Chemical modification of neamine

The aminocyclitol antibiotic neamine has been modified by changing the configuration of one or two hydroxyl groups of the aminocyclitol moiety to elucidate the relationship between configuration and antimicrobial activity. 5-Epi-, 6-epi-, and 5,6-diepineamine have been prepared and their antimicrobial activity has been determined against several micro-organisms.     

Chemical modification of aminocyclitol antibiotics

The aminocyclitol antibiotic neamine has been chemically modified at the hydroxyl group on C-6 of the 2-deoxystreptamine moiety. The partially acetylated neamine derivatives, 6,3′,4′-tri-O-acetyl- (3) and 5,3′,4′-tri-O-acetyl-1,3,2′,6′-tetra-N-(ethoxycarbonyl)neamine (4), were prepared by random hydrolysis of the 5,6-O-ethoxyethylidene derivative (2), followed by chromatographic purification. Condensation of 4 and 2,3,5-tri-O-benzoyl-d-ribofuranosyl chloride led to 6-O-(β-d-ribofuranosyl)neamine (7). Analogous condensation of 4 with 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide or 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl bromide afforded the corresponding 6-O-(d-hexopyranosyl)neamines.     

Oxidative Degradation of Squalene by Arthrobacter Species

An organism isolated from soil and identified as Arthrobacter sp. was studied for its squalene degradation. The degradation product from squalene, which accumulated in the culture broth, was isolated and identified as trans-geranylacetone by mass spectrometry, gas chromatography, infrared spectrometry, and nuclear magnetic resonance spectrometry. Addition of a high concentration of K₂HPO₄ to the culture medium resulted in accumulation of fairly large amounts of carboxylic acids in addition to geranylacetone. These carboxylic acids were identified as isovaleric, β,β′-dimethylacrylic, geranic, and (+)-(R)-citronellic acids. Among these acids, α,β-saturated carboxylic acids were found to be predominant in quantity.     

Persistent High Numbers of Clostridium perfringens in the Intestines of Japanese Aged Adults

TSN agar was applicable for enumeration of Clostridium perfringens in fecal samples of adults but not in those of infants. It was demonstrated using TSN agar that some healthy aged adults had persistently carried C. perfringens at levels ranging from 10⁷ to 10⁹, while some others ranged from 10³ to 10⁶ per ml volume of fecal sample although all of these adults had the same diets. In the test for agglutinability of isolates of C. perfringens collected from two elderly adults, a younger adult and a baby, it was demonstrated that most of the isolates obtained from an aged adult of high levels for 19 months belonged the same serotype, while rapid alteration of serotypes could be observed in three other persons with high or low levels. In spite of as many as 10⁹ C. perfringens per ml of feces, no trace of α-toxin could be detected in the fecal samples. In in vitro tests, fecal suspension suppressed the production of α-toxin although it allowed the organism to grow sufficiently.     

Dystrophin in rod spherules; submembranous dense regions facing bipolar cell processes

 It is known that the retina contains the protein dystrophin in the ribbon synapse, but the ultrastructural analysis is not yet fully elucidated. Our previous study reported that dystrophin is localized under the rod cell membranes in rat retinas. In the present study, we have investigated the relationship between dystrophin-rich regions of rod cell membranes and other neuronal processes in mouse retinas with a monoclonal antibody raised against the human dystrophin C-terminus. Immunoblotting, immunofluorescence stainings, and immunoelectron microscopy were employed. Immunoblotting analysis indicated that mouse retinas possessed some of the dystrophin isoforms of approximately 260 kDa, 140 kDa, and 70 kDa molecular weight. Confocal images showed a punctate appearance in the outer plexiform layer, as previously described. Immunoelectron microscopy showed that dystrophin immunoreactive products were always observed at submembranous dense regions of the rod spherule abutting bipolar processes. These results suggest that retinal dystrophin may be closely involved in signal transmission from rods to bipolar cells. Accepted: 7 May 1997     

In situ measurement of cell stiffness of Arabidopsis roots growing on a glass micropillar support by atomic force microscopy

Atomic force microscopy (AFM) can measure the mechanical properties of plant tissue at the cellular level, but for in situ observations, the sample must be held in place on a rigid support and it is difficult to obtain accurate data for living plants without inhibiting their growth. To investigate the dynamics of root cell stiffness during seedling growth, we circumvented these problems by using an array of glass micropillars as a support to hold an Arabidopsis thaliana root for AFM measurements without inhibiting root growth. The root elongated in the gaps between the pillars and was supported by the pillars. The AFM cantilever could contact the root for repeated measurements over the course of root growth. The elasticity of the root epidermal cells was used as an index of the stiffness. By contrast, we were not able to reliably observe roots on a smooth glass substrate because it was difficult to retain contact between the root and the cantilever without the support of the pillars. Using adhesive to fix the root on the smooth glass plane overcame this issue, but prevented root growth. The glass micropillar support allowed reproducible measurement of the spatial and temporal changes in root cell elasticity, making it possible to perform detailed AFM observations of the dynamics of root cell stiffness.     

