Ghrelin stimulates phagocytosis and superoxide production in fish leukocytes.

To clarify the role of ghrelin in the fish immune system, the in vitro effect of ghrelin was examined in phagocytic leukocytes of rainbow trout (Oncorhynchus mykiss). Administration of trout ghrelin and des-VRQ-trout ghrelin, in which three amino acids are deleted from trout ghrelin, increased superoxide production in zymosan-stimulated phagocytic leukocytes from the head kidney. Gene expression of growth hormone (GH) secretagogue-receptor (GHS-R) was detected by RT-PCR in leukocytes. Pretreatment of phagocytic leukocytes with a GHS-R antagonist, [D-Lys3]-GHRP-6, abolished the stimulatory effects of trout ghrelin and des-VRQ-trout ghrelin on superoxide production. Ghrelin increased mRNA levels of superoxide dismutase and GH expressed in trout phagocytic leukocytes. Immunoneutralization of GH by addition of anti-salmon GH serum to the medium blocked the stimulatory effect of ghrelin on superoxide production. These results suggest that ghrelin stimulates phagocytosis in fish leukocytes through a GHS-R-dependent pathway, and also that the effect of ghrelin is mediated, at least in part, by GH secreted by leukocytes.

Expression of prolactin-releasing peptide and prolactin in the euryhaline mudskippers (Periophthalmus modestus): prolactin-releasing peptide as a primary regulator of prolactin.

Prolactin (PRL)-releasing peptide (PrRP) is a strong candidate stimulator of pituitary PRL transcription and secretion in teleosts. However, the role in control of extrapituitary PRL expression is unclear even in mammals. To study the possible presence of PrRP-PRL axes not only in the brain-pituitary but also in peripheral organs, the expression patterns of PrRP, PRL and growth hormone (GH) were characterized in amphibious euryhaline mudskippers (Periophthalmus modestus). PrRP mRNA is abundantly expressed not only in the brain but also in the liver, gut and ovary, while less abundant expression was also detected in the skin and kidney. Corresponding to the distribution of PrRP mRNA, PRL mRNA was also detectable in these organs. During adaptation to different environments, the changes in mRNA levels of PrRP paralleled those in PRL in the brain-pituitary, liver and gut in an organ-specific manner. Brain PrRP mRNA and the pituitary PRL mRNA increased under freshwater and terrestrial conditions (P < 0.05); expression of PrRP and PRL in the gut of freshwater fish was higher (P < 0.05) than those in sea-water fish although there were no changes in fish kept out of water; no significant change was seen in the liver. Expressions of GH were not correlated with PrRP. In the gut, PrRP and PRL appear to be co-localized in the mucosal layer, especially in the mucous cells. Thus, PrRP may also be a local modulator of extrapituitary PRL expression and the PrRP-PRL axes in various organs may play an organ-specific role during environmental adaptation.

High susceptibility of chromosome 16 to radiation-induced chromosome rearrangements in human lymphocytes under in vivo and in vitro exposure.

The aim of the present study was to investigate whether chromosome 16p presents breakpoint regions susceptible to radiation-induced rearrangements. The frequencies of translocations were determined by fluorescence in situ hybridization (FISH) using cosmid probes C40 and C55 mapping on chromosome 16p, and a chromosome 16 centromere-specific probe (pHUR195). Peripheral lymphocytes were collected from normal individuals and from seven victims of 137Cs in the Goiania (Brasil) accident (absorbed doses: 0.8-4.6 Gy) 10 years after exposure. In vitro irradiated lymphocytes (3 Gy) were also analyzed. The mean translocation frequency/cell obtained for the 137Cs exposed individuals was 2.4-fold higher than the control value (3.6 x 10(-3) +/- 0.001), and the in vitro irradiated lymphocytes showed a seven-fold increase. The genomic translocation frequencies (FGs) were calculated by the formula Fp = 2.05 fp(1-fp)FG (Lucas et al., 1992). For the irradiated lymphocytes and victims of 137Cs, the FGs calculated on the basis of chromosome 16 were 2- to 8-fold higher than those for chromosomes 1, 4 and 12. Our results indicate that chromosome 16 is more prone to radiation-induced chromosome breaks, and demonstrate a non-random distribution of induced aberrations. This information is valuable for retrospective biological dosimetry in case of human exposure to radiation, since the estimates of absorbed doses are calculated by determining the translocation frequency for a sub-set of chromosomes, and the results are extrapolated to the whole genome, assuming a random distribution of induced aberrations. Furthermore, the demonstration of breakpoints on 16p is compatible with the reports about their involvement in neoplasias.

