Non-specific Dipeptidase Research Articles

Trans-10, cis-12 conjugated linoleic acid- and caloric restriction-mediated upregulation of CNDP2 expression in white adipose tissue in rodents, with implications in feeding and obesity

Certain dietary supplements such as trans-10, cis-12 conjugated linoleic acid (t10-c12 CLA), and diets including caloric-restricted diets can promote weight loss in certain animal models and humans. A very recent study showed that exercise induces the biosynthesis of N-lactoyl-phenylalanine (Lac-Phe), a circulating signaling metabolite that suppresses feeding and obesity selectively in mice fed with a high-fat diet, and that cytosolic nonspecific dipeptidase 2 (CNDP2) catalyzes the synthesis of Lac-Phe from lactate (Lac) and phenylalanine (Phe). In this in silico study, we found that two anti-obesity strategies, namely treatment with t10-c12 CLA and caloric restriction, increase CNDP2 expression in adipose tissue in mice and rats, respectively. We showed that the effect of t10-c12 CLA on CNDP2 expression might be isomer-specific. We hypothesized that these t10-c12 CLA treatment- or caloric-restricted diet-mediated increases in CNDP2 expression might contribute to their anti-obesity effects, possibly due to increased Lac-Phe levels and ultimately due to Lac-Phe-mediated decreases in daily food consumption, reduced body weight and fat mass. A better understanding of the regulation of CNDP2 expression in diverse tissues in mammals might be of high importance in the treatment of obesity, considering its role in the synthesis of Lac-Phe, a metabolite that decreases body weight and fat mass selectively in mice fed with a high-fat diet. Further research is needed to find out how these two strategies lead to the upregulation of CNDP2 expression and whether this increased expression of CNDP2 might translate to reduced body weight and fat mass through higher Lac-Phe levels.

Proteomic analysis of extracellular vesicles identified PI3K pathway as a potential therapeutic target for cabazitaxel-resistant prostate cancer.

Cabazitaxel (CBZ) is now widely used for prostate cancer (PC) patients resistant to docetaxel (DOC), however, most patients eventually acquire resistance. It will, therefore, be of great benefit to discover novel therapeutic target for the resistance. We aimed to identify candidate therapeutic targets for CBZ-resistance by proteomic analysis of extracellular vesicles (EVs) isolated from serum of DOC-resistant PC patients who later developed CBZ-resistance as well as those harvested from culture medium of DOC- and CBZ-resistant PC cell lines. Using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) conjugated to magnetic beads, EVs were purified from serum of PC patients with DOC-resistance that was collected before and after acquiring CBZ-resistance and conditioned medium of DOC-resistant (22Rv1DR) and CBZ-resistant (22Rv1CR) PC cell lines. Protein analysis of EVs was performed by nanoLC-MS/MS, followed by a comparative analysis of protein expression and network analysis. The cytotoxic effect of a phosphatidylinositol-3-kinase (PI3K) inhibitor, ZSTK474, was evaluated by WST-1 assay. The expression and phosphorylation of PI3K and PTEN were examined by western blot analysis. Among differentially regulated proteins, 77 and 61 proteins were significantly increased in EVs from CBZ-resistant PC cell line and patients, respectively. A comparison between the two datasets revealed that six proteins, fructose-bisphosphate aldolase, cytosolic nonspecific dipeptidase, CD63, CD151, myosin light chain 9, and peroxiredoxin-6 were elevated in EVs from both cell line and patients. Network analysis of the increased EV proteins identified pathways associated with CBZ-resistance including PI3K signaling pathway. ZSTK474 significantly inhibited growth of 22Rv1CR cells and improved their sensitivity to CBZ. In 22Rv1CR cells, PI3K was activated and PTEN that inhibits PI3K was deactivated. Proteomic analysis of serum EVs was successfully accomplished by using Tim-4 as a tool to isolate highly purified EVs. Our results suggest that the combination use of CBZ and PI3K inhibitor could be a promising treatment option for CBZ-resistant PC patients.

