Cloning and expression profile of the alanine aminotransferase gene from kuruma shrimp Penaeus japonicus exposed to different salinities.

Several crustaceans including shrimps change the amount of specific free amino acids to regulate the osmotic pressure in their bodies. Kuruma shrimp Penaeus japonicus also increases the concentration of alanine (Ala) in the abdominal muscle following the increase of environmental salinity. In the present study, to elucidate the mechanisms of changes in Ala accumulation of kuruma shrimp depending on salinity, we cloned the gene encoding alanine aminotransferase (ALT), an enzyme involved in Ala biosynthesis, and examined its expression profile. It was found that the full-length kuruma shrimp ALT1 cDNA consisted of 3,301 bp, encoding 514 amino acids, and that all amino acid residues important for ALT activity were conserved. Phylogenetic analysis also indicated that the ALT gene cloned in this study was classified as ALT1. Moreover, we examined the expression levels of the ALT1 gene in the abdominal muscle and the hepatopancreas of kuruma shrimp acclimated at 17‰, 34‰, and 40‰ salinities, resulting that the mRNA levels of the ALT1 genes in both tissues of the shrimp acclimated at 40‰ were significantly higher than those at 17‰ for 12 h (p < 0.05). The mRNA levels of the ALT1 gene in the abdominal muscle of the shrimp acclimated for more than 24 h tended to increase following the increase of environmental salinity. These results indicate that ALT1 is responsible for the increase of free Ala concentration in the abdominal muscle of kuruma shrimp to regulate osmotic pressure at high salinity.

Metastatic gallbladder cancer presenting as numb chin syndrome: A case report and literature review.

Gallbladder cancer (GBC) is an uncommon malignancy that is highly aggressive in the advanced stages. However, it rarely metastasizes to the mandible. Numb chin syndrome (NCS) is a rare neurological manifestation associated with various underlying causes, including occult primary cancers and distant metastases. It is often considered to be a significant indicator of malignancy, and thorough investigation is essential in the presence of unclear etiology. The current study reported on the case of a 69-year-old Japanese woman who presented with numbness and mild pain in the lower lip and chin area for three months. No other systemic symptoms were observed. Immunocytochemical examination revealed the presence of an adenocarcinoma and TNM staging as per the Union for International Cancer Control and the American Joint Committee on Cancer guidelines confirmed stage IVb GBC. Comprehensive full-body positron emission tomography-computed tomography examination using 18F-fluoro-2-deoxy-D-glucose revealed additional bone and soft-tissue metastases. Palliative chemotherapy and radiation treatment were initiated based on the advanced stage of disease at the time of diagnosis. However, the patient succumbed to multiple organ failure six months later. The simultaneous occurrence of GBC, mandibular metastasis and NCS is rare and associated with poor prognosis. Despite the widespread nature of the disease, it can often manifest as non-specific oral symptoms without any systemic indications. The current study emphasizes the critical importance of timely confirmatory testing for accurate diagnosis and initiation of appropriate management for such complex conditions.

A newly identified enzyme from Japanese common squid Todarodes pacificus has the ability to biosynthesize d-aspartate.

Amino acids exist in two chiral forms, namely L and D. Although l-amino acids are predominant in vivo, certain limited circumstances have reported the usage of d-amino acids. d-aspartate (Asp), among them, plays crucial physiological roles in living organisms and is biosynthesized from L-Asp by the enzyme named aspartate racemase (AspRase). D-Asp is known to accumulate in large amounts in the nervous system of cephalopods. To understand the function of D-Asp in nervous system in more detail, it is necessary to elucidate its metabolic pathway; however, AspRase gene has not been identified in cephalopods as in the case of mammals. In this study, we successfully identified a novel gene encoding AspRase from the optic ganglion of Japanese common squid Todarodes pacificus. Our discovery of the squid AspRase challenges the prevailing assumption that AspRases across different animals share similar structures. Surprisingly, the squid AspRase is a unique enzyme that differs significantly from known AspRases, being structurally and phylogenetically related to aspartate aminotransferase (AST) and possessing both AspRase and AST activities. The optimum pH and temperature for AspRase activity using L-Asp as a substrate are approximately 7.0 and 20 °C, respectively. Moreover, we have found that AspRase activity is enhanced in the presence of 2-oxoacids. These findings have far-reaching implications for the understanding of enzymology and suggest that yet-to-be-identified mammalian AspRases may also be phylogenetically related to AST, rather than conventional AspRases. Furthermore, our results provide valuable insights into the evolution of the D-Asp biosynthetic pathway.

