Analysis of IGHA1 and other salivary proteins post half marathon in female participants.

Background: High-intensity exercise (HIE), such as that in marathons and triathlons, suppresses transient local and systemic immunity. Serum and salivary immunoglobulin heavy constant alpha 1 (IGHA1) are major markers of immunosuppression by HIE. Although much is known about the systemic immunosuppressive response, little is known about its local response in the oral cavity, lungs, bronchial tubes, and skin. The oral cavity allows bacteria or viruses to enter the body. Saliva covers the epidermis of the oral cavity and plays an important role in the local stress response by preventing infection. In this study, we examined the properties of saliva secreted during the local stress response for half-marathon (HM) induced IGHA1 protein expression using quantitative proteomics. Methods: The Exercise Group (ExG) (19 healthy female university students) participated in a HM race. The Non-Exercise Group (NExG) (16 healthy female university students) did not participate in the ExG. The ExG saliva samples were collected 1 h pre and 2 h and 4 h post-HM. The NExG saliva samples were collected at the same time intervals. The saliva volume, protein concentration, and relative IGHA1 expression were analyzed. In addition, 1 h pre and 2 h post- HM saliva samples were analyzed by iTRAQ. The identified factors in iTRAQ were analyzed for the ExG and the NExG using western blotting. Results: We identified kallikrein 1 (KLK1), immunoglobulin kappa chain (IgK), and cystatin S (CST4) as suppression factors, as well as IGHA1, which has been reported to be an immunological stress marker. IGHA1 (p = 0.003), KLK1 (p = 0.011), IGK (p = 0.002), and CST4 (p = 0.003) were suppressed 2 h post-HM compared with their levels pre HM, and IGHA1 (p < 0.001), KLK1 (p = 0.004), and CST4 (p = 0.006) were suppressed 4 h post-HM. There was also a positive correlation between IGHA1, IGK, and CST4 levels at 2 and 4 h post-HM. In addition, KLK1 and IGK levels at 2 h post-HM were positively correlated. Conclusion: Our study demonstrated that the salivary proteome is regulated, and antimicrobial proteins are suppressed post-HM. These results suggest that oral immunity was transiently suppressed post-HM. The positive correlation of each protein at 2 and 4 h post-HM suggests that the suppressed state was similarly regulated up to 4 h after a HM. The proteins identified in this study may have applications as stress markers for recreational runners and individuals who perform moderate to HIE on a regular basis.

Homologue of mammalian apolipoprotein A-II in non-mammalian vertebrates.

Although apolipoprotein with molecular weight 14 kDa (apo-14 kDa) is associated with fish plasma high-density lipoproteins (HDLs), it remains to be determined whether apo-14 kDa is the homologue of mammalian apoA-II. We have obtained the full cDNA sequences that encode Japanese eel and rainbow trout apo-14 kDa. Homologues of Japanese eel apo-14 kDa sequence could be found in 14 fish species deposited in the DDBJ/EMBL/GenBank or TGI database. Fish apo-14 kDa lacks propeptide and contains more internal repeats than mammalian apoA-II. Nevertheless, phylogenetic analysis allowed fish apo-14 kDa to be the homologue of mammalian apoA-II. In addition, in silico cloning of the TGI, Ensembl, or NCBI database revealed apoA-IIs in dog, chicken, green anole lizard, and African clawed frog whose sequences had not so far been available, suggesting both apoA-I and apoA-II as fundamental constituents of vertebrate HDLs.

The utilization of lipovitellin during blue crab (Callinectes sapidus) embryogenesis.

Embryos of the blue crab Callinectes sapidus develop in egg sacs carried on the abdomen of the female. They develop over a period of 10-13 days at 28 degrees C and are nutritionally dependent on yolk until they emerge from the egg sacs as free-swimming zoeae. The principal component of blue crab yolk is lipovitellin (LpII), a water-soluble lipoprotein composed of approximately equal amounts of lipid and protein. We followed changes in the concentration of apoproteins of LpII during embryogenesis by ELISA and Western blots, using monoclonal antibodies against two LpII apoprotein associated peptides identified as Protein A (107 kDa) and Protein B (75 kDa). During embryogenesis there was a decrease in Protein B but an increase in two smaller peptides (52 and 35 kDa) that reacted with the Protein B antibody. Utilization of LpII during embryogenesis was also followed morphologically by immunohistochemistry. Utilization of LpII was slow in early embryonic stages, followed by rapid utilization in late embryonic stages, such that only traces of LpII were present at the end of embryogenesis. The cells of the developing hepatopancreas appear to play an important role in the utilization of LpII.

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.

Habitual exercise induced resistance to oxidative stress.

