Discovering proteins for chemoprevention and chemotherapy by curcumin in liver fluke infection-induced bile duct cancer.

Modulation or prevention of protein changes during the cholangiocarcinoma (CCA) process induced by Opisthorchis viverrini (Ov) infection may become a key strategy for prevention and treatment of CCA. Monitoring of such changes could lead to discovery of protein targets for CCA treatment. Curcumin exerts anti-inflammatory and anti-CCA activities partly through its protein-modulatory ability. To support the potential use of curcumin and to discover novel target molecules for CCA treatment, we used a quantitative proteomic approach to investigate the effects of curcumin on protein changes in an Ov-induced CCA-harboring hamster model. Isobaric labelling and tandem mass spectrometry were used to compare the protein expression profiles of liver tissues from CCA hamsters with or without curcumin dietary supplementation. Among the dysregulated proteins, five were upregulated in liver tissues of CCA hamsters but markedly downregulated in the CCA hamsters supplemented with curcumin: S100A6, lumican, plastin-2, 14-3-3 zeta/delta and vimentin. Western blot and immunohistochemical analyses also showed similar expression patterns of these proteins in liver tissues of hamsters in the CCA and CCA + curcumin groups. Proteins such as clusterin and S100A10, involved in the NF-κB signaling pathway, an important signaling cascade involved in CCA genesis, were also upregulated in CCA hamsters and were then suppressed by curcumin treatment. Taken together, our results demonstrate the important changes in the proteome during the genesis of O. viverrini-induced CCA and provide an insight into the possible protein targets for prevention and treatment of this cancer.

Protein profiling of paraquat-exposed rat lungs following treatment with Acai (Euterpe oleracea Mart.) berry extract.

Paraquat (1,1'-dimethyl-4,4'-bipyridinium chloride, PQ) is a non-selective herbicide, and PQ poisoning by accidental or intentional ingestion is a cause of numerous fatalities around the world every year. Although a great deal of research has been conducted into the development of an acceptable treatment for PQ poisoning, no effective guidelines for patients have been developed thus far. Acai berry extract and juice have been highlighted in this regard, due to their observed antioxidant effects in various diseases. Furthermore, the acai berry has been used in dietary supplements, as it contains a variety of nutrients, including proteins, lipids, vitamins A, C and E and polyphenols. In this study, we conducted proteomic analysis of PQ-poisoned rat lungs to evaluate the changes in protein expression induced by PQ and to identify any protective effects of acai berry on the PQ poisoning. Our data revealed that the expression of the calcium signaling-related proteins calcium binding protein 1 (CaBP1), FK506 binding protein 4 (FKBP4), S100A6 and secreted protein acidic and rich in cysteine (Sparc, also known as osteonectin) were induced by PQ treatment and downregulated by acai berry treatment. However, the levels of protein kinase C substrate 80K-H were shown to be downregulated as the result of PQ treatment. Our results indicated that these proteins may function as biomarkers for acute poisoning by PQ exposure. Further studies may be necessary to understand their clinical relevance with regard to PQ poisoning.

Serum S100A6 concentration predicts peritoneal tumor burden in mice with epithelial ovarian cancer and is associated with advanced stage in patients.

BACKGROUND: Ovarian cancer is the 5th leading cause of cancer related deaths in women. Five-year survival rates for early stage disease are greater than 94%, however most women are diagnosed in advanced stage with 5 year survival less than 28%. Improved means for early detection and reliable patient monitoring are needed to increase survival. METHODOLOGY AND PRINCIPAL FINDINGS: Applying mass spectrometry-based proteomics, we sought to elucidate an unanswered biomarker research question regarding ability to determine tumor burden detectable by an ovarian cancer biomarker protein emanating directly from the tumor cells. Since aggressive serous epithelial ovarian cancers account for most mortality, a xenograft model using human SKOV-3 serous ovarian cancer cells was established to model progression to disseminated carcinomatosis. Using a method for low molecular weight protein enrichment, followed by liquid chromatography and mass spectrometry analysis, a human-specific peptide sequence of S100A6 was identified in sera from mice with advanced-stage experimental ovarian carcinoma. S100A6 expression was documented in cancer xenografts as well as from ovarian cancer patient tissues. Longitudinal study revealed that serum S100A6 concentration is directly related to tumor burden predictions from an inverse regression calibration analysis of data obtained from a detergent-supplemented antigen capture immunoassay and whole-animal bioluminescent optical imaging. The result from the animal model was confirmed in human clinical material as S100A6 was found to be significantly elevated in the sera from women with advanced stage ovarian cancer compared to those with early stage disease. CONCLUSIONS: S100A6 is expressed in ovarian and other cancer tissues, but has not been documented previously in ovarian cancer disease sera. S100A6 is found in serum in concentrations that correlate with experimental tumor burden and with clinical disease stage. The data signify that S100A6 may prove useful in detecting and/or monitoring ovarian cancer, when used in concert with other biomarkers.