Hydrogen Peroxide-Triggered Conversion of Boronic Acid-Appended Insulin into Insulin and Its Application as a Glucose-Responsive Insulin Formulation.

p-Boronophenylmethoxycarbonyl (BPmoc) is a protecting group for amines that is removable by treatment with hydrogen peroxide (H2O2). We prepared BPmoc-modified insulin (BPmoc-Ins) and subcutaneously injected the formulation into diabetic rats. The results demonstrated that BPmoc effectively sealed the blood glucose (Glc)-lowering effects of Ins. Conversely, coinjection of BPmoc-Ins and Glc oxidase (GOx) resulted in reduced blood Glc levels, indicating that Ins was generated from BPmoc-Ins through the following reactions: oxidation of endogenous Glc by GOx; production of H2O2 accompanied by Glc oxidation; removal of BPmoc residues by H2O2. These results show the potential of BPmoc-Ins for a Glc-responsive Ins release system.

Cholesterol-binding ability of saponin from Japanese starfish.

Steroidal gylcosides are the predominant metabolites of starfish and are responsible for various biological activities. Some of these activities are recognized as a part of self-defense mechanism of starfish. Cholesterol-binding ability was evaluated with seven starfish crude extracts, where significantly (p < 0.05) highest ability (34%) was observed in Asterias amurensis and the lowest (16%) was attributed in Distolasterias nippon. To characterize the active compound exists in crude saponin from A. amurensis, the extract was subjected to thin layer chromatography following silica gel column chromatography. As the results, seven fractions (fr. A-G) were separated and frs. D and F demonstrated the highest cholesterol-binding ability (32% and 33%, respectively), equivalent to that of the A. amurensis extract. The isolated component (fr. F) was further separated (fr. F1-F3) for structural analysis. Based on cholesterol-binding ability result (29%), fr. F2 was analysed by matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy (MALDI-TOF MS) and then nuclear magnetic resonance spectroscopy (NMR). The compound was identified as thornasteroside A, one of the major bioactive compounds already found in A. amurensis. The discovery of a saponin with cholesterol-binding ability has important implications not only for the utilization of starfish but also for food and pharmaceutical research.

Changes in free amino acid concentrations and associated gene expression profiles in the abdominal muscle of kuruma shrimp (Marsupenaeus japonicus) acclimated at different salinities.

Shrimps inhabiting coastal waters can survive in a wide range of salinity. However, the molecular mechanisms involved in their acclimation to different environmental salinities have remained largely unknown. In the present study, we acclimated kuruma shrimp (Marsupenaeus japonicus) at 1.7%, 3.4% and 4.0% salinities. After acclimating for 6, 12, 24 and 72 h, we determined free amino acid concentrations in their abdominal muscle, and performed RNA sequencing analysis on this muscle. The concentrations of free amino acids were clearly altered depending on salinity after 24 h of acclimation. Glutamine and alanine concentrations were markedly increased following the increase of salinity. In association with such changes, many genes related to amino acid metabolism changed their expression levels. In particular, the increase of the expression level of the gene encoding glutamate-ammonia ligase, which functions in glutamine metabolism, appeared to be associated with the increased glutamine concentration at high salinity. Furthermore, the increased alanine concentration at high salinity was likely associated with the decrease in the expression levels of the the gene encoding alanine-glyoxylate transaminase. Thus, there is a possibility that changes in the concentration of free amino acids for osmoregulation in kuruma shrimp are regulated by changes in the expression levels of genes related to amino acid metabolism.

Novel Isoforms of N16 and N19 Families Implicated for the Nacreous Layer Formation in the Pearl Oyster Pinctada fucata.

