand binding were mutated to other uncharged, hydrophilic amino acids. The binding affinity of agonists at T88 mutant receptors was greatly diminished, although the receptors were well expressed and bound antagonists similar to the wild-type receptor. Thus, mutations that are specific for diminishing the affinity of ribose-containing ligands (i.e., adenosine agonists) have been identified in both TM3 and TM7. The T88A and T88S mutant receptor fully stimulated adenylyl cyclase, with the dose-response curves to CGS 21680 highly shifted to the right. A Q89A mutant gained affinity for all agonist and antagonist ligands examined in binding and functional assays. Q89 likely plays an indirect role in ligand binding. S90A, S91A, and S277C mutant receptors displayed only moderate changes in ligand affinity. A S281N mutant gained affinity for all adenosine derivatives (agonists), but antagonist affinity was generally diminished, with the exception of a novel tetrahydrobenzothiophenone derivative.

Adenosine↗
PubMed1995

Modelling the P2Y purinoceptor using rhodopsin as template.

The P2Y1 purinoceptor cloned from chick brain (Webb, T. et al (1993) FEBS Lett., 324, 219-225) is a 362 amino acid, 41 kDa protein. To locate residues tentatively involved in ligand recognition a molecular model of the P2Y purinoceptor has been constructed. The model was based on the primary sequence and structural homology with the G-protein coupled photoreceptor rhodopsin, in analogy to the method proposed by Ballesteros and Weinstein ((1995) Meth. Neurosci. 25, 366-428). Transmembrane helices were constructed from the amino acid sequence, minimized individually, and positioned in a helical bundle. The helical bundle was then minimized using the Amber forcefield in Discover (BIOSYM Technologies) to obtain the final model. Several residues that have been shown to be critical in ligand binding in other GPCRs are conserved in the P2Y1 purinoceptor. According to our model the side chains of these conserved residues are facing the internal cleft in which ligand binding likely occurs. The model also suggests four basic residues (H121 in TM3, H266 and K269 in TM6 and R299 in TM7) near the extracellular surface that might be involved in ligand binding. These basic residues might be essential in coordinating the triphosphate chain of the endogenous ligand adenosine 5'-triphosphate (ATP). Potential binding sites for agonists have been explored by docking several derivatives (including newly synthesized N6-derivatives) into the model. The N6-phenylethyl substituent is tolerated pharmacologically, and in our model this substituent occupies a region predominantly defined by aromatic residues such as F51 (TM1), Y100 (TM2) and F120 (TM3). The dimethylated analogue of ATP, N6,N6-dimethyl-adenosine 5'-triphosphate, is less well tolerated pharmacologically, and our model predicts that the attenuated activity is due to interference with hydrogen bonding capacity to Q296 (TM7).

Amino Acid Sequence↗
PubMed1996

Tissue-specific regulation of the WT1 locus.

The 11p13 Wilms' tumor locus consists of two coordinately regulated transcripts, WT1 and WIT-1. These genes are highly expressed in the developing urogenital system, beginning with the urogenital ridge at day 10.5, the metanephric blastema at day 11.5, and during glomerular formation at day 13.5, becoming ultimately restricted to the podocytes. Stromal cells of the gonad also show abundant expression. WT1 is expressed at lower levels in spleen, uterus, mesothelial linings of organs in the abdominal and thoracic cavities, and the ependymal layer of the ventral aspect of the spinal cord. WIT-1 mRNA is about 10-fold less abundant than WT1, but appears to be expressed in the same tissue-restricted manner. Expression of the WT1 protein is required for kidney development, although its physiological function remains to be determined. The function of WIT-1 is similarly unknown but one intriguing possibility is that it is an antisense regulator of WT1. An understanding of events controlling spatial and temporal regulation of these genes will greatly improve our ability to study the role of WT1 and WIT-1 in urogenital development. We have found that while chimeric reporter constructs containing 0.6-2.5 kb of the 5' region of the WT1 gene direct transcription in many different cell lines, we were unable to detect expression in 13.5-day mouse embryos. However, a cosmid containing about 42 kb encompassing this region was able to direct the expression of abundant levels of mRNA from the appropriate transcription initiation sites in both stable transfectants of mouse Leydig cells (TM3) or in transgenic embryos. We are currently localizing the DNA elements required for this expression.