Proteomic Analysis of Fresh and Liquid-Stored Boar Spermatozoa.

Simple SummaryMale gametes can be stored for a long period of time for the purpose of preserving genetic material. Cryopreservation and liquid preservation are two main storage procedures commonly used for boar semen. There is evidence in the literature suggesting that cryopreservation changes the profile of proteins that are linked to the motility and function of spermatozoa. It was postulated that they can affect motility, capacitation, oocyte binding ability, and the acrosome reaction of spermatozoa. On the other hand, little is known about changes in protein levels in sperm that occur during liquid storage. Therefore, the objective of this study was to investigate whether liquid storage also causes an alteration in the proteomic profile of stored spermatozoa. A comparative proteomic approach was used to analyze protein samples from fresh spermatozoa and spermatozoa stored for three days at 15–17 °C. Results obtained show that liquid preservation causes quantitative changes in the boar sperm proteome with the over-expression of three out of four proteins in the liquid-stored sperm. Our findings can help elucidate the events involved in liquid preservation. In this study comparative proteomics was used to define changes in the expression of the spermatozoa proteins during liquid storage. Semen from eight boars was analyzed on the day of collection and after liquid preservation at 15–17 °C for three days. Sperm parameters (concentration, motility, morphology, vitality) and percentage of non-capacitated and acrosomal-reacted spermatozoa were determined. Sperm proteins were extracted and separated by two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteomic profiles were computationally compared to highlight differentially expressed protein spots that were, in turn, identified by mass spectrometry. The intensities of four spots were significantly different between fresh and liquid stored sperm. Namely: ATP citrate lyase, chaperonin containing T-complex polypeptide 1 (TCP1) subunit ε and probable phospholipid-transporting ATP-ase were over-expressed in liquid stored sperm, whereas cytosolic non-specific dipeptidase was over-expressed in fresh sperm. These differentially expressed proteins could be used as plausible biomarkers for the evaluation of boar semen quality and spermatozoa survival after liquid storage and could help to address problems associated with sperm preservation.

A toolset to study functions of Cytosolic non-specific dipeptidase 2 (CNDP2) using Drosophila as a model organism

BackgroundExpression of the CNDP2 gene is frequently up- or down-regulated in different types of human cancers. However, how the product of this gene is involved in cell growth and proliferation is poorly understood. Moreover, our knowledge of the functions of the CNDP2 orthologs in well-established model organisms is scarce. In particular, the function of the D. melanogaster ortholog of CNDP2, encoded by the CG17337 gene (hereafter referred to as dCNDP2), is still unknown.ResultsThis study was aimed at developing a set of genetic and molecular tools to study the roles of dCNDP2. We generated a dCNDP2 null mutation (hereafter ∆dCNDP2) using CRISPR/Cas9-mediated homologous recombination (HR) and found that the ∆dCNDP2 mutants are homozygous viable, morphologically normal and fertile. We also generated transgenic fly lines expressing eGFP-tagged and non-tagged dCNDP2 protein, all under the control of the UAS promoter, as well as polyclonal antibodies specific to dCNDP2. Using these tools, we demonstrate that only one of the two predicted dCNDP2 isoforms is expressed throughout the different tissues tested. dCNDP2 was detected in both the cytoplasm and the nucleus, and was found to be associated with multiple sites in the salivary gland polytene chromosomes.ConclusionsThe dCNDP2 gene is not essential for fly viability under standard laboratory conditions. The subcellular localization pattern of dCNDP2 suggests that this protein might have roles in both the cytoplasm and the nucleus. The genetic and molecular tools developed in this study will allow further functional characterization of the conserved CNDP2 protein using D. melanogaster as a model system.

CNDP2 Acts as an Activator for Human Ovarian Cancer Growth and Metastasis via the PI3K/AKT Pathway.