We investigated whether habitual exercise (HE) modulates levels of oxidative DNA damage and responsiveness to oxidative stress induced by renal carcinogen Fe-nitrilotriacetic acid (Fe-NTA). During a ten week protocol, two groups of rats either remained sedentary or underwent swimming for 15--60 min per day, 5 days per week, with or without a weight equivalent to 5% of their body weight. Then we injected Fe-NTA and sacrificed the rats 1 h after the injection. We determined the activity of superoxide dismutase (SOD) in diaphragm and kidney, evaluated levels of 8-hydroxydeoxyguanosine (8OHdG), catalase, and glutathione peroxidase, and assayed OGG1 protein levels in kidney. SOD activity in the diaphragm and kidney was increased in HE rats. By itself, HE had no effect on the level of 8OHdG, but it did significantly suppress induction of 8OHdG by Fe-NTA, and the amount of suppression correlated with intensity of exercise. These results suggest that HE induces resistance to oxidative stress and, at least at the initiation stage, inhibits carcinogenesis.

Epilepsy and variation in the frontal lobe artery.

A patient with a rare variation of fronto-orbital artery (FOA) that developed generalized tonic and clonic seizures is reported. The epilepsy focus was in her left frontal region, where blood was supplied by the contralateral fronto-orbital artery. The region was vulnerable to ischemic changes due to a decrease in blood CO2 gas caused by an increase in endogenous progesterone in the luteal period. The anomaly illustrates an important mechanism of ischemia in epilepsy.

Unusual variant of the anterior cerebral artery.

A rare abnormality of the A1 segment of the anterior cerebral artery (ACA) is reported. The right ACA bifurcated into two parts at the middle point of the A1 segment, and these segments did not rejoin. The superior right A1 segment connected with the left A1 and formed a single pericallosal artery. The inferior right A1, from which the right ophthalmic artery originated, had no connection with the left A1.

Naringin attenuates the cytotoxicity of hepatotoxin microcystin-LR by the curious mechanisms to OATP1B1- and OATP1B3-expressing cells.

Microcystin-LR, which is an inhibitor of serine/threonine protein phosphatase (PP)1 and PP2A, induces liver injury by its selective uptake system into the hepatocyte. It is also thought that microcystin-LR induces reactive oxygen species (ROS). We tried to establish the chemical prevention of microcystin-LR poisoning. We investigated the effect of grapefruit flavanone glycoside naringin on cytotoxicity of microcystin-LR using human hepatocyte uptake transporter OATP1B3-expressing HEK293-OATP1B3 cells. We found cytotoxicity of microcystin-LR was attenuated by naringin in a dose dependent manner. The inhibition magnitude of total cellular serine/threonine protein phosphatase activity induced by microcystin-LR was suppressed by naringin. In addition, uptake of microcystin-LR into HEK293-OATP1B3 cells was inhibited by naringin. Furthermore, microcystin-LR induced phosphorylation of p53 was inhibited by naringin. Regardless of the difference in the exposure pattern of pre-processing and post-processing of naringin, the toxicity of microcystin-LR was comparable. These results suggested that naringin is promising remedy as well as preventive medicine for liver damage with microcystin-LR. In addition, involvement of ROS production after exposure to the sublethal concentrations of microcystin-LR in the onset of cytotoxicity was negligible. Therefore, inhibition of microcystin-LR uptake and the pathway other than ROS production would be involved in the effect of naringin on the attenuation of microcystin-LR toxicity.

Repair of the aortico-left ventricular tunnel originating from the right aortic sinus with severe aortic valve regurgitation.

A 31-year-old adult with an aortico-left ventricular tunnel (ALVT) arising from the right aortic sinus is reported. Preoperative transesophageal echocardiography demonstrated a ruptured sinus of Valsalva with severe aortic valve regurgitation which originated from the right coronary sinus entering the outlet portion of the left ventricular outflow tract. Operation revealed the aortic entrance of the tunnel was above the right coronary sinus. Direct closure of the orifice of the tunnel using three stitches of 4-0 polypropylene with felt and aortic valve replacement (AVR) was performed. At 10-month follow-up the patient is asymptomatic and receiving no oral medications except anticoagulants. We believe this to be the oldest case of ALVT managed with AVR.

Purification and identification of two carnosine-cleaving enzymes, carnosine dipeptidase I and Xaa-methyl-His dipeptidase, from Japanese eel (Anguilla japonica).

Three enzymes, carnosine dipeptidase I (EC 3.4.13.20, CNDP1), carnosine dipeptidase II (EC 3.4.13.18, CNDP2), and Xaa-methyl-His dipeptidase (or anserinase: EC 3.4.13.5, ANSN), are known to be capable of catalyzing the hydrolysis of carnosine (ß-alanyl-l-histidine), in vertebrates. Here we report the purification and identification of two unidentified carnosine-cleaving enzymes from Japanese eel (Anguilla japonica). Two different dipeptidases were successfully purified to homogeneity from the skeletal muscle; one exhibited a broad substrate specificity, while the other a narrow specificity. N-terminal amino-acid sequencing, deglycosylation analysis, and genetic analysis clearly revealed that the former is a homodimer of glycosylated subunits, encoded by ANSN, and the latter is another homodimer of glycosylated subunits, encoded by CNDP1; that is, Xaa-methyl-His dipeptidase, and carnosine dipeptidase I respectively. This is the first report on the identification of carnosine dipeptidase I from a non-mammal. Database search revealed presence of a CNDP1 ortholog only from salmonid fishes, including Atlantic salmon and rainbow trout, but not from other ray-finned fish species, such as zebrafish, fugu, and medaka whose genomes have been completely sequenced. The mRNAs of CNDP1 and ANSN are strongly expressed in the liver of Japanese eel, compared with other tissues, while that of CNDP2 is widely distributed in all tissues tested.

