Hepatic neuroendocrine carcinoma in a Japanese macaque (Macaca fuscata)

Primary neuroendocrine neoplasm of the liver is extremely rare in both humans and non‐human primates. The present report describes the clinical and pathological findings of an aged Japanese macaque (Macaca fuscata) with hepatic neuroendocrine carcinoma. To our knowledge, this is the first report of hepatic neuroendocrine neoplasm in macaques.     

Computational chemical analysis of Ru(II)‐Pheox–catalyzed highly enantioselective intramolecular cyclopropanation reactions

Computational chemical analysis of Ru(II)‐Pheox–catalyzed highly enantioselective intramolecular cyclopropanation reactions was performed using density functional theory (DFT). In this study, cyclopropane ring–fused γ‐lactones, which are 5.8 kcal/mol more stable than the corresponding minor enantiomer, are obtained as the major product. The results of the calculations suggest that the enantioselectivity of the Ru(II)‐Pheox–catalyzed intramolecular cyclopropanation reaction is affected by the energy differences between the starting structures 5l and 5i . The reaction pathway was found to be a stepwise mechanism that proceeds through the formation of a metallacyclobutane intermediate. This is the first example of a computational chemical analysis of enantioselective control in an intramolecular carbene‐transfer reaction using C₁‐symmetric catalysts.     

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.     

Determination of isonicotinic acid in the presence of isoniazid and acetylisoniazid Studies on isonicotinic acid formation from isoniazid in isolated rat hepatocytes

In comparison with the hepatocytes obtained from intact rats and rats pretreated with phenobarbital or 3-methylchoranthrene, the amount of isonicotinic acid (INA) formed from isoniazid (INH) increased substantially after incubation at 37°C using the pretreated hepatocytes. This suggests an oxidative pathway for INA formation from INH, apart from hydrolysis. In order to explore the exact mechanism of INA formation in the hepatocytes, an HPLC assay for INA in the presence of INH and acetylisoniazid was developed. In this assay, INA was extracted after the preparation of an ion pair with tetra-n-butylammonium hydroxide, and analysed using an ODS column and a mobile phase consisting of 0.067 M potassium dihydrogenphosphate solution-methanol (96:4 v/v). The method is simple, accurate and especially suitable for INA determination after incubation of INH in isolated rat hepatocytes.     

Urinary thromboxane A2/prostacyclin balance reflects the pathological state of a diabetic

Levels of the stable urinary metabolites of thromboxane A2 and prostacyclin, 11-dehydro-thromboxane B2 (11-dehydro-TXB2) and 2,3-dinor-6-keto-prostaglandin F1alpha (2,3-dinor-6-keto-PGF1alpha) were measured in diabetics to elucidate the relation between the thromboxane A2/prostacyclin (TX/PGI) balance and pathological states of diabetes mellitus. 11-Dehydro-TXB2 and 2,3-dinor-6-keto-PGF1alpha were derivatized to methyl ester-propylamide-dimethylisopropylsilyl ether and methyl ester-methoxime-dimethylisopropylsilyl ether derivatives, respectively, and applied to a gas chromatography/selected ion monitoring. The TX/PGI ratios of diabetics were higher than those of healthy volunteers, suggesting the hypercoagulative states of this disease. The ratios showed positive correlations with the levels of blood glucose. The levels of hemoglobin A1c and triglyceride were correlated weakly with the ratio. Some of the patients who had relatively low levels of blood glucose also showed high TX/PGI ratios. Furthermore, the ratio increased in the order of the groups 1, 2, and 3; group 1 contained patients who did not take medicine for diabetes, group 2 contained those who took oral hypoglycemic agents, and group 3 contained those who received insulin therapy. These observations indicate that the TX/PGI ratio reflects the pathological conditions of diabetes and is a useful marker, having few different features from other markers that are presently used.     

Evolution of Mhc–DRB Introns: Implications for the Origin of Primates

Introns are generally believed to evolve too rapidly and too erratically to be of much use in phylogenetic reconstructions. Few phylogenetically informative intron sequences are available, however, to ascertain the validity of this supposition. In the present study the supposition was tested on the example of the mammalian class II major histocompatibility complex (Mhc) genes of the DRB family. Since the Mhc genes evolve under balancing selection and are believed to recombine or rearrange frequently, the evolution of their introns could be expected to be particularly rapid and subject to scrambling. Sequences of intron 4 and 5 DRB genes were obtained from polymerase chain reaction-amplified fragments of genomic DNA from representatives of six eutherian orders—Primates, Scandentia, Chiroptera, Dermoptera, Lagomorpha, and Insectivora. Although short stretches of the introns have indeed proved to be unalignable, the bulk of the intron sequences from all six orders, spanning >85 million years (my) of evolution, could be aligned and used in a study of the tempo and mode of intron evolution. The analysis has revealed the Mhc introns to evolve at a rate similar to that of other genes and of synonymous sites of non-Mhc genes. No evidence of homogenization or large-scale scrambling of the intron sequences could be found. The Mhc introns apparently evolve largely by point mutations and insertions/deletions. The phylogenetic signals contained in the intron sequences could be used to identify Scandentia as the sister group of Primates, to support the existence of the Archonta superorder, and to confirm the monophyly of the Chiroptera. Received: 26 October 1998 / Accepted: 21 December 1998     

Phylogenetic analysis of saccharolytic oral treponemes isolated from human subgingival plaque.

A total of 74 strains of oral treponemes, which were isolated from subgingival plaque samples from patients with periodontitis, were taxonomically studied on the basis of biochemical characteristics, DNA-DNA hybridization, and 16S rRNA gene sequences. These organisms fermented carbohydrates and required rumen fluid or short-chain volatile fatty acids for growth. The isolates were divided into seven subgroups based on their biochemical characteristics. The levels of DNA relatedness among the representative strains of each subgroup and Treponema socranskii (including three subspecies) were greater than 78%, while the levels of DNA relatedness among these strains and other Treponema species, including T. denticola and "T. vincentii", were less than 15%. DNA-DNA hybridization indicated that all subgroups belonged to T. socranskii. This result correlated well with the cluster on the phylogenetic trees based on 16S rRNA sequences.     

Dissociation of double-headed cytoplasmic dynein into single-headed species and its motile properties

Cytoplasmic dynein is a minus-end directed microtubule motor and plays important roles in the transport of various intracellular cargoes. Cytoplasmic dynein comprises two identical heavy chains and forms a dimer (double-headed dynein); the total molecular weight of the cytoplasmic dynein complex is about 1.5 million. The dynein motor domain is structurally very different from those of kinesin and myosin, and our understanding of the mechanisms of dynein energy transduction is limited mainly because of the difficulty in obtaining a sufficient quantity of purified and active cytoplasmic dynein. We purified cytoplasmic dynein, which was free from dynactin and other dynein-associated proteins. The purified cytoplasmic dynein was active in an in vitro motility assay. The controlled dialysis of the purified dynein against 4 M urea resulted in its complete dissociation into monomeric species (single-headed dynein). The separation of the dynein heads by the treatment was reversible. The MgATPase activities of the single-headed and reconstituted double-headed dynein were comparable to that of intact dynein. The double-headed dynein bundled microtubules in the absence of ATP; the single-headed dynein did not. The single-headed dynein produced in vitro microtubule-gliding motility at velocities very similar to those of double-headed dynein at various ATP concentrations. These results indicate that a single cytoplasmic dynein heavy chain is sufficient to produce robust microtubule motility. Application of the double- and single-headed dynein molecules in various assay systems will elucidate the mechanism of action of the cytoplasmic dynein.