Although a wide variety of proteins and genes possibly related to the shell formation in bivalve have been identified, their functions have been only partially approved. We have recently performed deep sequencing of expressed sequence tags (ESTs) from the pearl oyster Pinctada fucata using a next-generation sequencer, identifying a dozen of novel gene candidates which are possibly associated with the nacreous layer formation. Among the ESTs, we focused on three novel isoforms (N16-6, N16-7, and N19-2) of N16 and N19 families with reference to five known genes in the families and determined the full-length cDNA sequences of these isoforms. Reverse transcription-polymerase chain reaction revealed that N16-6 was expressed in gill, gonad, adductor muscle, and mantle, whereas N16-7 exclusively in mantle. N19-2 was expressed in all tissues examined. In situ hybridization demonstrated their regional expression in mantle and pearl sac, which well corresponded to those shown by EST analysis previously reported. Shells in the pearl oyster injected with dsRNAs of N16-7 and N19-2 showed abnormal surface appearance in the nacreous layer. Taken together, novel isoforms in N16 and N19 families shown in this study are essential to form the nacreous layer.

Global gene expression analysis of the muscle tissues of medaka acclimated to low and high environmental temperatures.

Medaka (Oryzias latipes) is a temperate eurythermal fish that is able to survive over a wide range of water temperatures ranging from near zero to over 30°C throughout the year; it maintains its normal physiological and biochemical processes through temperature acclimation. To determine the mechanisms involved in temperature acclimation of fish, the fast skeletal muscle tissues of medaka underwent global gene expression analysis using next-generation sequencing. Ten individuals were placed into two aquariums at 24°C. While the water temperature of one aquarium was decreased to 10°C, that of the other aquarium was increased to 30°C; these temperatures were subsequently maintained for 5weeks. RNA sequencing (RNA-Seq) analyses revealed that 11 genes involved in energy metabolism and muscle atrophy were significantly highly expressed in the 10°C-acclimated fish. Meanwhile, significantly higher expression levels were observed for 20 genes encoding myofibrillar proteins and heat shock proteins in the 30°C-acclimated fish. Moreover, 1103 genes had at least fourfold differential expression between the acclimation groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses provided important information: although the expression of genes related to metabolic processes were activated, muscle atrophy occurred in the 10°C-acclimated fish, and muscle cells divided actively in the 30°C-acclimated fish and avoided thermal stress by expressing heat shock proteins. Therefore, RNA-Seq analyses with the available genome database will be useful for better understanding the molecular mechanisms involved in the temperature acclimation of fish.

Distant recurrence risk following early ipsilateral breast tumor recurrence.

At present, the risk factors for distant recurrence among patients with early ipsilateral breast tumor recurrence (IBTR) require further investigation. Early IBTR is defined as occurring within 3 years following the initial surgery. In the current study, 40 patients with early IBTR were examined to determine the risk factors for distant recurrence. A node-positive status at the time of primary surgery and the administration of adjuvant chemotherapy following the primary surgery were significantly correlated with poorer distant disease-free survival (P=0.001 and P=0.002, respectively). Multivariate analyses revealed that the nodal status at the time of primary surgery was an independent predictive factor for distant recurrence (P=0.050). Therefore, the results of the current study revealed that the nodal status at the time of primary surgery was an independent predictive factor for distant recurrence among patients with early IBTR.

Molecular characterization of aspartylglucosaminidase, a lysosomal hydrolase upregulated during strobilation in the moon jellyfish, Aurelia aurita.

The life cycle of the moon jellyfish, Aurelia aurita, alternates between a benthic asexual polyp stage and a planktonic sexual medusa (jellyfish) stage. Transition from polyp to medusa is called strobilation. To investigate the molecular mechanisms of strobilation, we screened for genes that are upregulated during strobilation using the differential display method and we identified aspartylglucosaminidase (AGA), which encodes a lysosomal hydrolase. Similar to AGAs from other species, Aurelia AGA possessed an N-terminal signal peptide and potential N-glycosylation sites. The genomic region of Aurelia AGA was approximately 9.8 kb in length and contained 12 exons and 11 introns. Quantitative RT-PCR analysis revealed that AGA expression increased during strobilation, and was then decreased in medusae. To inhibit AGA function, we administered the lysosomal acidification inhibitors, chloroquine or bafilomycin A1, to animals during strobilation. Both inhibitors disturbed medusa morphogenesis at the oral end, suggesting involvement of lysosomal hydrolases in strobilation.