Animals↗
PubMed1999

Genotoxic activity of different chromium compounds in larval cells of Drosophila melanogaster, as measured in the wing spot test.

Two chromium(VI) compounds (potassium chromate and potassium dichromate) and one chromium(III) compound, chromium chloride, were evaluated for genotoxic effects in the wing spot test of Drosophila melanogaster following standard procedures. This assay detects both somatic recombination and mutational events. The genotoxic effects were determined from the appearance of wing spots in flies transheterozygous for the third chromosome recessive markers multiple wing hairs (mwh) and flare-3 (flr(3)), as well as in flies heterozygous formwh and the multiply inverted TM3 balancer chromosome. Genetic changes induced in somatic cells of the wing's imaginal discs lead to the formation of mutant clones on the wingblade. Single spots are due to different genotoxic mechanisms: point mutation, deletion, chromosome breakage, and mitotic recombination; while twin spots are produced only by mitotic recombination. From our results it appears that both chromium(VI) compounds clearly increase the incidence of mutant clones by inducing high increases in the frequency of all types of clones recorded. On the contrary, chromium(III) did not increase the frequency of mutant clones. A high proportion of the total spot induction was due to mitotic recombination, confirming previously reported data on the strong recombinogenic activity of chromium(VI) compounds.

Animals↗
PubMed1999

Genotoxicity studies on the phenoxyacetates 2,4-D and 4-CPA in the Drosophila wing spot test.

The phenoxyacetates 2,4-D and 4-CPA were evaluated for genotoxicity using the Drosophila melanogaster wing spot test, which assesses for somatic mutation and recombination events. Third-instar larvae trans-heterozygous for two recessive mutations affecting the expression of wing trichomes, multiple wing hairs (mwh), and flare (flr) were treated by chronic feeding with different concentrations of the two chemicals. Feeding lasted until pupation of the surviving larvae and the genotoxic effects induced were evaluated in adults for the appearance of wing-blade cell clones with the mwh, flr, or mwh-flr phenotypes. Exposure to 2,4-D, at the highest concentration evaluated (10 mM), induced a weak but significant increase in the frequency of two of the categories of recorded spots: large single and total spots; in contrast, the 4-CPA treatments failed to induce any significant increase in the frequency of evaluated spots. When the heterozygous larvae for mwh and the multiple inverted TM3 balancer chromosome were treated with the chemicals, no increases were detected, either after the 2,4-D nor the 4-CPA treatments.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2000

Ets1 oncogene induction by ELF-modulated 50 MHz radiofrequency electromagnetic field.

We have analyzed gene expression in hemopoietic and testicular cell types after their exposure to 50 MHz radiofrequency (RF) non-ionizing radiation modulated (80%) with a 16 Hz frequency. The exposure system generates a 0.2 microT magnetic field parallel to the ground and a 60 V/m electric field orthogonal to the earth's magnetic field. Exposure conditions were selected so as to interfere with the calcium ion flow. Under these electromagnetic field (EMF) conditions, we observed an overexpression of the ets1 mRNA in Jurkat T-lymphoblastoid and Leydig TM3 cell lines. This effect was observed only in the presence of the 16 Hz modulation, corresponding to the resonance frequency for calcium ion with a DC magnetic field of 45.7 microT. We have also identified a putative candidate gene repressed after EMF exposure. The experimental model described in this paper may contribute to the understanding of the biological mechanisms involved in EMF effects.

Animals↗
PubMed2006

The predicted 3D structures of the human M1 muscarinic acetylcholine receptor with agonist or antagonist bound.