Increased Renal Iron Accumulation in Hypertensive Nephropathy of Salt-Loaded Hypertensive Rats

Although iron is reported to be associated with the pathogenesis of chronic kidney disease, it is unknown whether iron participates in the pathophysiology of nephrosclerosis. Here, we investigate whether iron is involved in the development of hypertensive nephropathy and the effects of iron restriction on nephrosclerosis in salt- loaded stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP were given either a normal or high-salt diet for 8 weeks. Another subset of SHRSP were fed a high-salt with iron-restricted diet. SHRSP given a high-salt diet developed severe hypertension and nephrosclerosis. As a result, survival rate was decreased after 8 weeks diet. Importantly, massive iron accumulation and increased iron content were observed in the kidneys of salt-loaded SHRSP, along with increased superoxide production, urinary 8-Hydroxy-2′-deoxyguanosine excretion, and urinary iron excretion; however, these changes were markedly attenuated by iron restriction. Of interest, expression of cellular iron transport proteins, transferrin receptor 1 and divalent metal transporter 1, was increased in the tubules of salt-loaded SHRSP. Notably, iron restriction attenuated the development of severe hypertension and nephrosclerosis, thereby improving survival rate in salt-loaded SHRSP. Taken together, these results suggest a novel mechanism by which iron plays a role in the development of hypertensive nephropathy and establish the effects of iron restriction on salt-induced nephrosclerosis.     

Common and Distinct Clinical Features in Adult Patients with Anti-Aminoacyl-tRNA Synthetase Antibodies: Heterogeneity within the Syndrome

Objective

To identify similarities and differences in the clinical features of adult Japanese patients with individual anti-aminoacyl-tRNA synthetase antibodies (anti-ARS Abs).

Methods

This was a retrospective analysis of 166 adult Japanese patients with anti-ARS Abs detected by immunoprecipitation assays. These patients had visited Kanazawa University Hospital or collaborating medical centers from 2003 to 2009.

Results

Anti-ARS Ab specificity included anti-Jo-1 (36%), anti-EJ (23%), anti-PL-7 (18%), anti-PL-12 (11%), anti-KS (8%), and anti-OJ (5%). These anti-ARS Abs were mutually exclusive, except for one serum Ab that had both anti-PL-7 and PL-12 reactivity. Myositis was closely associated with anti-Jo-1, anti-EJ, and anti-PL-7, while interstitial lung disease (ILD) was correlated with all 6 anti-ARS Abs. Dermatomyositis (DM)-specific skin manifestations (heliotrope rash and Gottron’s sign) were frequently observed in patients with anti-Jo-1, anti-EJ, anti-PL-7, and anti-PL-12. Therefore, most clinical diagnoses were polymyositis or DM for anti-Jo-1, anti-EJ, and anti-PL-7; clinically amyopathic DM or ILD for anti-PL-12; and ILD for anti-KS and anti-OJ. Patients with anti-Jo-1, anti-EJ, and anti-PL-7 developed myositis later if they had ILD alone at the time of disease onset, and most patients with anti-ARS Abs eventually developed ILD if they did not have ILD at disease onset.

Conclusion

Patients with anti-ARS Abs are relatively homogeneous. However, the distribution and timing of myositis, ILD, and rashes differ among patients with individual anti-ARS Abs. Thus, identification of individual anti-ARS Abs is beneficial to define this rather homogeneous subset and to predict clinical outcomes within the “anti-synthetase syndrome.”     

Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing

Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish.     

CD8+ T Cell Cross-Reactivity Profiles and HIV-1 Immune Escape towards an HLA-B35-Restricted Immunodominant Nef Epitope

Antigen cross-reactivity is an inbuilt feature of the T cell compartment. However, little is known about the flexibility of T cell recognition in the context of genetically variable pathogens such as HIV-1. In this study, we used a combinatorial library containing 24 billion octamer peptides to characterize the cross-reactivity profiles of CD8⁺ T cells specific for the immunodominant HIV-1 subtype B Nef epitope VY8 (VPLRPMTY) presented by HLA-B^*35∶01. In conjunction, we examined naturally occurring antigenic variations within the VY8 epitope. Sequence analysis of plasma viral RNA isolated from 336 HIV-1-infected individuals revealed variability at position (P) 3 and P8 of VY8; Phe at P8, but not Val at P3, was identified as an HLA-B^*35∶01-associated polymorphism. VY8-specific T cells generated from several different HIV-1-infected patients showed unique and clonotype-dependent cross-reactivity footprints. Nonetheless, all T cells recognized both the index Leu and mutant Val at P3 equally well. In contrast, competitive titration assays revealed that the Tyr to Phe substitution at P8 reduced T cell recognition by 50–130 fold despite intact peptide binding to HLA-B^*35∶01. These findings explain the preferential selection of Phe at the C-terminus of VY8 in HLA-B^*35∶01⁺ individuals and demonstrate that HIV-1 can exploit the limitations of T cell recognition in vivo.