Heme-binding properties of heme detoxification protein from Plasmodium falciparum

The heme detoxification protein of the malaria parasite Plasmodium falciparum is involved in the formation of hemozoin, an insoluble crystalline form of heme. Although the disruption of hemozoin formation is the most widely used strategy for controlling the malaria parasite, the heme-binding properties of heme detoxification protein are poorly characterized. In this study, we established a method for the expression and purification of the non-tagged protein and characterized heme-binding properties. The spectroscopic features of non-tagged protein differ from those of the His-tagged protein, suggesting that the artificial tag interferes with the properties of the recombinant protein. The purified recombinant non-tagged heme detoxification protein had two heme-binding sites and exhibited a spectrum typical of heme proteins. A mechanism for hemozoin formation is proposed.     

Screening and development of enzymes for determination and transformation of amino acids

ABSTRACT

The high stereo- and substrate specificities of enzymes have been utilized for micro-determination of amino acids. Here, I review the discovery of l-Phe dehydrogenase and its practical use in the diagnosis of phenylketonuria in more than 5,400,000 neonates over two decades in Japan. Screening and uses of other selective enzymes for micro-determination of amino acids have also been discussed. In addition, novel enzymatic assays with the systematic use of known enzymes, including assays based on a pyrophosphate detection system using pyrophosphate dikinase for a variety of l-amino acids with amino-acyl-tRNA synthetase have been reviewed. Finally, I review the substrate specificities of a few amino acid-metabolizing enzymes that have been altered, using protein engineering techniques, mainly for production of useful chemicals, thus enabling the wider use of natural enzymes.     

Mitochondrials complex I activity is reduced in latent adriamycin-induced cardiomyopathy of rat

We previously reported on the use of enzymatic analysis to impair fatty acid metabolism followed by reduced myocardial energy content, leading to severe heart failure in adriamycin (ADR)-treated rats. The aim of this study is to investigate whether impaired myocardial energy metabolism can also be detected by other methods; i.e. measuring mitochondrial complex I activity and myocardial ¹²⁵I-15-(p-iodophenyl)-3-(R,S)- methylpentadecanoic acid (BMIPP) accumulation in ADR-treated rats. Eight-week-old male Sprague-Dawley rats received 6 intraperitoneal injections of ADR (total 15 mg/kg: group ADR) or saline (control group) over 2 weeks. Left ventricular (LV) ejection fraction was assessed using echocardiography at 3- and 6-weeks after ADR injection (3 weeks and 6 weeks, respectively). Myocardial fatty acid utilization was assessed at 3 weeks and 6 weeks. The myocardial counts of BMIPP were measured after intravenous BMIPP (370 kBq) injection, and ¹²⁵I counts were measured to calculate the uptake ratio. The enzymatic activity of complex I was assessed by monitoring the oxidation of nicotinamide-adenine-dinucleotide-disodium-salt (NADH). In rats treated with ADR, significant decrease in LV ejection fraction was observed only at 6 weeks compared to control (72.5 vs. 84.5%, p < 0.01rpar;. LV ejection fraction at 3 weeks was identical between group ADR and control (81.8 vs. 84.4%). However, at 3 weeks, complex I activity was already reduced significantly in group ADR as compared to control group (p = 0.03), but the reduction in BMIPP accumulation was not (p = 0.15). Our data indicated that reduced complex I activity in a phenomenon occurred in early phase of ADR-induced cardiomyopathy, and it might play an important role in the progression of ADR-induced heart failure.     

SPECIES BOUNDARIES IN THE TOXIC DINOFLAGELLATE PROROCENTRUM LIMA (DINOPHYCEAE,PROROCENTRALES), BASED ON MORPHOLOGICAL AND PHYLOGENETIC CHARACTERS1

Wild and cultured specimens of Prorocentrum lima (Ehrenb.) F. Stein from 26 widely different areas in 13 countries were examined in order to determine consistent characters for delimiting species boundaries in this taxon. The morphological characters valve shape, valve size, valve ornamentation, number and shape of valve pores, number and shape of marginal pores, and periflagellar platelets were observed using LM and SEM, and two molecular genetic regions were sequenced. We identified stable morphological characters that were consistent among wild specimens and all cultures, which were valve shape, valve ornamentation, and number and arrangement of periflagellar platelets. All cultures of P. lima identified by these characters formed a monophyletic group in phylogenetic analyses based on the two genes, which, however, included the species Prorocentrum arenarium. P. arenarium was determined to be within the range of morphological variation of P. lima, and therefore we synonymize the two taxa. Within this monophyletic group, P. lima was divided into several subclades in the all phylogenetic analyses. There were no morphological characters specifically related to any one subclade. The subclades appeared to correlate broadly to sample collection regions, suggesting that geographically separated populations may have become genetically distinct within this epi‐benthic species. We have emended species boundaries in P. lima.     

