Kunihiko Futami

A Molecular Classification of Papillary Renal Cell Carcinoma

Despite the moderate incidence of papillary renal cell carcinoma (PRCC), there is a disproportionately limited understanding of its underlying genetic programs. There is no effective therapy for metastatic PRCC, and patients are often excluded from kidney cancer trials. A morphologic classification of PRCC into type 1 and 2 tumors has been recently proposed, but its biological relevance remains uncertain. We studied the gene expression profiles of 34 cases of PRCC using Affymetrix HGU133 Plus 2.0 arrays (54,675 probe sets) using both unsupervised and supervised analyses. Comparative genomic microarray analysis was used to infer cytogenetic aberrations, and pathways were ranked with a curated database. Expression of selected genes was validated by immunohistochemistry in 34 samples with 15 independent tumors. We identified two highly distinct molecular PRCC subclasses with morphologic correlation. The first class, with excellent survival, corresponded to three histologic subtypes: type 1, low-grade type 2, and mixed type 1/low-grade type 2 tumors. The second class, with poor survival, corresponded to high-grade type 2 tumors (n = 11). Dysregulation of G1-S and G2-M checkpoint genes were found in class 1 and 2 tumors, respectively, alongside characteristic chromosomal aberrations. We identified a seven-transcript predictor that classified samples on cross-validation with 97% accuracy. Immunohistochemistry confirmed high expression of cytokeratin 7 in class 1 tumors and of topoisomerase IIalpha in class 2 tumors. We report two molecular subclasses of PRCC, which are biologically and clinically distinct and may be readily distinguished in a clinical setting.

Deficiency of FLCN in Mouse Kidney Led to Development of Polycystic Kidneys and Renal Neoplasia

The Birt–Hogg–Dubé (BHD) disease is a genetic cancer syndrome. The responsible gene, BHD, has been identified by positional cloning and thought to be a novel tumor suppressor gene. BHD mutations cause many types of diseases including renal cell carcinomas, fibrofolliculomas, spontaneous pneumothorax, lung cysts, and colonic polyps/cancers. By combining Gateway Technology with the Ksp-Cre gene knockout system, we have developed a kidney-specific BHD knockout mouse model. BHDflox/flox/Ksp-Cre mice developed enlarged kidneys characterized by polycystic kidneys, hyperplasia, and cystic renal cell carcinoma. The affected BHDflox/flox/Ksp-Cre mice died of renal failure at approximate three weeks of age, having blood urea nitrogen levels over tenfold higher than those of BHD flox/+/Ksp-Cre and wild-type littermate controls. We further demonstrated that these phenotypes were caused by inactivation of BHD and subsequent activation of the mTOR pathway. Application of rapamycin, which inhibits mTOR activity, to the affected mice led to extended survival and inhibited further progression of cystogenesis. These results provide a correlation of kidney-targeted gene inactivation with renal carcinoma, and they suggest that the BHD product FLCN, functioning as a cyst and tumor suppressor, like other hamartoma syndrome–related proteins such as PTEN, LKB1, and TSC1/2, is a component of the mTOR pathway, constituting a novel FLCN-mTOR signaling branch that regulates cell growth/proliferation.

Disease resistance and hypocholesterolemia in yellowtail Seriola quinqueradiata fed a non-fishmeal diet

The physiology of yellowtail fed a non-fishmeal diet was examined, with a specific interest in the role of taurine in disease resistance and cholesterol metabolism. Decrease of disease resistance in fish fed a non-fishmeal diet was confirmed by mortality due to natural infection with pseudotuberculosis and artificial infection with Lactococcus garvieae. It is suggested that the most important symptoms related to decrease of disease resistance in fish fed a non-fishmeal diet is anemia. Anemia was improved by supple mentation with taurine. Significant elevation of relative expression of HMG-CoA reductase mRNA in fish fed a non-fishmeal diet suggests that cholesterol synthesis would be activated and not dysfunctional. Plasma cholesterol of these fish was elevated to the levels of control fish by supplementation of both cholesterol and taurine. These results suggest that hypocholesterolemia observed in fish fed a non-fishmeal diet compared with a fishmeal diet would be caused by insufficient dietary cholesterol and decrease of endogenous cholesterol due to the lack of dietary taurine.