Expression of the vasotocin gene in the hypothalamus of intact and osmotically stimulated bullfrogs during metamorphosis.

To study the ontogeny of the vasotocin (VT) system and its contribution to anuran metamorphosis, VT mRNA levels were determined by Northern blot analysis in metamorphosing bullfrog tadpoles. Effects of osmotic stimulation on VT mRNA levels were also analyzed in order to follow the development of osmotic responsiveness of VT neurons. The intensity of hybridization signals for VT mRNA gradually increased during prometamorphic development. The increase became marked thereafter until metamorphic climax. Plasma osmolality and hematocrit remained unchanged before metamorphosis, and increased after metamorphic climax, indicating that climactic tadpoles in a semi-terrestrial environment were in a dehydrated condition. These increases correlated well with the increase in VT mRNA level. Immersion of tadpoles in 30% seawater (approximately 350 mOsmol) for 3 days increased plasma osmolality at all stages. No significant changes were observed in the VT mRNA level in response to this treatment during premetamorphic stages. The VT mRNA levels were significantly higher in the treated tadpoles after preclimax stages. Hyperosmotic treatment also increased hematocrit until early metamorphic climax, but did not alter it in tadpoles at late metamorphic climax. These results suggest that the responsiveness of VT-producing neurons to hyperosmotic or hypovolemic stimulation, or both, is established by the time of the metamorphic climax in bullfrog. The marked increase in VT mRNA levels at metamorphic climax stages of intact individuals is probably induced by dehydration. VT-stimulated water absorption and reabsorption in the target organs probably prevented the increase in hematocrit at late metamorphic climax. Thus VT may contribute importantly to osmoregulatory mechanisms in relation to adaptation to a semi-terrestrial habitat through the metamorphosis.

A single and novel natriuretic peptide is expressed in the heart and brain of the most primitive vertebrate, the hagfish (Eptatretus burgeri).

In teleost fish and tetrapods, the natriuretic peptide (NP) family consists of ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide) and VNP (ventricular natriuretic peptide) that are secreted from the heart, and C-type natriuretic peptide (CNP) that is found in the brain. However, CNP is the only NP identified in the heart and brain of elasmobranchs, suggesting that it is the ancestral type of the NP family and that ANP, BNP and VNP appeared later in the vertebrate phylogeny. To delineate more clearly the molecular evolution of this hormone family, we determined the sequence of NP molecule(s) in evolutionarily the oldest vertebrate group, the cyclostomes. We have cloned a novel NP cDNA from the heart and brain of hagfish, Eptatretus burgeri, using the RACE method and degenerate primers that amplify all known types of NP cDNAs. The novel NP, named EbuNP after the scientific name of this hagfish, appears to be the only NP in the heart and brain, as no other NP cDNAs were amplified even after specific removal of the cloned EbuNP mRNA from the mRNA pool, except for a minor alternatively spliced EbuNP cDNA with a truncated 3'-untranslated sequence. The EbuNP was equally similar to known NPs but was not considered to be a CNP because of the presence of a C-terminal tail sequence. The EbuNP gene was abundantly expressed in the cardiac atrium, ventricle, portal heart and brain but scarcely in the intestine; no expression was observed in the gill and kidney. Mass spectrometry of affinity-purified EbuNP in plasma, heart and brain revealed a 68 amino acid peptide circulating in the blood and stored in the heart, which is cleaved at the typical cleavage signal of a processing enzyme, furin, as observed in mammalian BNP. The C-terminal Gly residue was used for amidation as is the case in eel ANP. The immunoreactive EbuNP was not detected in the brain, suggesting the presence of a different processing form in the brain. These results show that the molecular evolution of the NP family in vertebrates is more complex than previously thought.

Four natriuretic peptides (ANP, BNP, VNP and CNP) coexist in the sturgeon: identification of BNP in fish lineage.