Distant Recurrence Risk after Late Ipsilateral Breast Tumor Recurrence: Results of a Retrospective, Single-Institution Study.

OBJECTIVES: There is limited information regarding risk factors for distant recurrence in patients with late ipsilateral breast tumor recurrence (IBTR). METHODS: Late IBTR was defined as IBTR occurring >5 years after the initial surgery. Seventy-eight patients with late IBTR were reviewed to examine the risk factors of distant recurrence. RESULTS: The negative estrogen receptor and positive HER2 statuses of IBTR were significantly correlated with poorer distant disease-free survival (p=0.03 and 0.02, respectively). Multivariate analyses demonstrated that the HER2 status of IBTR was an independent predictive factor of distant recurrence (p=0.04). CONCLUSIONS: The results of our retrospective, single-institution study revealed that the HER2 status of IBTR was an independent predictive factor of distant recurrence in patients with late IBTR.

Association of human epidermal growth factor receptor 2 status with ipsilateral breast tumor recurrence and resistance to endocrine therapy.

The effect of prior endocrine therapy on tumor biology and clinical outcomes of locoregional recurrence remains unclear. A total of 76 patients, who underwent salvage breast surgery for estrogen receptor-positive ipsilateral breast tumor recurrence (IBTR) following breast-conserving surgery for primary breast cancer, were retrospectively reviewed to investigate the association of human epidermal growth factor receptor 2 (HER2) status with the characteristics of IBTR and clinical outcomes following resection of IBTR. There was a tendency for more patients with HER2-positive IBTR to show resistance to endocrine therapy (71.4%) compared with those with HER2-negative IBTR (37.3%) (P=0.085). Patients with HER2-positive IBTR exhibited a higher rate of recurrence (62.5%) compared with those with HER2-negative IBTR (25.0%) (P=0.027). These results suggested that HER2-positive IBTR may be associated with resistance to endocrine therapy and a poorer clinical outcome.

Dynamic changes in the accumulation of metabolites in brackish water clam Corbicula japonica associated with alternation of salinity.

The brackish water clam Corbicula japonica inhabits rivers and brackish waters throughout Japan where the major fishing grounds in the Ibaraki Prefecture, Japan, are located at the Hinuma Lake and Hinuma River. Water salinity in the Lake Hinuma is low and stable due to the long distance from the Pacific Ocean, whereas that in the downstream of the river varies daily due to a strong effect of tidal waters. In the present study, we dissected the gill and foot muscle of brackish water clam collected from these areas, and subjected them to metabolome analysis by capillary electrophoresis-time-of-flight mass spectrometry. More than 200 metabolites including free amino acids, peptides and organic acids were identified, and their amounts from the foot muscle tend to be higher than those from the gill. The principal component analysis revealed that the amount of each metabolite was different among sampling areas and between the gill and foot muscle, whereas no apparent differences were observed between male and female specimens. When the metabolites in the female clam at high salinity were compared with those at low salinity, concentrations of ß-alanine, choline, γ-aminobutyric acid, ornithine and glycine betaine were found to be changed in association with salinity. We also compared various metabolites in relation to metabolic pathways, suggesting that many enzymes were involved in their changes depending on salinity.

Treatment sequence of aromatase inhibitors and radiotherapy and long-term outcomes of breast cancer patients.

BACKGROUND: The optimal sequence of radiotherapy (RT) and hormone therapy using aromatase inhibitors (AI) in patients with breast cancer treated with breast-conserving surgery is unclear. Several short-term analyses have shown that there are no differences in breast cancer outcomes according to the treatment sequence. However, long-term outcomes have not been reported. PATIENTS AND METHODS: We retrospectively analyzed disease-free survival events in 315 consecutive breast cancer patients who underwent breast-conserving surgery, RT, and received adjuvant AI at our Institute between 2001 and 2009. We compared the outcomes between treatment sequences of AI and RT (concurrent vs. sequential). RESULTS: With a median follow-up of 5.6 years, no significant differences between the 2 groups in terms of disease-free survival (unadjusted p=0.6; adjusted p=0.5) were observed. CONCLUSION: Similarly to previous short-term reports, AI administration after RT and AI concurrently with RT are both reasonable treatment options for early-stage breast cancer patients treated with breast-conserving surgery.