Molecular cloning of fresh water and deep-sea rod opsin genes from Japanese eel Anguilla japonica and expressional analyses during sexual maturation1

We have determined the complete cDNA sequences of fresh water rod opsin gene (fwo) and deep‐sea rod opsin gene (dso) from Japanese eel Anguilla japonica. The cDNA clones of fwo and dso consisted of 1437 and 1497 nucleotides, respectively. The predicted opsins of both genes consisted of 352 amino acid residues. Southern blot and PCR analyses of genomic DNA indicated that the Japanese eel genome contains only one fwo and one dso and they are intronless. Quantitative RT‐PCR analyses revealed that the expression of fwo decreases with sexual maturation while that of dso increases.

Differential expression of max and two types of c-myc genes in a tetraploid fish, the common carp (Cyprinus carpio).

We cloned the full-length cDNA of max gene from the common carp (Cyprinus carpio). The cDNA clone of carp max consists of 1209 bp and contained an ATG-initiated ORF consisting of 156 aa. The carp MAX share 76.7-93.8% aa identity with those of human, mouse, rat, chicken, Xenopus and zebrafish, respectively. The 15 bp alternative splicing was observed in the loop region of helix-loop-helix and is not previously described in mammalian max sequences. Transcripts of max gene were observed in all of the tissues of carp investigated in this study. The highest expression was found in the ovary, and the transcripts in hepatopancreas and heart were low. Two carp c-myc genes (CAM1 and CAM2) showed differential expression pattern. The expression of max was concomitant with CAM2 expression, but not with CAM1. It has been reported that MYC/MAX heterodimer as a regulator of gene expression has been maintained throughout vertebrate evolution, and the expression of c-myc has been concomitant with max expression. In addition, according to phylogenetic analysis, CAM1 is evolving faster than CAM2 after gene duplication. Therefore, this result suggests that CAM1 may evolve to obtain a new function different from c-myc.

Development of a serology-based assay for efficacy evaluation of a lactococcicosis vaccine in Seriola fish

Lactococcicosis is an infection caused by the bacterium Lactococcus garvieae and creates serious economic damage to cultured marine and fresh water fish industries. The use of the assay currently applied to evaluate the potency of the lactococcicosis vaccine is contingent upon meeting specific parameters after statistical analysis of the percent survival of the vaccinated yellowtail or greater amberjack fish after challenge with a virulent strain of L. garvieae. We found that measuring the serological response with a quantitative agglutinating antibody against the L. garvieae antigen (phenotype KG+) was an effective method of monitoring the potency of lactococcicosis vaccines. Vaccinated fish had significantly higher antibody titers than control fish when the L. garvieae Lg2-S strain was used as an antigen. Furthermore, the titer of the KG + agglutinating antibody was correlated with vaccine potency, and the cut-off titer was determined by comparing the data with those from the challenge test. An advantage of the proposed serology-based potency assay is that it will contribute to reduced numbers of animal deaths during vaccine potency evaluations.

Deformation and blemishing of pearls caused by bacteria

Bacteria cause deformation and blemishing in pearls, and we investigate this relationship. We examined pearls derived from Pinctada margaritifera, Pinctada maxima, and Pinctada fucata, and determined the location of bacteria using histopathological and immunohistochemical techniques and 16S ribosomal RNA (rRNA) sequence analyses. The most remarkable change was the inflammatory reaction located between the pearl nucleus and the nacreous layer, composed of hemocytic infiltration with melanization, periostracum, and fibrous aragonite-like structures. These anomalous changes were limited to abnormal sites, and such inflammatory reaction sites are a major factor in the formation of pearl abnormalities. Bacteria were detected from the inflammatory sites and are suspected as the causative agent. Most of these bacteria were anaerobic.

