Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing

Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION? sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.     

Structural study on the active site of porcine pepsin and Rhizopus chinensis acid protease. Spin labeling with diazoketone reagents

To investigate the active site structures of porcine pepsin and Rhizopus chinensis acid protease (RAP), spin label techniques were applied for these enzymes. Comparison of spin labeled porcine pepsin and RAP suggested that the active site cleft of porcine pepsin was narrower at the top, but wider at the bottom than that of RAP. Addition of pepstatin restricted the motion of the labeled nitroxide radicals. Under alkaline conditions, the enzymes changed their conformation discontinuously and irreversibly to open the active site clefts and to lose the binding ability for pepstatin. The denaturation points of both the enzymes were determined to be pH 6.2.     

Availability of Energy in Ester and Ether Derivatives of Glycols by Growing Chicks

Biological availability of 33 esters, 17 ethers and 2 acetals of ethanediol, 1,2-propanediol, 1,3-butanediol and 1,4-butanediol was compared by mini-test with chicks. Chicks can utilize esters of ethanediol, 1,2-propanediol and 1,3-butanediol with acetic acid and fatty acids of carbon chain length from 5 to 12 with more improved palatability than that of free acids, while availability of esters of these glycols with propionic and butyric acids was low. Esters of 1,4-butanediol and ether derivatives of these glycols was not available, except ethyl ether of di-ethanediol which was partially available. Acetacetal of ethanediol was partially available but n-butyracetal was not.     

Isolation of variant carrot cell lines with altered pigmentation

Cultured carrot cells were treated with a known mutagenic compound, N-methyl-N′-nitro-N-nitroso-guanidine, and plated on a nutrient agar medium. Four variant cell lines whose pigmentation properties differed from stock calluses have been isolated. The contents of major carotenoid components, β-carotene and lycopene, of these cells were determined and compared with those of parent strains.     

1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids

Summary Two-dimensional (2D)¹H NMR experiments using deuterium labeling have been carried out to investigate the solution structure of ribonuclease HI (RNase HI) fromEscherichia coli (E. coli), which consists of 155 amino acids. To simplify the¹H NMR spectra, two fully deuterated enzymes bearing several prototed amino acids were prepared from an RNase HI overproducing strain ofE. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, He. Val. and Leu. The other was labeled by only protonated His and Ile. The 2D¹H NMR spectra of these deuterated R Nase H1 proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.     

Polyamine enhancement of the synthesis of adenylate cyclase at the translational level and the consequential stimulation of the synthesis of the RNA polymerase sigma 28 subunit

The effects of polyamines on the synthesis of various final sigma subunits of RNA polymerase were studied using Western blot analysis. Synthesis of final sigma(28) was stimulated 4.0-fold and that of final sigma(38) was stimulated 2.3-fold by polyamines, whereas synthesis of other final sigma subunits was not influenced by polyamines. Stimulation of final sigma(28) synthesis was due to an increase in the level of cAMP, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. Polyamines were found to increase the translation of adenylate cyclase mRNA by facilitating the UUG codon-dependent initiation. Analysis of RNA secondary structure suggests that exposure of the Shine-Dalgarno sequence of mRNA is a prerequisite for polyamine stimulation of the UUG codon-dependent initiation.     

Identification of Novel HLA-A*24:02-Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies

The homeobox protein, PEPP2 (RHOXF2), has been suggested as a cancer/testis (CT) antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs) is unknown. In this study, we revealed that PEPP2 gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2^271-279). The CTLs induced by PEPP2^271-279 recognized PEPP2-positive target cells in an HLA-A*24:02-restricted manner. We also found that a demethylating agent, 5-aza-2’-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2’-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients.     

A new species of Trichogramma (Hymenoptera: Trichogrammatidae) parasitic on eggs of the alderfly Sialis melania (Neuroptera: Sialidae) from Japan,with comments on its phylogeny and male wing polymorphism

A new species of Trichogramma that parasitizes Sialis melania eggs is described as Trichogramma tajimaense Yashiro, Hirose and Honda, sp. nov. from Japan. Its phylogenetic position is based on a DNA‐based analysis, and data regarding its male wing polymorphism are also presented. The view that T. tajimaense is closely related to T. semblidis, another parasitoid of Sialis eggs, is supported by the results of a phylogenetic analysis, as well as by the biological and morphological similarities between both species. Trichogramma tajimaense is also similar in male wing polymorphism to T. kurosuae, a gregarious egg parasitoid of the lepidopteran Ivela auripes, as both Trichogramma species exhibit male wing trimorphism (fully alate, brachypterous and apterous forms) in contrast to the male wing dimorphism (fully alate and apterous forms) of T. semblidis. However, no phylogenetic analysis reveals a close relationship between T. tajimaense and T. kurosuae, and a difference exists between these two species in the mean percentage of flightless (brachypterous and apterous) males that emerge from a host egg mass; 96% of T. tajimaense males are incapable of flight, whereas about 50% of T. kurosuae males are flightless. Because all or almost all males of T. semblidis parasitizing Sialis eggs are apterous, T. tajimaense is more similar to T. semblidis than to T. kurosuae in the proportion of flightless males. In addition, male wing polymorphisms of Trichogramma in relation to mating systems could also show a similarity between T. tajimaense and T. semblidis when considering both species as quasi‐gregarious parasitoids of Sialis eggs.