Introduction:The mechanism of tumorigenesis and metastasis of ovarian cancer has not yet been elucidated. This study aimed to investigate the role and molecular mechanism of cytosolic nonspecific dipeptidase 2 in tumorigenesis and metastasis.Methods:Cytosolic nonspecific dipeptidase 2 expression in human ovarian cancer tissues and cell lines was assessed with methyl thiazolyl tetrazolium (MTT), clone formation, and transwell assays performed to evaluate the ability of ovarian cancer cells to proliferate and migrate. Nude mice tumor formation experiments were also performed by subcutaneously injecting cells with stable cytosolic nonspecific dipeptidase 2 knockdown and control SKOV3 cells into BALB/c female nude mice to detect changes in PI3K/AKT pathway-related proteins by Western blotting.Results:Cytosolic nonspecific dipeptidase 2 was highly expressed in human ovarian cancer tissues, with its expression associated with pathological data, including ovarian cancer metastasis. A cytosolic nonspecific dipeptidase 2 stable knockdown or ectopic expression ovarian cancer cell model was established and demonstrated that cytosolic nonspecific dipeptidase 2 could promote the proliferation of ovarian cancer cells. Transwell cell migration and invasion assays confirmed that cytosolic nonspecific dipeptidase 2 enhanced cell metastasis in ovarian cancer. Furthermore, in vivo xenograft experiments demonstrated that cytosolic nonspecific dipeptidase 2 can promote the development and progression of ovarian cancer, increasing the expression of phosphorylated PI3K and AKT.Conclusions:Cytosolic nonspecific dipeptidase 2 promotes the occurrence and development of ovarian cancer through the PI3K/AKT signaling pathway.

Gene expression of digestive enzymes during the initial ontogeny of Mexican snook Centropomus poeyi

Mexican snook ( Centropomus poeyi ) is one of the most important commercial species in Southeast Mexico, which has been overexploited by fisheries. For this reason, some recent studies related to its culture have been done. However, the basic biological knowledge of the digestive physiology is still unknown. In this study, the gene expression of trypsin, (TRY), bile-salt dependent lipase (BAL), amylase (AMY), and cytosolic non-specific dipeptidase (CNP) was investigated during the larval period from embryo (0 days after hatching, DAH), eleuteroembryo (1 DAH) until 25 DAH using RTq-PCR approach and was normalized with the elongation factor 1-α gene (EF1-α). TRY gene expression showed the maximum peak at 17 DAH; meanwhile, BAL gene expression showed variations during larviculture, however, three peaks were observed at 10, 17 and 25 DAH, which appears to correspond to changes in live food supply (rotifers and Artemia nauplii). AMY gene expression was low detected from hatching onwards and showed a slight peak at 10 DAH when larvae were fed with Artemia nauplii. Finally, CNP gene expression had presented three peaks at 2 DAH (yolksac absorption), 19 and 23 DAH when larvae were fed with Artemia nauplii. We conclude that C. poeyi larvae showed early and high digestive enzyme expressions, especially TRY, which indicate that could be possible to start the weaning process at 25 DAH, which could be shorter compared than other Centropomid species.

The Combined Effects of Genetic Variation in the CNDP1 and CNDP2 Genes and Dietary Carbohydrate and Carotene Intake on Obesity Risk