Annexin XI co-localises with calcyclin in proliferating cells of the embryonic mouse testis.

Mammalian sex determination relies on the expression of SRY, which triggers a tightly regulated cascade of gene expression leading to male differentiation. Many elements of this pathway remain to be identified. Here, we characterise Annexin XI (Anxa11), a gene whose major site of embryonic expression was within the undifferentiated and differentiating testis. Lower level expression was also observed in both sexes in the Müllerian and Wolffian ducts, the somitic dermamyotome, and the dorsal intermediate zone of the neural tube. Anxa11 transcripts were detected in the indifferent gonad from 10.5 days post coitum (dpc), becoming male specific as development proceeded. Expression was within the testis cords, initially in germ cells, and then in both Sertoli and germ cells. Annexin XI protein was seen in the testis cords from 12.5 dpc, localising to the cytoplasm of the Sertoli cells. Expression of calcyclin (S100a6), shown previously to interact with annexin XI in vitro, was also observed in proliferating cells of the embryonic testis, supporting a possible in vivo interaction.

Calcyclin mediates serum response element (SRE) activation by an osteoblastic extracellular cation-sensing mechanism.

UNLABELLED: The molecular mechanism of sensing extracellular cations in osteoblasts is controversial. Using an expression-cloning strategy, the calcium-binding protein calcyclin was found to mediate the response of MC3T3-E1 osteoblasts to extracellular cations, but not the calcimimetic NPS-568, indicating the presence of another cation-sensing mechanism. Further understanding of calcyclin function in osteoblasts may identify novel targets for regulating bone formation. INTRODUCTION: Extracellular calcium and other cations seem to regulate the function of osteoblasts through a distinct calcium-sensing mechanism that is coupled to activation of c-fos gene transcription. The identity of this calcium-sensing mechanism is unknown. METHODS: To identify molecules that participate in this extracellular cation-sensing pathway, we developed an expression cloning strategy in COS-7 cells using cation stimulation of a serum response element (SRE) luciferase reporter derived from the c-fos promoter to screen a mouse MC3T3-E1 osteoblast cDNA library. RESULTS AND CONCLUSIONS: We identified calcyclin (S100A6), a calcium-binding protein of the EF-hand type belonging to the S100 family, as being responsible for transferring a cation-sensing response from osteoblasts to COS-7 cells. Transfection of the calcyclin cDNA into COS-7 and HEK-293 cells confirmed that the overexpression of calcylin caused these cells to gain the ability to sense extracellular cations, including aluminum, gadolinium, calcium, and magnesium. Conversely, we found that an antisense calcyclin construct reduced calcyclin expression and partially inhibited the cation-sensing response in MC3T3-E1 osteoblasts. These results implicate calcyclin in the activation of SRE and establish a role for calcyclin as an accessory protein involved in the cation-sensing pathway in osteoblasts.

Expression of members of the S100 Ca2+-binding protein family in guinea-pig smooth muscle.

The calcium-binding proteins of the S100 family show tissue-specific expression. In this study, the mRNA and protein expression of five S100 calcium-binding proteins was investigated in different guinea-pig smooth muscle preparations. Transcripts of cDNA of S100A1, S100A4, S100A6 and S100A10 were amplified from smooth muscle RNA of neonatal and adult urinary bladder, ileum, aorta and from freshly isolated and cultured urinary bladder smooth muscle cells. S100B was not detectable in smooth muscle RNA, but was seen in control RNA isolated from brain. The pattern of S100 mRNA expression did not change during postnatal development and cell culture of smooth muscle. The structural homology of guinea-pig S100 proteins reverse transcription-polymerase chain reaction (RT-PCR) sequences compared to other species was 85-93% (human), 83-88% (rat) and 81-87% (mouse). Protein expression of S100A4, S100A6 and S100A10 was investigated in aorta, ileum, bladder and cultured bladder smooth muscle cells by Western blot analysis using polyclonal antibodies against guinea-pig-specific S100 immunogenic peptide sequences raised in rabbits. The results show that the proteins S100A4, S100A6 and S100A10 are expressed in the smooth muscle of ileum, bladder and aorta. S100A4 and S100A6 proteins are also expressed in cultured smooth muscle cells. The results of this study suggest that the calcium-binding proteins S100A1, S100A4, S100A6 and S100A10, but not S100B, are expressed in guinea-pig smooth muscle, and could be potentially involved in the regulation of cytoplasmic Ca(2+)-concentration and/or in signal transduction in smooth muscle.