The natriuretic peptide (NP) family is composed of three members: atrial, brain/ventricular and C-type NPs (ANP, BNP/VNP and CNP respectively) in tetrapods and teleostean fish, but only CNP in elasmobranch fish. In order to trace the process of divergence of the NP family in early vertebrate evolution, we attempted to detect NPs in the primitive ray-finned fish, the sturgeon (Acipenser transmontanus). Unexpectedly, we isolated four distinct NP cDNAs from the heart and brain of this chondrostean fish. The single NP from the brain was CNP, as judged from the lack of C-terminal 'tail' sequence extending from the intramolecular ring. Two of the three cardiac NPs were ANP and VNP, as judged by the presence of an amidation signal at its C-terminus (ANP) and a long and conserved C-terminal tail sequence (VNP) respectively. The third cardiac NP was most probably BNP because it possessed all the features characteristic of BNP including: (1) the presence of dibasic amino acids within the intramolecular ring; (2) the presence of AUUUA repeats in the 3'-untranslated region of its mRNA; (3) equivalent expression of its mRNA in the atrium and ventricle and appreciable expression in the brain. Based on the sturgeon BNP sequence, we further isolated BNP cDNA from the heart of tilapia and pufferfish for the first time in teleostean fish. Phylogenetic analysis of the precursors showed that newly identified NPs belong to each group of the four NPs. The current identification of both VNP and BNP in the sturgeon clearly showed that BNP and VNP are coded by distinct genes, and that the NP family consists of at least four members in the ray-finned fish. VNP has not been molecularly identified in mammals but its presence is suggested from physiological studies; heterologous fish VNP exhibited more potent vasorelaxant activity than homologous mammalian ANP in the isolated coronary artery of dogs.

Characterization and localization of mRNA encoding the salmon-type gonadotrophin-releasing hormone precursor of the masu salmon.

Gonadotrophin-releasing hormone (GnRH) is considered to have an important role in the control of reproduction in salmonid fish, although we do not have any direct evidence. To clarify this problem by molecular techniques, we first determined the nucleotide sequence of the mRNA encoding the precursor of salmon-type GnRH (sGnRH) from the masu salmon, Oncorhynchus masou. The masu salmon sGnRH precursor was composed of a signal peptide, sGnRH and a GnRH-associated peptide (GAP) which was connected to sGnRH by a Gly-Lys-Arg sequence. The amino acid sequence of sGnRH and Gly-Lys-Arg were highly conserved when compared with the corresponding regions of African cichlid sGnRH and mammalian GnRH precursors. However, the GAP region was markedly divergent, with a 66% amino acid similarity to African cichlid GAP and an 8.3-15% similarity to mammalian GAPs. Northern blot analysis indicated the presence of a single mRNA species of about 600 bases in the olfactory bulb and telencephalon and in the diencephalon. The signal was more intense in the former regions. An in-situ hybridization study further revealed that sGnRH neurones were distributed in the olfactory nerve, the ventral part of the olfactory bulb, the ventral part of the telencephalon, the lateral preoptic area and the preoptic nucleus. The sGnRH neurones were thus longitudinally scattered between the olfactory nerve and the lateral preoptic area in the rostroventral part of brain. The intensity of the hybridization signals and the size of hybridization-positive somata were much greater in the olfactory nerve and the rostral olfactory bulb than in the other regions. Preoptic sGnRH neurones were scarcely detected in immature masu salmon, whereas they were more frequently observed in maturing animals. It is possible that the olfactory and the preoptic sGnRH neurones have different physiological roles in salmonid fish.

Expression of provasopressin gene during ontogeny in the hypothalamus of developing mice.

We investigated the ontogeny of provasopressin gene expression in neurosecretory neurons of the supraoptic and paraventricular nuclei of developing mice by semi-quantitative in situ hybridization and immunohistochemical techniques in combination with stereometry of vasopressin-immunoreactive neurons. Provasopressin mRNA was detected in paraffin sections using a mixture of radiolabeled synthetic oligonucleotide probes complementary to the mRNA loci encoding vasopressin (2-9) and vasopressin neurophysin (1-8). Vasopressin immunoreactivity was located with a polyclonal anti-vasopressin antiserum and a monoclonal anti-vasopressin-neurophysin antibody either with or without enhancing technique for the diaminobenzidine reaction. Autoradiographic hybridization signals that indicate the localization of provasopressin mRNA were first detected on embryonic day 15 in the supraoptic nucleus and embryonic day 18 in the paraventricular nucleus. Vasopressin immunoreactivity was first found in the median eminence on embryonic day 14, and then in the supraoptic and paraventricular nuclei on embryonic days 15 and 16, respectively. The provasopressin mRNA levels were markedly increased in both the supraoptic and the paraventricular nuclei just after birth. The immunoreactivity of vasopressin neurons was drastically decreased in both nuclei on postnatal days 1 and 2, suggesting marked vasopressin release in the neonates. Cross-sectional areas of vasopressin-immunoreactive somata and their cell nuclei gradually increased in both the supraoptic and the paraventricular nuclei during the perinatal period by day 5, and then attained adult size between days 10 and 20. During this phase, the level of provasopressin mRNA remained low compared with that in the adult magnocellular neurosecretory cells. These results indicate that the expression of provasopressin gene is markedly increased in both the supraoptic and the paraventricular nuclei soon after birth. Secretory activity of vasopressin neurons is elevated in neonatal mice. Vasopressin may have an important osmoregulatory role in neonatal mice undergoing drastic changes in water metabolism following birth.