Regional lymphatic recurrence after salvage surgery for ipsilateral breast tumor recurrence of breast cancer without local treatment for regional lymphatic basin.

BACKGROUND: There is limited information regarding rates of and risk factors for regional lymphatic recurrence (RLR) after salvage surgery for ipsilateral breast tumor recurrence (IBTR) without local treatment for the regional lymphatic basin. METHODS: One hundred two patients, who underwent salvage breast surgery without local treatment for the regional lymphatic basin (surgery or radiotherapy) for IBTR that relapsed after breast-conserving surgery for primary breast cancer, were reviewed retrospectively to examine the rate of and risk factors for RLR. RESULTS: Of the 102 patients, 9 patients (8.8%) had RLR with a median follow-up period of 3.7 years after salvage breast surgery for IBTR. The estrogen receptor (ER) status and lymphovascular invasion of the recurrent breast tumor were both independent predictive factors of RLR (P = 0.04 and 0.02, respectively). CONCLUSIONS: The rate of RLR was not low in patients with IBTR who received salvage breast surgery only without any treatment for the regional lymphatic basin. The ER status and lymphovascular invasion of the recurrent breast tumor were predictive factors of RLR.

Novel genes participating in the formation of prismatic and nacreous layers in the pearl oyster as revealed by their tissue distribution and RNA interference knockdown.

In our previous publication, we identified novel gene candidates involved in shell formation by EST analyses of the nacreous and prismatic layer-forming tissues in the pearl oyster Pinctada fucata. In the present study, 14 of those genes, including two known genes, were selected and further examined for their involvement in shell formation using the RNA interference. Molecular characterization based on the deduced amino acid sequences showed that seven of the novel genes encode secretory proteins. The tissue distribution of the transcripts of the genes, as analyzed by RT-PCR and in situ hybridization, was mostly consistent with those obtained by the EST analysis reported previously. Shells in the pearl oysters injected with dsRNAs targeting genes 000027, 000058, 000081, 000096, 000113 (nacrein), 000118, 000133 and 000411 (MSI60), which showed expression specific to the nacreous layer forming tissues, showed abnormal surface appearance in this layer. Individuals injected with dsRNAs targeting genes 000027, 000113 and 000133 also exhibited abnormal prismatic layers. Individuals injected with dsRNAs targeting genes 000031, 000066, 000098, 000145, 000194 and 000200, which showed expression specific to prismatic layer forming tissues, displayed an abnormal surface appearance in both the nacreous and prismatic layers. Taken together, the results suggest that the genes involved in prismatic layer formation might also be involved in the formation of the nacreous layers.

Preferences for oral versus intravenous adjuvant chemotherapy among early breast cancer patients.

PURPOSE: The purpose of this study was to evaluate preferences for oral versus intravenous adjuvant chemotherapy among early breast cancer patients (UMIN-CTR number UMIN000004696). PATIENTS AND METHODS: Eighty-two postmenopausal women with estrogen receptor-positive, human epidermal growth-factor receptor 2-negative breast cancer who had completed adjuvant chemotherapy were asked about their preferred route of administration of chemotherapy and the reason. Women also answered questions about their physical and psychological status and quality of life during chemotherapy. RESULTS: Patients who had received oral chemotherapy preferred it more frequently than those who had received intravenous chemotherapy (100% versus 37%, respectively, chi-square =15.5; P<0.001). Patients who preferred the same route of administration of chemotherapy as they had previously received showed a significantly better psychological status during chemotherapy compared with those who preferred a different route. CONCLUSION: Our study showed that preferences for oral and intravenous chemotherapy strongly depended on the actual prior administration of chemotherapy and patients' own experiences during chemotherapy.

Cloning of skeletal myosin heavy chain gene family from adult pleopod muscle and whole larvae of shrimps.