Construction of cDNA representational difference analysis based on two cDNA libraries and identification of garlic inducible expression genes in human gastric cancer cells.

AIM: To elucidate molecular mechanism of chemopreventive efficacies of garlic against human gastric cancer (HGC). METHODS: HGC cell line BGC823 was treated with Allitridi (a kind of garlic extract) and Allitridi-treated and parental BGC823 cDNA libraries were constructed respectively by using lambdaZAP II vector. cDNA Representational Difference Analysis (cDNA RDA) was performed using Bam H I cutting-site and abundant cDNA messages provided by the libraries. Northern blot analysis was applied to identify the obtained difference products. RESULTS: Two specific cDNA fragments were obtained and characterized to be derived from homo sapiens folate receptor alpha (FRalpha) gene and calcyclin gene respectively. Northern blot results showed a 4-fold increase in FRalpha gene expression level and 9-fold increase in calcyclin mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: The method of cDNA RDA based on cDNA libraries combines the high specificity of cDNA RDA with abundant cDNA messages in cDNA library; this expands the application of cDNA library and increases the specificity of cDNA RDA. Up-regulation of FRalpha gene and calcyclin gene expressions induced by Allitridi provide valuable molecular evidence for the efficacy garlic in treating HGC as well as other diseases.

Gastrin induces over-expression of genes involved in human U373 glioblastoma cell migration.

Astrocytic tumors are the most common and the most malignant primary tumors of the central nervous system. We had previously observed that gastrin could significantly modulate both cell proliferation and migration of astrocytoma cells. We have investigated in the present study which genes could be targeted by gastrin in tumor astrocyte migration. Using a subtractive hybridization PCR technique we have cloned genes differentially over-expressed in human astrocytoma U373 cells treated or not with gastrin. We found about 70 genes over-expressed by gastrin. Among the genes overexpressed by gastrin, we paid particular attention to tenascin-C, S100A6 and MLCK genes because their direct involvement in cell migration features. Their gastrin-induced overexpression was quantitatively determined by competitive RT-PCR technique. We also showed by means of a reporter gene system that S100A6 and tenascin-C respective promoters were upregulated after gastrin treatment. These data show that gastrin-mediated effects in glioblastoma cells occur through activation of a number of genes involved in cell migration and suggest that gastrin could be a target in new therapeutic strategies against malignant gliomas.

Immunization with an antigen identified by cytokine tumor vaccine-assisted SEREX (CAS) suppressed growth of the rat 9L glioma in vivo.

We have reported previously that s.c. immunization of rats with IL-4 transduced 9L gliosarcoma cells (9L-IL-4) induced a potent antitumor immunity against intracranial, parental 9L tumors. Subcutaneous implantation of 9L-IL-4 influenced the systemic humoral response, which was demonstrated by Th2-type isotype-switching and the induction of cellular immune responses, which played a critical role in the rejection of tumors. Serological analyses of recombinant cDNA expression libraries (SEREX), has recently emerged as a powerful method for serological identification of tumor-associated antigens (TAAs) and/or tumor rejection antigens (TRAs). Because IL-4 is known to activate B cells and to promote humoral responses, and inasmuch as induction of humoral responses by central nervous system tumors has been reported to be minimal, we investigated whether the induction of a potent humoral immune response against 9L TAAs or TRAs in rats immunized s.c. with 9L-IL4 could be demonstrated. Screening of 5 x 10(5) independent clones of 9L-expression cDNA library for the presence of reactive antibodies in the serum from a 91-IL-4 immunized rat led to the identification of three different TAAs. One 9L TAA (clone 29) was demonstrated to be calcyclin, a member of the S-100 family of calcium-binding proteins. The second 9L TAA (clone 37) was demonstrated to be the rat homologue of the J6B7 mouse immunomodulatory molecule. The third TAA (clones 158 and 171) was determined to be the rat homologue of the mouse Id-associated protein 1 (MIDA1), a DNA-binding, protein-associated protein. Northern blotting demonstrated that message for calcyclin was overexpressed in 9L cells. Message encoding MIDA1 was highly expressed in parental 9L cells and thymus and, to a lesser degree, in testis, suggesting that MIDA1 was comparable with the cancer/testis category of TAAs. Sera obtained from animals bearing 9L-IL-4 were found to have a higher a frequency and titer of antibodies to these antigens when compared with sera obtained from rats bearing sham-transduced 9L (9L-neo) cells. To determine whether immunization with these TAAs induced antitumor immunity, animals were immunized by intradermal injection with expression plasmids encoding calcyclin or MIDA1. Subsequent challenge of rats with parental 9L resulted in significant suppression of tumor growth in animals immunized with MIDA1, but not with calcyclin. These results indicate that MIDA1 is an effective 9L TRA and will be useful for the investigation of specific antitumor immunity in this glioma model. Furthermore, these results suggest that this approach, termed "cytokine-assisted SEREX (CAS)," may serve as an effective strategy for identification of TRAs for in animal-glioma models of cytokine gene therapy, and potentially in humans undergoing cytokine gene therapy protocols as well.