Vitamin D metabolism in osteogenesis imperfecta during calcitonin therapy.

The effect of calcitonin on plasma concentrations of 25-hydroxyvitamin D (25-OHD) and 1,25-dihydroxyvitamin D [1,25-(OH)2D] was studied in six patients with osteogenesis imperfecta. The mean pretreatment value of plasma 1,25-(OH)2D was significantly higher than the mean value of age-matched control subjects (P less than .05). High or normal plasma levels of 1,25-(OH)2D before calcitonin therapy were decreased after 1 month of therapy and remained normal thereafter in all six patients. Plasma 25-hydroxyvitamin D concentrations, which were normal before calcitonin injection, remained normal during calcitonin administration. These results indicate that there may be acute and chronic effects of calcitonin on vitamin D metabolism.

Is the primitive regulation of pituitary prolactin (tPRL177 and tPRL188) secretion and gene expression in the euryhaline tilapia (Oreochromis mossambicus) hypothalamic or environmental?

We examined the effects of environmental salinity on circulating levels of the two prolactins (tPRL177 and tPRL188) and levels of pituitary tPRL177 and tPRL188 mRNA in the euryhaline tilapia, Oreochromis mossambicus. Fish were sham-operated or hypophysectomized and the rostral pars distalis (RPD) autotransplanted onto the optic nerve. Following post-operative recovery in (1/4) seawater, tilapia were transferred to fresh water (FW), (1/4) seawater (SW) or SW. Serum tPRL177 and tPRL188 levels in sham-operated and RPD-autotransplanted fish were highest in FW and decreased as salinity was increased. tPRL177 and tPRL188 mRNA levels in RPD implants as well as in pituitaries from the sham-operated fish were also highest in FW and decreased with increasing salinity. Serum osmolality increased with salinity, with the highest levels occurring in the seawater groups. We conclude that some plasma factor (probably plasma osmolality), in the absence of hypothalamic innervation, exerts a direct regulatory action on prolactin release and gene expression in the pituitary of O. mossambicus. This regulation is in accord with the actions of the two prolactins in the freshwater osmoregulation of the tilapia.

Androgenic regulation of expression of beta-tubulin messenger ribonucleic acid in motoneurons of the spinal nucleus of the bulbocavernosus.

Expression of beta-tubulin mRNA was examined in androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using cDNA encoding mouse beta-tubulin. Hybridizable beta-tubulin mRNA was localized in the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression level of beta-tubulin mRNA in the SNB motoneurons, whereas the change was prevented by testosterone treatment. On the contrary, castration or testosterone treatment did not induce any changes in the expression level of beta-tubulin mRNA in the androgen-insensitive motoneurons of the retrodorsolateral nucleus. These results suggest that androgen regulates the expression of beta-tubulin gene in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally-induced neuronal plasticity in the SNB motoneurons.

Effect of androgen on the expression of gap junction and beta-actin mRNAs in adult rat motoneurons.

Expression of gap junction and beta-actin mRNAs was examined in androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using complementary DNAs encoding rat liver gap junction protein (connexin 32) and chick beta-actin. Hybridizable gap junction and beta-actin mRNAs were localised on the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression levels of both gap junction and beta-actin mRNAs in the SNB motoneurons, whereas these changes were prevented by testosterone treatment. On the contrary, castration or testosterone treatment did not induce any changes in the expression levels of gap junction and beta-actin mRNAs in the motoneurons of the retrodorsolateral nucleus (RDLN), which accumulate androgen less frequently and sparsely than those in the SNB. These results suggest that androgen regulates the expression of both gap junction and beta-actin genes in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally induced neuronal plasticity in the SNB motoneurons.

Androgen regulates gap junction mRNA expression in androgen-sensitive motoneurons in the rat spinal cord.

