Reaction time and replenishment time of SP and CGRP after incision in rat skin

Background. The skin neurogenic inflammation is mainly related to Substance P (SP) and Calcitonin Gene-related Peptide (CGRP). There is no data on their availability in the dynamics of skin nerve endings, concerning their release and replenishment after a nociceptive stimulus, so this was investigated. Materials and methods. 25 rats were randomly distributed in 5 groups. The animals of the control group (CG) determined the baseline levels of neuropeptides in the skin. The groups S0 and S30 did not receive any cutaneous stimulus at 30 and 60 minutes, respectively. In the group S1, an “incision stimulus” was made at 30 minutes. In the group S31, a nociceptive stimulus was performed by subdermal scratching at 30 minutes and, at 60 minutes, the “incision stimulus” was carried out in the same location (“nociceptive hyperstimulation”). The skin samples of the other animals were harvested from the back 1 minute after their death. SP, pro-CGRP and CGRP were quantified by Western Blotting. Results. The “incision stimulus” released SP, S1 compared to S0 (p <0.05) detected in the first minute, and the replenishment time was more than 30 minutes. Also, it cleaved pro-CGRP, S1 compared to S31 (p <0.05) in the first minute, and its replenishment time less than 30 minutes. Release of CGRP was not detected. Conclusion. The incision released SP already detected in the first minute; its replenishment time is more than 30 minutes. The incision decreased pro-CGRP, also detected in the first minute; and its replenishment time is less than 30 minutes.     

Identification of the Microbiota in Carious Dentin Lesions Using 16S rRNA Gene Sequencing

While mutans streptococci have long been assumed to be the specific pathogen responsible for human dental caries, the concept of a complex dental caries-associated microbiota has received significant attention in recent years. Molecular analyses revealed the complexity of the microbiota with the predominance of Lactobacillus and Prevotella in carious dentine lesions. However, characterization of the dentin caries-associated microbiota has not been extensively explored in different ethnicities and races. In the present study, the bacterial communities in the carious dentin of Japanese subjects were analyzed comprehensively with molecular approaches using the16S rRNA gene. Carious dentin lesion samples were collected from 32 subjects aged 4–76 years, and the 16S rRNA genes, amplified from the extracted DNA with universal primers, were sequenced with a pyrosequencer. The bacterial composition was classified into clusters I, II, and III according to the relative abundance (high, middle, low) of Lactobacillus. The bacterial composition in cluster II was composed of relatively high proportions of Olsenella and Propionibacterium or subdominated by heterogeneous genera. The bacterial communities in cluster III were characterized by the predominance of Atopobium, Prevotella, or Propionibacterium with Streptococcus or Actinomyces. Some samples in clusters II and III, mainly related to Atopobium and Propionibacterium, were novel combinations of microbiota in carious dentin lesions and may be characteristic of the Japanese population. Clone library analysis revealed that Atopobium sp. HOT-416 and P. acidifaciens were specific species associated with dentinal caries among these genera in a Japanese population. We summarized the bacterial composition of dentinal carious lesions in a Japanese population using next-generation sequencing and found typical Japanese types with Atopobium or Propionibacterium predominating.     

Structure-based de novo design of non-nucleoside adenosine deaminase inhibitors

We searched for non-nucleoside inhibitors of adenosine deaminase by rational structure-based de novo design and succeeded in the discovery of 1-(1-hydroxy-4-phenyl-2-butyl)imidazole-4-carboxamide (FR221647: K(i)=5.9 microM to human ADA) as a novel inhibitor with moderate activity and good pharmacokinetics compared with the known inhibitors pentostatin and EHNA.     

Molecular cloning of the prothoracicotropic hormone from the tobacco hornworm,Manduca sexta

A cDNA encoding a putative precursor of prothoracicotropic hormone (PTTH) from the tobacco hornworm, Manduca sexta, was isolated and sequenced. This clone contains an open reading frame encoding a 226-amino acid prepropeptide hormone. The deduced amino acid sequence is composed of a signal sequence, a precursor domain and a mature hormone and shows similarities to the other PTTHs that have been cloned from closely related lepidopteran species, Bombyx mori, Samia cynthia ricini, Antheraea peryni, and Hyalophora cecropia. Although these cDNAs showed slightly less similarities in predicted amino acid sequences, seven cysteine residues and the hydrophobic regions within those mature peptides were conserved. In situ hybridization using a cDNA probe encoding the Manduca PTTH showed that PTTH mRNA was in two pairs of neurosecretory cells in the Manduca brain. The recombinant putative Manduca PTTH produced in E. coli was biologically active, both causing a larval molt in neck-ligated Manduca 4th instar larvae (ED(50)=50 pM) and the adult molt of diapausing Manduca pupae (ED(50)=79 pM), but was unable to stimulate molting of debrained Bombyx pupae.     