S100A6 and S100A11 are specific targets of the calcium- and zinc-binding S100B protein in vivo.

In solution, S100B protein is a noncovalent homodimer composed of two subunits associated in an antiparallel manner. Upon calcium binding, the conformation of S100B changes dramatically, leading to the exposure of hydrophobic residues at the surface of S100B. The residues in the C-terminal domain of S100B encompassing Phe(87) and Phe(88) have been implicated in interaction with target proteins. In this study, we used two-hybrid technology to identify specific S100B target proteins. Using S100B as bait, we identify S100A6 and S100A11 as specific targets for S100B. S100A1, the closest homologue of S100B, is capable of interaction with S100B but does not interact with S100A6 or S100A11. S100B, S100A6, and S100A11 isoforms are co-regulated and co-localized in astrocytoma U373 cells. Furthermore, co-immunoprecipitation experiments demonstrated that Ca(2+)/Zn(2+) stabilizes S100B-S100A6 and S100B-S100A11 heterocomplexes. Deletion of the C-terminal domain or mutation of Phe(87) and Phe(88) residues has no effect on S100B homodimerization and heterodimerization with S100A1 but drastically decreases interaction between S100B and S100A6 or S100A11. Our data suggest that the interaction between S100B and S100A6 or S100A11 should not be viewed as a typical S100 heterodimerization but rather as a model of interaction between S100B and target proteins.

Calcyclin gene expression is increased by mechanical strain in fibroblasts and lung.

Mechanical tension extending throughout the structural elements of the lung is a potential stimulus for cell proliferation and gene expression. Pulmonary fibroblasts located in the interstitial space of the capillary wall throughout the lung parenchyma and within the large vessels and airways are uniquely situated to sense changes in mechanical force. Therefore, we used the polymerase chain reaction-based method of differential display analysis to screen for altered gene expression in fetal human lung fibroblasts exposed to increased cyclic stretch. IMR-90 cells were seeded at 3 x 10(4) cells/cm(2) on laminin-coated plates. Cells were subsequently exposed to mechanical strain on a Flexercell apparatus, resulting in a maximal elongation of 20% at a rate of 60 cycles/min over a period of 48 h. A complementary DNA corresponding to the cell cycle-regulated gene calcyclin was identified in mechanically strained fibroblasts. Increased calcyclin messenger RNA levels were confirmed by Northern blot analysis. Further, calcyclin gene expression was upregulated in isolated-perfused rat lungs exposed to increased mechanical strain by ventilation at high states of lung inflation for 4 h. These data suggest that calcyclin gene expression plays a role in the response of pulmonary fibroblasts to increased mechanical tension and may alter the regulation of the fibroblast cell cycle.

Identification and initial characterization of calcyclin and phospholipase A2 in equine conceptuses.