Expression of gap junction mRNA was examined in the androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) by in situ hybridization histochemistry using cDNA encoding rat liver gap junction protein (connexin 32). Hybridizable gap junction mRNA was localized on the somata and proximal dendrites of SNB motoneurons. The removal of androgen by castration dramatically reduced the expression level of gap junction mRNA in the SNB motoneurons, whereas this change was prevented by testosterone treatment. These results are the first demonstration of hormonal regulation of gap junction mRNA expression in the central nervous system.

Cellular localization of gap junction mRNA in the neonatal rat brain.

Cellular localization of grap junction mRNA was examined in the neonatal rat brain by in situ hybridization histochemistry using cDNA of rat liver gap junction (connexin 32). Hybridizable gap junction mRNA was localized on neural cells in the hippocampus. Gap junction mRNA was also found to be localized on neural cells of the parenchyma and ependymal layers in many other regions. Immunohistochemical and ultrastructural observations of the hippocampus supported the presence of gap junctions in the examined region. These results are the first demonstration of morphologically identified gap junctions and cellularly localized gap junction mRNA in the neonatal rat brain.

Interaction effects of 5-azacytidine with topoisomerase II inhibitors on CHO cells, as detected by cytogenetic analysis.

Different cell treatment protocols with the hypomethylating agent 5 azacytidine (5-aza C) were used in exponentially growing Chinese hamster ovary (CHO) cells in order to test its influence on the induction of chromosomal aberrations (CAs) induced by topoisomerase II inhibitors, ellipticine (EPC) and teniposide (VM-26). Cells pre-treated with 1 microg/ml 5-aza C for 1 h during the S-phase and post-treated in the last 2 h of incubation with 0.6 microg/ml EPC or 0.04 microg/ml VM-26 showed a reduction of 48% and 45%, respectively, in the frequencies of CAs as compared to the sum value of the frequencies obtained for each drug alone. 5-aza C added to the cultures for the last 2 h before cell fixation after a 30-min pulse treatment with EPC or VM-26 caused a 38% and 28% reduction, respectively. Simultaneous treatments with 5-aza C plus EPC, or 5-aza C plus VM-26 during the last 2 h of incubation (G2-phase), showed a significant effect of CA reduction (24%) only for the combination of 5-aza C + EPC. Preliminary assays with 5-aza C alone added to the cultures at different times demonstrated its effectiveness in inducing chromosome damage during the S-phase. Since S-phase-treated CHO cells showed a higher degree of reduction in the frequencies of CAs induced by EPC and VM-26, we suggest that 5-aza C incorporation into DNA may change the topo II cleavage sites, protecting the DNA from the induction of damage, or that the hypomethylation induced by incorporation of 5-aza C into DNA may change the chromatin structure facilitating the access to DNA repair enzymes. An alternative possibility is that 5-azaC can reactivate methylated genes involved in the repair of DNA double-strand breaks induced by topo II inhibitors.

Cloning and sequence analyses of vasotocin and isotocin precursor cDNAs in the masu salmon, Oncorhynchus masou: evolution of neurohypophysial hormone precursors.

We have cloned and determined the nucleotide sequences of cDNAs encoding precursors of neurohypophysial hormones, vasotocin (VT) and isotocin (IT), from the hypothalamus of masu salmon, Oncorhynchus masou. The deduced amino acid sequences of masu salmon VT and IT precursors (proVT-I and proIT-I) are highly homologous to those of chum salmon proVT-I and proIT-I, respectively. The VT and IT precursors are composed of a signal peptide, hormone and neurophysin (NP), the middle portion of which is highly conserved among vertebrates. Both the NPs extend about 30 amino acids at the C-terminal. The extended C-terminals have a leucin-rich segment in the carboxyl-terminal, as copeptin of vasopressin precursor. Southern bot analysis showed the presence of two types of proVT genes (proVT-I and proVT-II) and proIT genes (proIT-I and proIT-II) in an individual masu salmon, as in a chum salmon. Southern blot analysis with proVT probes further suggested that at least two different types of proVT-I genes exist in a single masu salmon. Northern blot analysis indicated that proVT-I and proIT-I genes are expressed in the hypothalamus, whereas proVT-II and proIT-II genes are not expressed. Evolutionary distance between proVT-I and proIT-I genes was statistically estimated based on synonymous nucleotide substitution in the coding region of the cDNAs. The magnitude of distance between masu salmon proVT-I and proIT-I genes suggested that the highly conserved central portion of NPs resulted from a gene conversion event. Between masu salmon and chum salmon, evolutionary distance for proVT-I genes is about 6-fold larger than that for proIT-I genes.

Schwannoma in the sigmoid colon: report of a case.