Background/Aims: It is possible that carnosinase (CNDP1) and cellular nonspecific dipeptidase (CNDP2) have important roles in protecting cells and tissues against the damage of oxidative stress. Oxidative stress and subsequent inflammation are key factors in the development of common chronic metabolic diseases, such as obesity. We aimed to investigate the combined effects of genetic variations in CNDP1 and CNDP2 and dietary carbohydrate and carotene intake on obesity risk. Methods: A total of 1,059 Japanese men were randomly selected from participants who visited a medical center for routine medical checkups. We analyzed the relationships between the genotypes of 4 single-nucleotide polymorphisms (SNPs) (rs12605520, rs7244647, rs4891558, and rs17089368) in the CNDP1/CNDP2 locus and body mass index or prevalence of obesity/overweight taking into account dietary carbohydrate and carotene intake. Results: We found that 2 SNPs (rs7244647 in CNDP1 and rs4891558 in CNDP2) were associated with obesity risk. In addition, these associations were observed only in the group with high carbohydrate and low carotene intake but not in the group with low carbohydrate and high carotene intake. Conclusions: Our findings indicate that the combination of genetic variations in CNDP1 and CNDP2 and dietary carbohydrate/carotene intake modulate obesity risk.

Chronic Exposure to Proline Causes Aminoacidotoxicity and Impaired Beta-Cell Function: Studies In Vitro.

Pancreatic islet-cell dysfunction is a hallmark in the development of diabetes, but the reasons for the primary β-cell defect are still elusive. Elevated circulating proline levels have been found in subjects with insulin resistance, obesity, and type 2 diabetes. Therefore, we assessed β-cell function, gene expressions, and cell death after long-term exposure of pancreatic β-cells to excess proline in vitro. Isolated mouse islets and INS-1E cells were incubated with and without excess proline. After 72 h, we examined: (1) β-cell function, including basal insulin secretion (BIS) and glucose-stimulated insulin secretion (GSIS), (2) transcription factors related to insulin gene expression and enzymes involved in the tricarboxylic acid cycle and cholesterol biogenesis, (3) cellular triglycerides (TG) and cholesterol content, (4) the death of INS-1E cells and 3H thymidine incorporation, and (5) protein expression of INS-1E cells in response to proline by proteomics. We found that high doses of proline increased BIS and decreased GSIS in both isolated mouse islets and INS-1E cells. MafA, insulin 1, and the cytochrome c oxidase subunit VIa polypeptide 2 mRNA expressions were all downregulated, indicating that proline impaired insulin gene transcription and mitochondrial oxidative phosphorylation. In contrast, mevalonate decarboxylase gene expression was upregulated, and simultaneously, cholesterol content in INS-1E cells was enhanced. Protein profiling of INS-1E cells revealed that cytosolic non-specific dipeptidase and α enolase were differentially expressed. Our results indicate that proline-induced insulin transcription and mitochondrial oxidative phosphorylation impairment may contribute to the β-cell dysfunction observed in type 2 diabetes. Caution should be applied in interpreting the pathophysiological role of proline since very high proline concentrations were used in the experiments.

N-lactoyl-amino acids are ubiquitous metabolites that originate from CNDP2-mediated reverse proteolysis of lactate and amino acids.

Despite technological advances in metabolomics, large parts of the human metabolome are still unexplored. In an untargeted metabolomics screen aiming to identify substrates of the orphan transporter ATP-binding cassette subfamily C member 5 (ABCC5), we identified a class of mammalian metabolites, N-lactoyl-amino acids. Using parallel protein fractionation in conjunction with shotgun proteomics on fractions containing N-lactoyl-Phe-forming activity, we unexpectedly found that a protease, cytosolic nonspecific dipeptidase 2 (CNDP2), catalyzes their formation. N-lactoyl-amino acids are ubiquitous pseudodipeptides of lactic acid and amino acids that are rapidly formed by reverse proteolysis, a process previously considered to be negligible in vivo. The plasma levels of these metabolites strongly correlate with plasma levels of lactate and amino acid, as shown by increased levels after physical exercise and in patients with phenylketonuria who suffer from elevated Phe levels. Our approach to identify unknown metabolites and their biosynthesis has general applicability in the further exploration of the human metabolome.