The physiological and biological properties of skeletal muscle in crustacea have not been well understood compared with those of vertebrates. The present study focused on myosin, the major protein in skeletal muscle, from shrimps. In our previous study, two full-length genes encoding myosin heavy chain (MHC), a large subunit of the myosin molecule, were cloned from abdominal fast skeletal muscle of kuruma Marsupenaeus japonicus, black tiger Penaeus monodon and Pacific white Penaeus vannamei shrimps, and named as MHCa and MHCb. In this study, we renamed these as MHC1 and MHC2, respectively, due to the presence of various isoforms newly identified. Partial MHC sequences were identified from pleopod muscle of these shrimps. Two MHCs, named MHC3 and MHC4, were identified from pleopod muscle of kuruma shrimp, whereas two MHCs, named MHC4 and MHC5, were cloned from Pacific white shrimp pleopod. MHC3 was cloned only from black tiger shrimp pleopod. Partial MHC sequences from zoea, mysis, and postlarvae of black tiger and Pacific white shrimps were also determined. The phylogenetic tree demonstrated that most MHCs from pleopod muscle and larval MHCs formed clades with MHC1 and MHC2, respectively. These MHCs were considered to be of fast type, since MHC1 and MHC2 are fast-type MHCs according to our previous study. MHC5 obtained from pleopod muscle of Pacific white shrimp in this study was monophyletic with American lobster Homarus americanus S2 slow tonic MHC previously reported, indicating that MHC5 from Pacific white shrimp is of slow type.

Cloning, expression, and localization of two types of fast skeletal myosin heavy chain genes from black tiger and Pacific white shrimps.

The physiology and biochemistry of skeletal muscles in shrimps have been poorly understood compared with those from vertebrates. The present study was conducted focusing on myosin, the major protein in skeletal muscle, from adult specimens of black tiger Penaeus monodon and Pacific white Penaeus vannamei shrimps. Two genes encoding myosin heavy chain (MHC), a large subunit of the myosin molecule, were cloned from abdominal fast skeletal muscle and defined as MHCa and MHCb according to our previous study on kuruma shrimp Marsupenaeus japonicus. Random cloning demonstrated that the MHCb gene (MHCb) was expressed more abundantly than MHCa. The full-length cDNA clones of MHCa and MHCb from black tiger shrimp consisted of 5,926 and 5,914 bp, respectively, which encoded 1,914 and 1,909 amino acids, respectively, whereas those from Pacific white shrimp consisted of 5,923 and 5,908 bp, respectively, which encoded 1,913 and 1,909 amino acids, respectively. Both MHCa and MHCb were considered to be fast muscle type due to their strict localization in fast muscle. The amino acid identities between MHCa and MHCb of black tiger shrimp were 77%, 60%, and 73% in the regions of subfragment-1 (S1), subfragment-2 (S2) and light meromyosin (LMM), respectively, with 71% in total, whereas those of Pacific white shrimp were 78%, 60%, and 73% in the regions of S1, S2, and LMM, respectively, with 72% in total. In situ hybridization and northern blot analysis using different regions from abdominal muscle demonstrated different localizations of MHCa and MHCb transcripts in this muscle, suggesting their distinct physiological functions.

The occurrence of two types of fast skeletal myosin heavy chains from abdominal muscle of kuruma shrimp Marsupenaeus japonicus and their different tissue distribution.

Shrimps belong to the class Crustacea, which forms a large, diverse group in the invertebrates. However, the physiology and biochemistry of their skeletal muscles have been poorly understood compared with those from vertebrates including mammals and fish. The present study focused on myosin, the major protein in skeletal muscle, from adult specimens of kuruma shrimp Marsupenaeus japonicus. Two types of the gene encoding myosin heavy chain (MHC), a large subunit of the myosin molecule, were cloned from abdominal fast skeletal muscle and defined as MHCa and MHCb. Protein analysis revealed that the MHCa isoform was expressed at a higher level than the MHCb isoform. The full-length cDNA clones of MHCa and MHCb consisted of 5929 bp and 5955 bp, respectively, which encoded 1912 and 1910 amino acids, respectively. Both were classified into fast muscle type by comparison with the partially deduced amino acid sequences of fast-type and slow-type (S(1), slow twitch) MHCs reported previously for the American lobster Homarus americanus. The amino acid identities between MHCa and MHCb of kuruma shrimp were 78%, 60% and 72% in the regions of subfragment-1, subfragment-2 and light meromyosin, respectively, and 71% in total. In situ hybridisation using anti-sense RNA-specific probes, along with northern blot analysis using different tissues from abdominal muscle, revealed the different localisation of MHCa and MHCb transcripts in abdominal fast skeletal muscle, suggesting their distinct physiological functions.