Schwann cell tumor occurring in the intestines is rare. A 68-year-old female came to our hospital because of hematemesis. Barium enema and colonoscopic examination revealed submucosal tumor in the sigmoid colon. Laboratory data showed mild anemia. No other abnormal finding was found in the blood chemistry. Tumor marker levels of carcinoembryonic antigen (CEA), CA19-9, alpha feto protein (AFP) and neuron specific enolase (NSE) were within normal limits. The exploratory laparotomy confirmed a large sigmoid colon tumor. She received sigmoid colectomy. The resected specimen was a submucosal tumor with central depression, measuring 4.7 x 3.5 x 3.0 cm in size. The cut surface of the tumor was yellowish hue with necrosis. Histological examination showed spindle-shaped tumor cells with palisading comma-shaped nuclei and the nuclear pleomorphism. Immunohistochemical examination revealed that the tumor was positive for S-100 protein staining, and negative for Actin and for H.H.F. staining. These findings showed that this tumor was of Schwann cell origin. We report here the case in detail of a schwannoma in the sigmoid colon.

[Dose escalation study of high dose etoposide in autologous hematopoietic stem cell transplantation].

Eight cases with poor prognosis hematological malignancies (non-Hodgkin lymphoma, 6 cases; acute non-lymphocytic leukemia, 2 cases) and nine cases with non-hematological malignancies were treated with high dose etoposide (VP16) containing regimen followed by autologous hemopoietic stem cell transplantation. Results were as follows; 1) all of three chemotherapy sensitive relapse patients with hematological malignancies continue complete remission without any cyto-reductive therapy 2) one of four refractory relapse patients continue remission 3) partial anti-tumor effect was noted in non-hematological malignancies, however, only two cases continue complete remission. Remission duration of other responders was not so long. The results disclosed the dose-limiting factor of high-dose VP16 therapy as reversible stomatitis with no related mortality, and maximal tolerated dose appears to be 60 mg/kg over 72 hr with 45 mg/kg as a safe and recommended therapeutic dose in future clinical trial. The clinical effect of dose escalation was not clearly demonstrated.

[Successful neoadjuvant chemotherapy in a patient with advanced gastric cancer with periaortic lymph node metastasis].

We described a case of advanced gastric cancer accompanied by metastasis to the periaortic lymph node. Two cycles of the neoadjuvant chemotherapy consisting of 5-FU and low-dose CDDP (FP therapy) were given. 5-FU (800mg/body/day) was administered as a continuous intravenous infusion for five days, and CDDP (10mg/body/day) was given as an intravenous infusion for an hour on days 1-5. The FP therapy resulted in a significant effect in the metastatic periaortic lymph node. Then, total gastrectomy with combined resection of spleen was done. Histological examination of the resected specimen revealed the histological effect showing Grade 3 in the primary site and Grade 2 in the periaortic lymph node. The patient is alive with no evidence of recurrence 13 months after operation. Thus, FP therapy is thought to be effective against advanced gastric cancer.

Effects of melatonin on DNA damage induced by cyclophosphamide in rats.

The antioxidant and free radical scavenger properties of melatonin have been well described in the literature. In this study, our objective was to determine the protective effect of the pineal gland hormone against the DNA damage induced by cyclophosphamide (CP), an anti-tumor agent that is widely applied in clinical practice. DNA damage was induced in rats by a single intraperitoneal injection of CP (20 or 50 mg/kg). Animals received melatonin during the dark period for 15 days (1 mg/kg in the drinking water). Rat bone marrow cells were used for the determination of chromosomal aberrations and of formamidopyrimidine DNA glycosylase enzyme (Fpg)-sensitive sites by the comet technique and of Xpf mRNA expression by qRT-PCR. The number (mean ± SE) of chromosomal aberrations in pinealectomized (PINX) animals treated with melatonin and CP (2.50 ± 0.50/100 cells) was lower than that obtained for PINX animals injected with CP (12 ± 1.8/100 cells), thus showing a reduction of 85.8% in the number of chromosomal aberrations. This melatonin-mediated protection was also observed when oxidative lesions were analyzed by the Fpg-sensitive assay, both 24 and 48 h after CP administration. The expression of Xpf mRNA, which is involved in the DNA nucleotide excision repair machinery, was up-regulated by melatonin. The results indicate that melatonin is able to protect bone marrow cells by completely blocking CP-induced chromosome aberrations. Therefore, melatonin administration could be an alternative and effective treatment during chemotherapy.