Quantitative Proteomics Approach to Screening of Potential Diagnostic and Therapeutic Targets for Laryngeal Carcinoma

To discover candidate biomarkers for diagnosis and detection of human laryngeal carcinoma and explore possible mechanisms of this cancer carcinogenesis, two-dimensional strong cation-exchange/reversed-phase nano-scale liquid chromatography/mass spectrometry analysis was used to identify differentially expressed proteins between the laryngeal carcinoma tissue and the adjacent normal tissue. As a result, 281 proteins with significant difference in expression were identified, and four differential proteins, Profilin-1 (PFN1), Nucleolin (NCL), Cytosolic non-specific dipeptidase (CNDP2) and Mimecan (OGN) with different subcellular localization were selectively validated. Semiquantitative RT-PCR and Western blotting were performed to detect the expression of the four proteins employing a large collection of human laryngeal carcinoma tissues, and the results validated the differentially expressed proteins identified by the proteomics. Furthermore, we knocked down PFN1 in immortalized human laryngeal squamous cell line Hep-2 cells and then the proliferation and metastasis of these transfected cells were measured. The results showed that PFN1 silencing inhibited the proliferation and affected the migration ability of Hep-2 cells, providing some new insights into the pathogenesis of PFN1 in laryngeal carcinoma. Altogether, our present data first time show that PFN1, NCL, CNDP2 and OGN are novel potential biomarkers for diagnosis and therapeutic targets for laryngeal carcinoma, and PFN1 is involved in the metastasis of laryngeal carcinoma.

Underexpressed CNDP2 Participates in Gastric Cancer Growth Inhibition through Activating the MAPK Signaling Pathway

Increasing evidence suggests that cytosolic non-specific dipeptidase 2 (CNDP2) appears to do more than just perform an enzymatic activity; it is functionally important in cancers as well. Here, we show that the expression of CNDP2 is commonly down-regulated in gastric cancer tissues. The ectopic expression of CNDP2 resulted in significant inhibition of cell proliferation, induction of cell apoptosis and cell cycle arrest, and suppressed gastric tumor growth in nude mice. We further revealed that the reintroduction of CNDP2 transcriptionally upregulated p38 and activated c-Jun NH2-terminal kinase (JNK), whereas the loss of CNDP2 increased the phosphorylation of extracellular signal-related kinase (ERK). These results suggest that CNDP2 acts as a functional tumor suppressor in gastric cancer via activation of the mitogen-activated protein kinase (MAPK) pathway.

Proteomic analysis of seminal plasma in men with different spermatogenic impairment

Seminal plasma is a potential source of biomarkers for many disorders of the male reproductive system including male infertility. The identification and characterisation of differentially expressed proteins in seminal plasma of man with normal and impaired spermatogenesis can help in the elucidation of the molecular basis of male infertility. We compared the protein expression profiles of seminal plasma from four different groups of men as follows: normozoospermic, asthenozoospermic, oligozoospermic and azoospermic groups, using two-dimensional differential gel electrophoresis (2-D DIGE). We found eight proteins with statistically significant increased expression in azoospermia compared with at least one of the other studied groups. The differentially expressed spots were fibronectin, prostatic acid phosphatase (PAP), proteasome subunit alpha type-3, beta-2-microglobulin, galectin-3-binding protein, prolactin-inducible protein and cytosolic nonspecific dipeptidase. Notably, PAP was increased in patients with azoospermia compared with that of all other groups. We have observed no statistically significant differences in protein expression between three of the groups: normozoospermic, oligozoospermic and asthenozoospermic. We suggest that the identified panel of proteins in our study especially PAP have a strong potential to be used as azoospermia markers. However, further investigations will be necessary to validate these markers in samples of larger and independent patient cohorts and to clarify their role in the pathogenesis of male infertility.