Histological observation of the urogenital papillae in the bi-directional sex-changing gobiid fish, Trimma okinawae

The gobiid fish Trimma okinawae changes its sex bi-directionally according to its social status. Morphological changes in the urinogenital papillae (UGP) of this fish have been reported during sex change. However, there have been no detailed observations of such changes. Here, we histologically examined the UGP structure of male- and female-phase fish. UGPs of fish in female and male phase contained both oviducts and sperm ducts. Both ducts were coalesced into one duct within the posterior region of the UGP. Female-phase fish had many longitudinal folds in the hypertrophied tunica mucosa of the oviduct, which was found to be responsible for the transport of eggs and the removal of follicular cells from the oocyte. In contrast, male-phase fish had an immature oviduct and a mature sperm duct in the UGP. In the male-phase fish, the co-existence of spermatozoa and fibrillar secretions was observed in the sperm duct during spermiation.     

Mass mortality of the Japanese pearl oyster Pinctada fucata martensii in relation to water temperature, chlorophyll a and phytoplankton composition

Mass mortalities of the Japanese pearl oyster Pinctada fucata martensii have widely occurred in western Japan since 1994. The causes of these mass mortalities are at present not thoroughly understood. In this study, we investigated oyster survival in relation to some environmental factors such as water temperature, concentration of chlorophyll a and density or composition of phytoplankton. The examined mass mortality occurred from September to December 1998, and the color on the adductor muscle of the oysters was red-brown, suggesting an infectious disease. Oysters that became moribund during the experiment lost weight, while the weight of unaffected oysters increased. The cell density of Nitzschia spp., an inedible algae for the oyster, in Uchiumi Bay increased before and during the mass mortality event. From the results of our study, we hypothesize that P. fucata martensii was weakened by starvation because of the dominance of inedible food and then contracted an infectious disease that resulted in mortality.     

Permanence of single-species stage-structured models

In this paper, we consider population survival by using single-species stage-structured models. As a criterion of population survival, we employ the mathematical notation of permanence. Permanence of stage-structured models has already been studied by Cushing (1998). We generalize his result of permanence, and obtain a condition which guarantees that population survives. The condition is applicable to a wide class of stage-structured models. In particular, we apply our results to the Neubert-Caswell model, which is a typical stage-structured model, and obtain a condition for population survival of the model.The research is partially supported by the Ministry of Education, Science and Culture, Japan, under Grant in Aid for Scientific Research (A) 13304006.     

Direct phenotypic analysis of human MHC class I antigen presentation: visualization,quantitation, and in situ detection of human viral epitopes using peptide-specific,MHC-restricted human recombinant antibodies

The advent in recent years of the application of tetrameric arrays of class I peptide-MHC complexes now enables us to detect and study rare populations of Ag-specific CD8(+) T cells. However, available methods cannot visualize or determine the number and distribution of these TCR ligands on individual cells nor detect APCs in tissues. In this study, we describe for the first time studies of human class I peptide-MHC ligand presentation. These studies were facilitated by applying novel tools in the form of peptide-specific, HLA-A2-restricted human recombinant Abs directed toward a viral epitope derived from human T cell lymphotropic virus type I. Using a large human Ab phage display library, we isolated a large panel of recombinant Fab Abs that are specific for a particular peptide-MHC class I complex in a peptide-dependent, MHC-restricted manner. We used these Abs to visualize the specific complex on APCs and virus-infected cells by flow cytometry, to quantify the number of, and visualize in situ, a particular complex on the surface of APCs bearing complexes formed by naturally occurring active intracellular processing of the cognate viral Ag. These findings demonstrate our ability to transform the unique fine specificity, but low intrinsic affinity of TCRs into high affinity soluble Ab molecules endowed with a TCR-like specificity toward human viral epitopes. These molecules may prove to be crucial useful tools for studying MHC class I Ag presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases, and autoimmune disorders.