Changes in fused cells induced by hvj (sendai virus): redistribution of cytoplasmic organelles and cytoskeletal reorganization

To understand the relationship between the location of organelles and cellular function, we examined the dynamic state of cytoplasmic organelles and cytoskeleton in polynuclear Ehrlich ascites tumor (EAT) cells fused with hemagglutinating virus of Japan (HVJ; Sendai virus) by confocal laser scanning microscopy. Irregular fused cells gradually became spherical during culture, and nuclei and mitochondria were redistributed in the fused cell; nuclei formed a cluster surrounded by mitochondria. F-actin, vimentin, and microtubules were also reorganized with the redistribution of cell organelles. Further, when the morphological change was inhibited by L4-1, a chlorophyll-like substance derived from silkworm faeces, or pyropheophorbide-a, the arrangement of organelles and cytoskeleton remained disturbed, suggesting that the movement of the cytoskeleton is closely associated with cell shape and the distribution of cytoplasmic organelles.     

Improved capacity of a monkey-tropic HIV-1 derivative to replicate in cynomolgus monkeys with minimal modifications

Human immunodeficiency virus type 1 (HIV-1) hardly replicates in Old World monkeys. Recently, a mutant HIV-1 clone, NL-DT5R, in which a small part of gag and the entire vif gene are replaced with SIVmac239-derived ones, was shown to be able to replicate in pigtail monkeys but not in rhesus monkeys (RM). In the present study, we found that a modified monkey-tropic HIV-1 (HIV-1mt), MN4-5S, acquired the ability to replicate efficiently in cynomolgus monkeys as compared with the NL-DT5R, while neither NL-DT5R nor MN4-5S replicated in RM cells. These results suggest that multiple determinants may be involved in the restriction of HIV-1 replication in macaques, depending on the species of macaques. The new HIV-1mt clone will be useful for studying molecular mechanisms by which anti-viral host factors regulate HIV-1 replication in macaques.     

Identification of three new autoantibodies associated with systemic lupus erythematosus using two proteomic approaches

Our objective was to identify new serum autoantibodies associated with systemic lupus erythematosus (SLE), focusing on those found in patients with central nervous system (CNS) syndromes. Autoantigens in human brain proteins were screened by multiple proteomic analyses: two-dimensional polyacrylamide gel electrophoresis/Western blots followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis and immunoprecipitation followed by liquid chromatography-tandem mass spectrometry shotgun analysis. The presence of serum IgG autoantibodies against 11 selected recombinant antigens was assessed by Western blot and enzyme-linked immunosorbent assay (ELISA) in the sera of 106 SLE patients and 100 normal healthy controls. The O.D. values in sera from SLE patients were significantly higher than those of controls for the antigens crystallin αB (p = 0.0002), esterase D (p = 0.0002), APEX nuclease 1 (p < 0.0001), ribosomal protein P0 (p < 0.0001), and PA28γ (p = 0.0005); the first three are newly reported. The anti-esterase D antibody levels were significantly higher in the CNS group than in the non-CNS group (p = 0.016). Moreover, when the SLE patients were categorized using CNS manifestations indicating neurologic or psychiatric disorders, the anti-APEX nuclease 1 antibody levels were significantly elevated in SLE patients with psychiatric disorders (p = 0.037). In conclusion, the association of SLE with several new and previously reported autoantibodies has been demonstrated. Statistically significant associations between anti-esterase D antibodies and CNS syndromes as well as between anti-APEX nuclease 1 antibodies and psychiatric disorders in SLE were also demonstrated. The combined immunoproteomic approaches used in this study are reliable and effective methods for identifying SLE autoantigens.     

The epistatic relationship between BRCA2 and the other RAD51 mediators in homologous recombination

RAD51 recombinase polymerizes at the site of double-strand breaks (DSBs) where it performs DSB repair. The loss of RAD51 causes extensive chromosomal breaks, leading to apoptosis. The polymerization of RAD51 is regulated by a number of RAD51 mediators, such as BRCA1, BRCA2, RAD52, SFR1, SWS1, and the five RAD51 paralogs, including XRCC3. We here show that brca2-null mutant cells were able to proliferate, indicating that RAD51 can perform DSB repair in the absence of BRCA2. We disrupted the BRCA1, RAD52, SFR1, SWS1, and XRCC3 genes in the brca2-null cells. All the resulting double-mutant cells displayed a phenotype that was very similar to that of the brca2-null cells. We suggest that BRCA2 might thus serve as a platform to recruit various RAD51 mediators at the appropriate position at the DNA-damage site.     

The combination of genetic variations in the PRDX3 gene and dietary fat intake contribute to obesity risk

Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and the antioxidant capacity of the cell. This imbalance and an excess of ROS induce tissue/cellular damage, which are implicated in chronic inflammation disorders such as obesity, insulin resistance, and metabolic syndromes. Peroxiredoxins (Prxs) are the most abundant and ancient cellular antioxidant proteins that help to control intracellular peroxide levels and ROS-dependent signaling. Of the six mammalian isoforms, Prx III is specifically localized in mitochondria. In this study, we detected novel associations between genetic variations of the PRDX3 gene and BMI and obesity risk in the general Japanese population. In addition, these associations were observed only in the subjects with high dietary fat intake, but not in the subjects with low dietary fat intake. These findings indicate that the interaction between genetic variations in the PRDX3 gene and dietary fat intake is important for modulation of BMI and obesity risk.