The muscarinic acetylcholine G-protein-coupled receptors are implicated in diseases ranging from cognitive dysfunctions to smooth-muscle disorders. To provide a structural basis for drug design, we used the MembStruk computational method to predict the 3D structure of the human M1 muscarinic receptor. We validated this structure by using the HierDock method to predict the binding sites for three agonists and four antagonists. The intermolecular ligand-receptor contacts at the predicted binding sites agree well with deductions from available mutagenesis experiments, and the calculated relative binding energies correlate with measured binding affinities. The predicted binding site of all four antagonists is located between transmembrane (TM) helices 3, 4, 5, 6, and 7, whereas the three agonists prefer a site involving residues from TM3, TM6, and TM7. We find that Trp 157(4) contributes directly to antagonist binding, whereas Pro 159(4) provides an indirect conformational switch to position Trp 157(4) in the binding site (the number in parentheses indicates the TM helix). This explains the large decrease in ligand binding affinity and signaling efficacy by mutations of Trp 157(4) and Pro 159(4) not previously explained by homology models. We also found that Asp 105(3) and aromatic residues Tyr 381(6), Tyr 404(7), and Tyr 408(7) are critical for binding the quaternary ammonium head group of the ligand through cation-pi interactions. For ligands with a charged tertiary amine head group, we suggest that proton transfer from the ligand to Asp 105(3) occurs upon binding. Furthermore, we found that an extensive aromatic network involving Tyr 106(3), Trp 157(4), Phe 197(5), Trp 378(6), and Tyr 381(6) is important in stabilizing antagonist binding. For antagonists with two terminal phenyl rings, this aromatic network extends to Trp 164(4), Tyr 179(extracellular loop 2), and Phe 390(6) located at the extracellular end of the TMs. We find that Asn 382(6) forms hydrogen bonds with selected antagonists. Tyr381(6) and Ser 109(3) form hydrogen bonds with the ester moiety of acetylcholine, which binds in the gauche conformation.

Amino Acid Substitution↗
PubMed1991

Multiple genes for cell surface cAMP receptors in Dictyostelium discoideum.

We have cloned and characterized three genes (CAR1, CAR2, CAR3) encoding potential cell surface, cyclic adenosine 3':5' monophosphate (AMP) receptors from Dictyostelium discoideum. The three proteins are predicted to be substantially similar in amino acid sequence throughout most of their transmembrane (TM) and loop domains but are distinctly different in their carboxyl terminal segments. In addition, all three genes possess an intron which interrupts an equivalent codon of TM3. CAR1 is expressed early in development when the cAMP relay system is being established. As development proceeds multiple size forms of CAR1 RNA are detected which apparently result from differences in their 5'-untranslated regions. Late in development levels of CAR1 RNA decrease. In contrast, CAR2 encodes a single sized RNA which is expressed only during postaggregative development. CAR3 expression is approximately 10% of CAR1 during early development, is maximal during tight aggregate formation but declines thereafter. Only one size class of CAR3 mRNA is detected throughout development. Because RNA for each of the three genes is present in postaggregative cells, it was of interest to determine the cell type distribution of each RNA. Gene-specific probes were hybridized to RNAs isolated from cells of Percoll gradient-enriched prespore and prestalk fractions and relative levels of hybridization compared. CAR1 and CAR3 show approximately the same pattern of accumulation; a 3-4 fold enrichment in prestalk cells. CAR2, however, is highly enriched in prestalk cells, more than 10 fold relative to prespore cells.

Base Sequence↗
PubMed2005

Genotoxicity testing of Plantago major extracts in somatic cells of Drosophila melanogaster.

Plantago major is used in many parts of the world for the treatment of diseases and to promote the healing of wounds. In the present study, the somatic mutation and recombination test (SMART) in Drosophila melanogaster was used to evaluate the genotoxic activity of an aqueous extract of P. major. The following Drosophila crosses were made: standard (ST) cross, in which virgin flare females (flr3/TM3, Bds) were mated with mwh/mwh males, and high-bioactivation (HB) cross, in which virgin ORR females (ORR/ORR; flr3/TM3, Bds) were mated with mwh/mwh males. Each cross produced two types of descendents, marker-transheterozygous (MH) (mwh +/+ flr3) and balancer-heterozygous (BH) (mwh +/+ TM3, Bds) flies. Three-day-old larvae of both types of descendents were treated with undiluted and diluted (1:1 and 1:2 in water) aqueous extracts of P. major. The extracts were genotoxic in both crosses, producing similar induced frequencies in ST and HB flies. Comparison of the frequencies of wing spots in the BH and MH descendents indicated that recombination was a major response. The results indicate that, under these experimental conditions, aqueous extracts of P. major are genotoxic (recombinagenic).

Animals↗
PubMed2004

Alterations in tropomyosin isoform expression in human transitional cell carcinoma of the urinary bladder.

