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.

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.

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.

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.

Phenylenediamine derivatives induce GDF-15/MIC-1 and inhibit adipocyte differentiation of mouse 3T3-L1 cells.

Phenylenediamine derivatives can function as a hydrogen donor and reportedly exert various biological actions including cytoprotective effects against oxidative stress, possibly by acting as an antioxidant. Previous studies showed that feeding of such compounds to mice reduced their body weight, but the precise mechanism remains unknown at present. Here, we found that these compounds inhibited the in vitro differentiation of mouse preadipocytes, 3T3-L1 cells, into adipocytes, suggesting that, at least in part, reduced generation of adipocytes might contribute to the observed weight loss in mice. Next, we performed array analysis and found that the expression of GDF-15/MIC-1, which is a TGFß superfamily cytokine, and Trib 3, an intracellular downstream effector of the cytokines, was up-regulated by these derivatives. Thus, we identified the compounds as inducers of GDF-15/MIC-1 and suggest that such induction may have led to inhibition of adipocyte differentiation, which could account for the weight-loss effect of these compounds.