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.

p53 Plays an important role in cell fate determination after exposure to microcystin-LR.

BACKGROUND: Microcystin-LR, a cyclic heptapeptide, possesses the ability to inhibit the serine/threonine protein phosphatases PP1 and PP2A and, consequently, exhibits acute hepatocytotoxicity. Moreover, microcystin-LR induces cellular proliferation, resulting in tumor-promoting activity in hepatocytes. However, mechanisms that regulate the balance between cell death and proliferation after microcystin-LR treatment remain unclear. OBJECTIVE: We examined the contribution of the transcription factor p53, as well as that of the hepatic uptake transporter for microcystin-LR, organic anion transporting polypeptide 1B3 (OATP1B3), to the cellular response to microcystin-LR exposure. METHODS: We analyzed intracellular signaling responses to microcystin-LR by immunoblotting and real-time reverse-transcriptase polymerase chain reaction techniques using HEK293 human embryonic kidney cells stably transfected with SLCO1B3 (HEK293-OATP1B3). In addition, we analyzed the effect of attenuation of p53 function, via the p53 inhibitor pifithrin-alpha, and knockdown of p53 mRNA on the cytotoxicity of microcystin-LR using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Microcystin-LR induced the phosphorylation and accumulation of p53 in HEK293-OATP1B3 cells, which resulted in up-regulation of the expression of p53 transcript targets, including p21 and seven in absentia homolog 1 (siah-1). In addition, microcystin-LR activated Akt signaling through the phosphorylation of Akt and glycogen synthase kinase 3beta. Although Akt signaling was activated, the accumulation of p53 led cells to apoptosis after treatment with 50 nM microcystin-LR for 24 hr. Both pharmacological inhibition of transcription factor activity of p53 by pifithrin-alpha and knockdown of p53 with small hairpin RNA attenuated the susceptibility of HEK293-OATP1B3 cells to microcystin-LR. CONCLUSIONS: This study demonstrates the importance of p53 in the regulation of cell fate after exposure to microcystin-LR. Our results suggest that, under conditions of p53 inactivation (including p53 mutation), chronic exposure to low doses of microcystin-LR may lead to cell proliferation through activation of Akt signaling. Results of this study may contribute to the development of chemoprevention and chemotherapeutic approaches to microcystin-LR poisoning.

Occurrence of a novel acetylated amino acid, N(alpha)-acetylhistidine, in skeletal muscle of freshwater fish and other ectothermic vertebrates.

The occurrence of N(alpha)-acetylhistidine (NAH) in skeletal muscle of 91 species of freshwater fish and 9 species of other ectothermic vertebrates was investigated, with consideration of phylogenetic relationships. Of the 91 freshwater fish species examined, 13 species (7 cichlids, 5 anabantids, and 1 catfish) contained considerable amounts (>1 micromol/g) of NAH in their skeletal muscles. The highest level (10.37 micromol/g) of NAH was found in the tissue of Betta splendens (Siamese fighting fish). Moreover, the NAH contents in the tissues of Trichogaster trichopterus (three spot gourami), Kryptopterus bicirrhis (glass catfish), Oreochromis niloticus (Nile tilapia), Mikrogeophagus ramirezi (ram cichlid) and Parachromis managuensis (Guapote tigre) were 3.17-6.16 micromol/g. The skeletal muscle of amphibians (5 species) and reptiles (4 species) had a low level (<0.25 micromol/g) of NAH. The present findings clearly demonstrate NAH as the fifth imidazole-related compound, in addition to histidine, carnosine, anserine and ophidine (balenine), recognized as a major non-protein nitrogenous constituent in the skeletal muscle of vertebrate animals.

Apolipoprotein complexity in Japanese eel Anguilla japonica: truncated apolipoprotein A-I and apolipoprotein A-I-like protein in plasma lipoproteins.

A truncated apolipoprotein (apo) A-I with a molecular weight (M(r)) of 26 kDa was first isolated from the plasma high density lipoproteins of an atypical Japanese eel (Anguilla japonica). Interestingly, this eel contained a very small amount of intact apoA-I (M(r)28 kDa) in the plasma, although serine protease inhibitors were present throughout the plasma preparation. The N-terminal sequence of 20 amino acids in truncated apoA-I was completely identical with that of intact apoA-I. Another apolipoprotein with M(r)28 kDa, whose N-terminal amino acid sequence differed from apoA-I, was also found in high density lipoprotein and low density lipoprotein. The apolipoprotein profiles of Japanese eel plasma appear to be complicated.