Determination of heterogeneous transcription start points of two c-myc genes from the common carp (Cyprinus carpio)

We determined the heterogeneous transcription start points (tsp) of two c-myc genes from the common carp (Cyprinus carpio), tetraploid teleost, by the oligo-capping method and showed the existence of the first exon. This is the first report on the existence of the first exons of the fish c-myc gene. Transcription of the two carp c-myc genes started from at least four sites in CAM1, locating from -752 to -381bp upstream of the translation start site, and from 12 sites in CAM2, locating from -586 to -413bp upstream respectively. The first introns of CAM1 and CAM2 were deduced to be 335 and 356bp, respectively. They shared 86.9% nt identity, lower than those of the second exons (94.1%), and third exons (92.3%), which suggest that the first exons evolved faster. No nt identities were found between the c-myc first exons of carp and other vertebrates. The putative promoter regions in CAM1 and CAM2 contained no obvious TATA or CCAAT boxes in the expected positions.

Application of ELISA-based kit for detecting AOZ and determining its clearance in eel tissues

Furazolidone, an antibacterial drug that was once widely used in the livestock industry and aquaculture, is now prohibited in numerous countries. It is difficult to detect residual furazolidone because it is readily metabolized in animal tissues but, by using and liquid chromatography coupled with tandem mass spectrometry, its metabolite, 3-amino-2-oxazolidinone (AOZ) can be detected. Here we describe the validity of an enzyme-linked immunosorbent assay (ELISA) kit to detect AOZ in Japanese eel Anguilla japonica tissue. ELISA is capable of detecting AOZ at 1.0 μg/kg in an eel sample with excellent accuracy and precision. Our results show that ELISA is suitable for regulatory purposes and for studying the fate of AOZ residues in eel treated with furazolidone. To measure the persistence of AOZ in eel tissues, eels (1.4–6.5g) were immersed in tanks containing 2 and 10 mg furazolidone/L for 3 h, and then maintained in a tank supplying well water for the next 160 days. The half-lives of AOZ, calculated from the linear terminal part of the excretion curve, were 25.0 days in muscle and 21.6 days in liver from fish exposed to 2 mg/L furazolidone. In the eels treated with 10 mg/L furazolidone, by contrast, high levels of AOZ were detected in liver and muscle, but the half-lives of AOZ were similar to those in fish treated with 2 mg/L furazolidone. The half-lives of AOZ in eel tissues were prolonged by the condition of low water temperature.

Anthelmintic activity of Rosmarinus officinalis against Dactylogyrus minutus (Monogenea) infections in Cyprinus carpio

Monogenean parasites are important ectoparasites of fish, and are responsible for severe economic impacts in the aquaculture industry. They are usually treated with chemicals, but the chemicals can have harmful side effects in the fish and may pose threats to human health. Rosemary (Rosmarinus officinalis) is a common medicinal herb, with antimicrobial and antitumor properties. Here, we examined the anthelmintic activity of rosemary extract against the monogenean (Dactylogyrus minutus) in vitro and in vivo using bath treatment and oral administration. The in vitro experiments showed that parasite survival was affected by both rosemary extract concentration and the solvent (water and ethanol). Parasites were dead at 61.8±5.6 and 7.8±1.4min when exposed to 100 and 200g aqueous rosemary extract solution/L of water respectively. It took 166.7±48.2 and 5.4±1.01min to kill the parasites when exposed to 1 and 32g ethanol rosemary extract solution/L of water respectively. Moreover, pure component of rosemary extract obtained commercially used in in vitro experiments showed that 1,8-Cineole was the most toxic component of the main components tested. Parasite intensity and prevalence in fish exposed to 50 and 100g aqueous rosemary solution/L water for 30min were significantly lower than they were in controls (p<0.05). In oral treatment experiments, diets of Cyprinus carpio were supplemented with eight different concentrations of aqueous rosemary extract. The intensity of parasites was significantly less in fish fed for 30days with feed containing 60, 80 and 100ml aqueous extract/100g feed than in control (p<0.05). Together these results indicate that rosemary is a promising candidate for prevention and control of monogenean infection.