For development to proceed normally, the appropriate genes must be expressed in the correct tissues and in the correct time frame. Knowledge of gene expression during development provides information about the changes taking place within the conceptus as well as possible reasons for pregnancy failure. However, little is known about gene expression during development in the equine conceptus. In this study, we examined differences in gene expression between day 12 and day 15 equine conceptuses by suppression subtractive hybridization. This technique was used to isolate transcripts that are more abundantly expressed in day 15 conceptuses compared to day 12 conceptuses. Between day 12 and 15 of pregnancy in horses, maternal recognition of pregnancy occurs, gastrulation is taking place, and mesoderm is beginning to form. Fifty cDNA clones were isolated, sequenced, and compared to known sequences in the GenBank database. Two cDNA clones identified that were of primary interest were calcyclin and phospholipase A2. Calcyclin is a calcium-binding protein of the S-100 protein family that has been found in mouse decidua and trophoblast. Calcyclin was found to be expressed in both day 12 and 15 equine conceptuses, with approximately a 30-fold increase in transcript abundance between days 12 and 15. Phospholipase A2 is an enzyme that cleaves phospholipids to release fatty acids and is involved in arachidonic acid release needed for prostaglandin, thromboxane, and leukotriene synthesis. Multiple forms of PLA2, that appear to be differentially regulated in day 12 and 15 conceptuses, were detected by northern blotting.

Molecular cloning and expression of a mouse brain cDNA encoding a novel protein target of calcyclin.

A protein target of mouse calcyclin, p30, which we call calcyclin-binding protein (CacyBP), was identified in mouse brain and Ehrlich ascites tumor (EAT) cells. The amino acid sequence of the CacyBP chymotryptic peptide was used to prepare synthetic oligonucleotides that served as a probe to screen the mouse brain cDNA library. A 1.4-kb positive clone was detected, isolated, and sequenced. The analyzed clone contains an open reading frame encoding a protein of a molecular mass of approximately 26 kDa. The nucleotide and predicted amino acid sequences indicate that CacyBP is a novel protein. The results obtained from northern blots show that the CacyBP gene is expressed predominantly in mouse brain and EAT cells. Using a pGEX vector the recombinant CacyBP was expressed in Escherichia coli, and its properties were analyzed. The recombinant protein interacts with calcyclin at a physiologically relevant range of Ca2+ in solution during affinity chromatography and on blots. Because CacyBP, like calcyclin, is present in the brain, the interaction of these two proteins might be involved in calcium signaling pathways in neuronal tissue.

Cytotoxic principles of a Bangladeshi crude drug, akond mul (roots of Calotropis gigantea L.).

Three cardenolide glycosides, calotropin (1), frugoside (2), and 4'-O-beta-D-glucopyranosylfrugoside (3), were obtained as the cytotoxic principles of "akond mul" (roots of Calotropis gigantea L.). The cytotoxicity of these compounds against various cell lines of human and mouse origin was tested. They showed similar cell line selectivity to those of cardiac glycosides such as digoxin and ouabain: they are toxic to cell lines of human origin, but not to those from mouse at 2 micrograms/ml.

Functional quantitative analysis of the genome in cultured human mesangial cells. Technical note.

For normal physiological function, each cell tightly regulates gene expression in a specific fashion so that critical proteins are synthesized in a well-coordinated manner. Therefore, it is very important to uncover which genes are expressed in specific cells. Recent technological advances combined with rapid large-scale DNA sequencing and computerized data processing have allowed us to investigate the expression levels of a variety of transcripts in the mesangial cells, a target of injury in many forms of glomerulonephritis. Utilizing a large scale sequencing of a 3'-directed cDNA library, which allows us to avoid variable cloning efficiencies reflecting the size of cDNA, we investigated expression profiles of various molecules in cultured human mesangial cells. Among the 1,193 sequenced clones, 688 (57.7%) appeared more than once (redundant sequence group), representing 203 different species. Thirty-nine of these appeared more than three times. The most abundant mRNA was that of fibronectin, which consisted of 3.9% of the total mRNA population. Except for mitochondrial or ribosomal genes, calcyclin came next (2.5%), followed by two cytoskeletal genes, gamma-actin gene and calpactin 1 light chain gene, in addition to an amyloid precursor protein homolog (0.7%). In conclusion, we performed a molecular biological quantification of transcripts in mesangial cells. Fibronectin was the most abundantly expressed, followed by calcyclin, gamma-actin, calpactin 1 light chain, and an amyloid precursor protein homolog. We also discovered some candidate genes specific for human mesangial cells. The expression profile of the transcripts serves as an important tool in understanding the biological properties of mesangial cells.

PRAP, a prolactin receptor associated protein: its gene expression and regulation in the corpus luteum.