Purification and identification of a novel primitive secretory enzyme catalyzing the hydrolysis of imidazole-related dipeptides in the jawless vertebrate Lethenteron reissneri

Imidazole-related dipeptides, such as carnosine and anserine, occur widely in skeletal muscles of jawed vertebrates. All of the known enzymes that catalyze the hydrolysis of these dipeptides belong to the M20A metallopeptidase subfamily; two secretory enzymes, serum carnosinase (EC 3.4.13.20) and anserinase (EC 3.4.13.5), and one non-secretory enzyme, cytosolic nonspecific dipeptidase (EC 3.4.13.18). Here we report the enzymatic characterization and molecular identification of an unidentified enzyme, which catalyzes the hydrolysis of these dipeptides, from the skeletal muscle of Far Eastern brook lamprey ( Lethenteron reissneri). A 60-kDa subunit protein of the enzyme was purified to near homogeneity. We cloned two M20A genes from the skeletal muscle of Far Eastern brook lamprey; one was a secretory-type gene encoding for the 60-kD protein, and another was a non-secretory-type gene presumably encoding for cytosolic nonspecific dipeptidase. Our findings indicate that the purified enzyme is a N-glycosylated secretory M20A dipeptidase distributed specifically in the jawless vertebrate group, and may be derived from a common ancestor gene between serum carnosinase and anserinase. We propose that this dipeptidase is a novel secretory M20A enzyme and is classified as neither serum carnosinase nor anserinase.

Molecular Identification of Carnosine Synthase as ATP-grasp Domain-containing Protein 1 (ATPGD1)

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyryl-L-histidine) are abundant dipeptides in skeletal muscle and brain of most vertebrates and some invertebrates. The formation of both compounds is catalyzed by carnosine synthase, which is thought to convert ATP to AMP and inorganic pyrophosphate, and whose molecular identity is unknown. In the present work, we have purified carnosine synthase from chicken pectoral muscle about 1500-fold until only two major polypeptides of 100 and 90 kDa were present in the preparation. Mass spectrometry analysis of these polypeptides did not yield any meaningful candidate. Carnosine formation catalyzed by the purified enzyme was accompanied by a stoichiometric formation, not of AMP, but of ADP, suggesting that carnosine synthase belongs to the "ATP-grasp family" of ligases. A data base mining approach identified ATPGD1 as a likely candidate. As this protein was absent from chicken protein data bases, we reconstituted its sequence from a PCR-amplified cDNA and found it to fit with the 100-kDa polypeptide of the chicken carnosine synthase preparation. Mouse and human ATPGD1 were expressed in HEK293T cells, purified to homogeneity, and shown to catalyze the formation of carnosine, as confirmed by mass spectrometry, and of homocarnosine. Specificity studies carried out on all three enzymes were in agreement with published data. In particular, they acted with 15-25-fold higher catalytic efficiencies on beta-alanine than on gamma-aminobutyrate. The identification of the gene encoding carnosine synthase will help for a better understanding of the biological functions of carnosine and related dipeptides, which still remain largely unknown.

Proteomic analysis of down-regulated proteins in colonic mucosa of chronic slow transit constipation rats

Objective To investigate the alternations of proteins in the colonic mucosa of chronic slow transit constipation (STC) rats with a 2-DE-based proteomic method and analyze the function of these down-regulated proteins so as to provide theoretical basis for the pathogenesis of intestinal mucosa of chronic STC rats. Methods STC model was established by feeding rats with 8 mg/(kg·d) diphenoxylate for 120 d. An experimental model of chronic STC rat was used for separation of proteomics from colonic mucosa using two-dimensional electrophoresis (2-DE). Proteins altered in expressional level were identified by Image Master 2DElite, mass spectrometry, and bibliometrics were applied to identify the differential protein expression and their clinical significance and function were analyzed. Results Obvious differential protein expression was observed in the pathogenesis of STC, including mast cell protease (A1), non-specific dipeptidase (A2) and chondrosome succinate dehydrogenase precursor (A3). The expressions of A1, A2 and A3 were down-regulated in the gel graph of STC rats. Conclusion The down-regulation of chondrosome succinate dehydrogenase, mast cell protease as well as non-specific dipeptidase in rat colon suggests the functional impairment of the oxidoreduction of mitochondrion is very important in the genesis and development of STC. The immunological reaction of STC rats is weakened, and the function of digesting and absorbing protein may be damaged to some extent.