Previous studies of transformed rodent fibroblasts have suggested that specific isoforms of the actin-binding protein tropomyosin (TM) could function as suppressors of transformation, but an analysis of TM expression in patient tumor tissue is limited. The purpose of our study was to characterize expression of the different TM isoforms in human transitional cell carcinoma of the urinary bladder by immunohistochemistry and Western blot analysis. We found that TM1 and TM2 protein levels were markedly reduced and showed >60% reduction in 61% and 55% of tumor samples, respectively. TM5, which was expressed at very low levels in normal bladder mucosa, exhibited aberrant expression in 91% of tumor specimens. The Western blot findings were confirmed by immunohistochemical analysis in a number of tumors. We then investigated the mechanism underlying TM expression deregulation, in the T24 human bladder cancer cell line. We showed that levels of TM1, TM2 and TM3 are reduced in T24 cells, but significantly upregulated by inhibition of the mitogen-activated protein kinase-signaling pathway. In addition, inhibition of this pathway was accompanied by restoration of stress fibers. Overall, changes in TM expression levels seem to be an early event during bladder carcinogenesis. We conclude that alterations in TM isoform expression may provide further insight into malignant transformation in transitional cell carcinomas of the bladder and may be a useful target for early detection strategies.

Blotting, Western↗
PubMed1988

A carbohydrate epitope expressed uniquely on the cell surface of Drosophila neurons is altered in the mutant nac (neurally altered carbohydrate).

Antibodies against horseradish peroxidase (anti-HRP) recognize neural specific cell surface antigens in Drosophila and other insects. The nature of these antigens was investigated in Drosophila and found to include a complex set of developmentally regulated proteins. Their common epitope appears to be a carbohydrate that shares features with the sugar moiety of pineapple stem bromelain, a plant glycoprotein whose carbohydrate structure has been determined. A mutation was identified that eliminates staining by the antibody in imaginal and adult neural tissue. Tissue specific glycoconjugates, although widespread in the animal kingdom, are little understood. This mutation provides a unique opportunity to address the consequences of altering a neural specific carbohydrate moiety in an otherwise intact and behaving animal. The mutation maps to 84F. A second mutation, contained on the third chromosome balancer, TM3, eliminates anti-HRP staining in embryos. These mutations appear to be separate genes.

Animals↗
PubMedDate not supplied

Effects of gossypol on rat Sertoli and Leydig cells in primary culture and established cell lines.

In order to evaluate the direct effect of gossypol on testicular cells, we used primary cultures of rat Leydig and Sertoli cells. No alteration in Leydig cell survival, morphology, or testosterone production was seen during three days of culture with up to 3 microgram/ml gossypol. With higher concentrations (3 to 7 microgram/ml) of gossypol, there was a reduction in cell survival but no change in androgen secretion. In contrast, there was a marked change in Sertoli cell morphology after five days of gossypol treatment. Large vacuoles and electron dense granules appeared in the cytoplasm, but these effects were reversed within six days of removing gossypol from the medium. There was a significant decrease in androgen binding protein (ABP) secretion by Sertoli cells in the presence of gossypol. We also tested the effect of gossypol on the growth of three established cell lines. Two Sertoli-derived cell lines, TM4 and TR-ST, were more sensitive than a Leydig-derived cell line (TM3). These results suggest that, of the somatic cell types in the testis, the Sertoli cells are most sensitive to gossypol.

Androgen-Binding Protein↗
PubMed1999

Cloning of two chemokine receptor homologs (CXC-R4 and CC-R7) in rainbow trout Oncorhynchus mykiss.

Two rainbow trout chemokine receptors have been sequenced, with homology to CXC-R4 and CC-R7 molecules. The CXC-R4 sequence consisted of 1681 nucleotides, which translated into a mature protein of 357 amino acids, with 80.7% similarity to human CXC-R4. The CC-R7 sequence consisted of 2287 nucleotides, which translated into a 368-amino acid mature protein with 64.5% similarity to human CC-R7. Both sequences contained seven hydrophobic regions, representing the seven transmembrane domains (TM) typical of G-protein-coupled receptors. Extracellular cysteines, transmembrane prolines, and the DRY motif immediately following TM3 were conserved. Phylogenetic tree analysis revealed a tight clustering of trout CXC-R4 with CXC-R3-5 genes. Trout CC-R7 clustered with CC-R6-7 and CXC-R1-2. Reverse transcriptase-polymerase chain reaction analysis demonstrated a wide tissue distribution of CXC-R4 and CC-R7 message in trout, being present in head-kidney leukocytes, blood, gill, brain, spleen, and liver.

Amino Acid Sequence↗