We have recently identified, characterized, and cloned a luteal microsomal 32-kDa phosphoprotein that we named PRAP (for PRL-receptor associated protein), and we have demonstrated that PRAP binds to the intracellular domain of the short but not the long form of the PRL receptor. In this study, we used PRAP cDNA to examine the tissue specificity, the developmental expression, and the hormonal regulation of PRAP gene expression. Northern blot analysis revealed that in the corpus luteum, PRAP cDNA hybridized to multiple transcripts (5.5 kb, 4.3 kb, and 1.8 kb), with the smallest transcript (1.8 kb) corresponding to the size of the cDNA clone. However, none of these transcripts were detected in any other tissues examined. PRAP appears to be tightly regulated by steroids and PRL. When pregnant rats were treated with aminoglutethimide, a steroid synthesis inhibitor, all three PRAP transcripts became barely detectable. Similar results were obtained when all luteotropic support was removed by hypophysectomy and hysterectomy. Estradiol up-regulated PRAP expression and, more specifically, the two lower transcripts. PRL had no stimulatory effect on PRAP messenger RNA (mRNA) expression but caused a substantial increase in the level of PRAP protein when administered to hypophysectomized pregnant rat, suggesting that PRL may stabilize this protein. Similar dissociation between levels of mRNA and protein were observed during luteal development. Although both PRAP mRNA and protein were barely detectable in early pregnancy, their expression increased abruptly from midpregnancy; however, whereas levels of PRAP mRNA declined from day 18, those of the protein remained elevated until parturition. In summary, results of this study have defined the tissue specificity and developmental expression of PRAP mRNA during pregnancy. The data have also revealed that the gene expression of this protein is up-regulated by estradiol, suggesting a pivotal role for PRAP in the synergistic action of estradiol and PRL on the function of the rat corpus luteum.

Immunoreactivities of p11 and calcyclin in baker's yeast, wheat germ and lobster.

After effectively eliminating the nonspecific cross-immunoreactivity with the affinity columns of anti-IgG agarose and IgG agarose, the potent immunoreactivities of p11 and calcyclin in wheat germ, lobster tail muscle, and three strains of baker's yeast were analyzed by Western blotting using mouse anti-p11 and rabbit anti-calcyclin. The occurrence of multiple bands may be due to either autolyses and/or the interactions between the p11 (or calcyclin) and other endogenous biological molecules. The results suggest not only a ubiquitous distribution and a universal Ca(2+)-mediating regulatory role of p11 and calcyclin in eukaryotes, but also an evolutionary conservation of these (S-100)-related proteins.

Molecular cloning of chicken calcyclin (S100A6) and identification of putative isoforms.

A full-length cDNA encoding smooth muscle calcyclin (S100A6) was cloned from chicken gizzard, using reverse transcription--polymerase chain reaction techniques. The deduced amino acid sequence contains 92 residues with 12 substitutions and a 2 amino acid C-terminal extension when compared with human calcyclin. Calcyclin was purified from chicken gizzard by Ca(2+)-dependent hydrophobic chromatography, heat treatment, and anion-exchange chromatography, N-terminal sequencing of two CNBr peptides confirmed its identity as calcyclin. Two isoforms of calcyclin (A and B), which differ with respect to the presence or absence of a C-terminal lysine, were identified and the native protein was shown to exist as noncovalently associated homodimers (AA and BB) and heterodimers (AB). Incubation of purified calcyclin AA with an extract of chicken gizzard did not result in degradation of calcyclin A or appearance of calcyclin B, suggesting that calcyclin B is a bona fide isoform rather than a proteolytic fragment generated during purification. Western blotting of chicken tissues with anti-(gizzard calcyclin) indicated abundant expression of calcyclin in smooth muscle tissues, including esophagus, large intestine, and trachea, with lower levels in lung, heart, kidney, and brain, and none detectable in liver or skeletal muscle.

Expressed sequence tags (EST) identify genes preferentially expressed in catfish chemosensory tissues.

Expressed sequence tags (ESTs) for the catfish (Ictalurus punctatus) were identified and characterized by shotgun sequencing coupled to Northern analysis. We have identified and characterized a number of cDNA clones from a catfish olfactory mucosal library that show differential tissue expression including several that are enriched in chemosensory tissue. Among the novel cDNA clones studied were an olfactory specific beta-tubulin and a novel member of the S-100 family of calcium-binding proteins that is highly expressed in barbel, olfactory mucosa and gill, but not in brain. Several clones of low abundance mRNAs were also identified, including one manifesting a basic-helix-loop-helix (b-HLH) motif that is typical of many transcription factors. Additional cDNA clones whose mRNAs are differentially expressed, but are of unknown function, were also obtained. These results demonstrate the case with which novel gene products enriched in chemosensory tissues can be identified.