Colocalization of a carnosine-splitting enzyme, tissue carnosinase (CN2)/cytosolic non-specific dipeptidase 2 (CNDP2), with histidine decarboxylase in the tuberomammillary nucleus of the hypothalamus

Carnosine is a naturally occurring dipeptide (β-alanyl- l-histidine) present in mammalian tissues such as the brain and skeletal muscles. Carnosine is not only a radical scavenger but also a possible neurotransmitter-like molecule that regulates neuronal functions such as hypothalamic control of the autonomic nervous system. CN2 (CNDP2) is a cytosolic enzyme that can hydrolyze carnosine to yield l-histidine and β-alanine. In order to understand the functions of carnosine and CN2 in the brain, we have investigated the immunohistochemical localization of CN2 in the hypothalamus. CN2-immunoreactivity was highly concentrated in neuronal cells in the dorsal part of the tuberomammillary nucleus of the posterior hypothalamus. Since the tuberomammillary nucleus is the exclusive origin of histaminergic neurons, we further investigated whether CN2 is present in the histaminergic neurons. We found that CN2-immunoreactivity was colocalized with that of histidine decarboxylase, which is the key enzyme for histamine biosynthesis specifically expressed in the histaminergic neurons of the tuberomammillary nucleus. These results suggest that CN2 is highly expressed in the histaminergic neurons in the tuberomammillary nucleus, implying that it may supply histidine to histaminergic neurons for histamine synthesis.

Proteomic characterization of the site-dependent functional difference in the rat small intestine

To investigate the site-dependent functional difference in the small intestine, proteomic analysis was carried out on the three distinct parts of the rat small intestine. Male Wistar rats (7 weeks old) were fed a semi-purified diet ad libitum for 1 week. Intestinal tissues from the proximal, middle and distal regions of the small intestine were subjected to two-dimensional polyacrylamide gel electrophoresis, and the abundance of each spot was determined fluorometrically. MALDI-TOF/MS and LC-MS/MS analysis of the tryptic peptides were performed to identify the proteins. Many of the 180 identified proteins showed a distinctive distribution pattern along the small intestine. Glutathione S-transferase, Catechol O-methyltransferase and Villin 2 decreased gradually from the jejunum to the ileum, in contrast, non-specific dipeptidase and Keratin 19 increased gradually toward the ileum. The voltage-dependent anion channel 2 was most abundant in the duodenum while the L- and I-Fatty acid binding protein (FABP) and Cellular retinol binding protein (CRBP-II) were in the jejunum, and the Bile acid binding protein (BABP) was only observed in the ileum. The findings of these and of another proteins identified in this study may contribute to further understanding of the small intestinal function, and to clinical applications of small intestinal diseases.

PEPA‐1* Genotype Affects Return Rate for Hatchery Steelhead

AbstractAllozymes continue to be useful as genetic markers in a variety of studies; however, their utility often hinges on the selective neutrality of the allelic variation. Our study tested for neutrality between the two most common alleles (*100 and *110) at the cytosol nonspecific dipeptidase locus (PEPA‐1*) in steelhead Oncorhynchus mykiss from Dworshak National Fish Hatchery in Idaho. We tested for differential growth and survival among fish with the *100/100, *100/ 110, and *110/110 genotypes rearing in a hatchery or a natural stream. We repeated the study for two year‐classes, using heterozygous (*100/110) adults to make the experimental crosses. This design avoided differences in family contribution among genotypes because each cross produced all three genotypes. We divided the progeny from each family into two groups. One group was reared in a hatchery for 1 year and then released for migration to the sea and subsequent return to the hatchery as adults. The other group was released into a natural stream and monitored for 3 years. We found no significant differences in size or survival among PEPA‐1* genotypes for either the naturally reared fish or the hatchery‐reared fish immediately prior to release as smolts. For females, survival to returning adult also was similar among genotypes; however, hatchery‐reared males with the *110/110 genotype returned at a higher rate than did males with the *100/ 100 genotype; heterozygous males were intermediate. These results indicate that selection occurs at the PEPA‐1* locus or at one or more loci tightly linked to it. The finding of nearly equal frequencies for these two alleles in the source population suggests that selection differentials among genotypes reverse or vary from year to year; otherwise, steady directional selection would drive the *100 allele to low frequencies or extinction. Locus PEPA‐1* seems inappropriate for genetic marks in studies of steelhead that span the full life cycle and probably should be avoided for any portion of the life cycle. Inferences about gene flow and population structure from studies that are substantially influenced by PEPA‐1* allele frequencies might be misleading.

Purification and sequence identification of anserinase

Anserinase (Xaa-methyl-His dipeptidase, EC 3.4.13.5) is a dipeptidase that mainly catalyzes the hydrolysis of Nalpha-acetylhistidine in the brain, retina and vitreous body of all poikilothermic vertebrates. The gene encoding anserinase has not been previously identified. We report the molecular identification of anserinase, purified from brain of Nile tilapia Oreochromis niloticus. The determination of the N-terminal sequence of the purified anserinase allowed the design of primers permitting the corresponding cDNA to be cloned by PCR. The anserinase cDNA has an ORF of 1485 nucleotides and encodes a signal peptide of 18 amino acids and a mature protein of 476 amino acids with a predicted molecular mass of 53.3 kDa. Sequence analysis showed that anserinase is a member of the M20A metallopeptidase subfamily in MEROPS peptidase database, to which 'serum' carnosinase (EC 3.4.13.20) and cytosolic nonspecific dipeptidase (EC 3.4.13.18, CNDP) belong. A cDNA encoding CNDP-like protein was also isolated from tilapia brain. Whereas anserinase mRNA was detected only in brain, retina, kidney and skeletal muscle, CNDP-like protein mRNA was detected in all tissues examined.

Sequence Identification and Characterization of Human Carnosinase and a Closely Related Non-specific Dipeptidase

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyric acid-L-histidine) are two naturally occurring dipeptides with potential neuroprotective and neurotransmitter functions in the brain. Peptidase activities degrading both carnosine and homocarnosine have been described previously, but the genes linked to these activities were unknown. Here we present the identification of two novel cDNAs named CN1 and CN2 coding for two proteins of 56.8 and 52.7 kDa and their classification as members of the M20 metalloprotease family. Whereas human CN1 mRNA and protein are brain-specific, CN2 codes for a ubiquitous protein. In contrast, expression of the mouse and rat CN1 orthologues was detectable only in kidney. The recombinant CN1 and CN2 proteins were expressed in Chinese hamster ovary cells and purified to homogeneity. CN1 was identified as a homodimeric dipeptidase with a narrow substrate specificity for Xaa-His dipeptides including those with Xaa = beta Ala (carnosine, K(m) 1.2 mM), N-methyl beta Ala, Ala, Gly, and gamma-aminobutyric acid (homocarnosine, K(m) 200 microM), an isoelectric point of pH 4.5, and maximal activity at pH 8.5. CN2 protein is a dipeptidase not limited to Xaa-His dipeptides, requires Mn(2+) for full activity, and is sensitive to inhibition by bestatin (IC(50) 7 nM). This enzyme does not degrade homocarnosine and hydrolyzes carnosine only at alkaline pH with an optimum at pH 9.5. Based on their substrate specificity and biophysical and biochemical properties CN1 was identified as human carnosinase (EC ), whereas CN2 corresponds to the cytosolic nonspecific dipeptidase (EC ).