Stability of the O-octanoyl group of rat ghrelin during chemical synthesis: Counter-ion-dependent alteration of an ester bond breakage

Rat ghrelin, a 28-amino acid residue peptide with an octanoyl group at the side chain of Ser3, was synthesized chemically by applying Fmoc/^tBu strategy. An ester linkage between octanoic acid and the hydroxyl function of Ser3 was found to be maintained without serious damage during the final deprotection with trifluoroacetic acid (TFA). The most notable finding was the counter-ion-dependent stability change of the octanoyl moiety in the molecule. After consolidation of the counter-ion to TFA (TFA form), the octanoyl group persisted stably upon dissolution in water, whereas in the case of the acetate-form peptide, both de-octanoylation and dehydration (formation of the dehydro-Ala residue) occurred in aqueous solution at the same Ser3 residue. The amounts of these degraded products varied with factors such as solvent, temperature and times of lyophilization. These experimental findings lay the basis for performing the bioassay of ghrelin, which has an octanoyl moiety involved in its numerous biological activities thus far revealed.     

Extracellular DJ-1 induces sterile inflammation in the ischemic brain

Inflammation is implicated in the onset and progression of various diseases, including cerebral pathologies. Here, we report that DJ-1, which plays a role within cells as an antioxidant protein, functions as a damage-associated molecular pattern (DAMP) and triggers inflammation if released from dead cells into the extracellular space. We first found that recombinant DJ-1 protein induces the production of various inflammatory cytokines in bone marrow–derived macrophages (BMMs) and dendritic cells (BMDCs). We further identified a unique peptide sequence in the αG and αH helices of DJ-1 that activates Toll-like receptor 2 (TLR2) and TLR4. In the ischemic brain, DJ-1 is released into the extracellular space from necrotic neurons within 24 h after stroke onset and makes direct contact with TLR2 and TLR4 in infiltrating myeloid cells. Although DJ-1 deficiency in a murine model of middle cerebral artery occlusion did not attenuate neuronal injury, the inflammatory cytokine expression in infiltrating immune cells was significantly decreased. Next, we found that the administration of an antibody to neutralize extracellular DJ-1 suppressed cerebral post-ischemic inflammation and attenuated ischemic neuronal damage. Our results demonstrate a previously unknown function of DJ-1 as a DAMP and suggest that extracellular DJ-1 could be a therapeutic target to prevent inflammation in tissue injuries and neurodegenerative diseases.

Intracellular expression of the antioxidant protein DJ-1 has previously been shown to be neuroprotective. This study reveals that extracellularly released DJ-1 from necrotic neurons is a trigger of sterile inflammation that promotes neuronal injury and neurological deficits after ischemic stroke.     

In vivo genotoxicity of ortho-phenylphenol, biphenyl, and thiabendazole detected in multiple mouse organs by the alkaline single cell gel electrophoresis assay

In Japan, ortho-phenylphenol (OPP), biphenyl (BP), and thiabendazole (2-(4'-thiazolyl)benzimidazole, TBZ) are commonly used as a postharvest treatment to preserve imported citrus fruits during transport and storage. We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of those agents in mouse stomach, liver, kidney, bladder, lung, brain, and bone marrow. CD-1 male mice were sacrificed 3, 8, and 24 h after oral administration of the test compounds. OPP (2000 mg/kg) induced DNA damage in the stomach, liver, kidney, bladder, and lung, BP (2000 mg/kg) and TBZ (200 mg/kg) induced DNA damage in all the organs studied. For OPP, increased DNA damage peaked at 3–8 h and tended to decrease at 24 h. For BP, on the contrary, increased DNA migration peaked at 24 h. That delay may have been due to the fact that OPP is metabolized by cytochrome 450 and prostaglandin H synthase to phenylbenzoquinone (PBQ), a DNA binding metabolite, and BP is metabolized to PBQ via OPP and m-phenylphenol. The positive response to TBZ, an aneugen, supports the in vivo DNA-damaging action of TBZ.     

Elderly People With Low Body Weight May Have Subtle Low‐grade Inflammation

Low‐grade inflammation, which plays important roles in the development of fatal diseases, is commonly observed in obese people. However, this has not been evaluated in lean people, who have relatively increased mortality risk compared with people of normal weight. Here, we elucidate the association between systemic low‐grade inflammation and low body weight, with particular emphasis on aging. We examined the relationship between circulating C‐reactive protein (CRP) and BMI in a cross‐sectional study of 2,675 apparently healthy adults who had undergone a medical check‐up. Overall, subjects with low BMI (<21.0 kg/m², n = 585) showed a favorable cardiovascular profile without being undernourished. In the elderly (≥55 years old), logarithmic CRP (LogCRP) showed a sigmoid curve against BMI with a base at BMI 21.0–22.9 kg/m², but not against waist circumference (WC), even in nonsmokers. In contrast, in middle‐aged people, LogCRP showed an almost linear relationship with both BMI and WC. LogCRP levels in elderly nonsmokers with low BMI, but not normal or high BMI, were significantly higher than those in middle‐aged with corresponding BMI (P < 0.05). After adjustment for age, sex, smoking status, and weight change over the past 2 years, the adjusted means of LogCRP still had a similar sigmoid curve against BMI in the elderly. These results suggest that elderly people with low body weight may have subtle low‐grade inflammation irrespective of a favorable cardiovascular risk, which remains to be confirmed in further studies.     

Changes of phorbol ester binding sites in rat brain following intracerebroventricular administration of thyrotropin-releasing hormone (TRH): An in vitro macroautoradiographic investigation

We examined the influence of the intracerebroventricular (icv) administration of thyrotropin-releasing hormone (THH) on protein kinase C (PKC) activities in various rat forebrain regions in order to cast light on the mechanism of extra-pituitary non-endocrine physiological actions of TRH in the central nervous system. An in vitro macroautoradiographic method, with [³H]phorbol 12, 13-dibutyrate (PDBu) as the radioactive ligand, was used to investigate quantitative alterations of PKC activities. The optical densities for PDBu binding sites in the striatum and hippocampal formation were significantly increased after the icv administration of TRH, while those in the frontal cortex and septum were unchanged. These findings suggest that TRH may exert some of its non-endocrine functions through striatal and hippocampal neurons which used PKC in their second messenger systems.     

Preparation and regeneration of protoplasts of three fungi of Boletaceae

Protoplasts of three fungi of Boletaceae,Suillus luteus, S. grevillei, andBoletinus cavipes, were prepared with yields of 45, 8.0, and 1.8×10⁷/g fresh mycelia under the optimal conditions, respectively. Nucleate protoplasts accounted for 42% of the whole preparation ofS. luteus and 32% of that ofS. grevillei, and 21% of the nucleate protoplasts ofS. luteus and 35% of those ofS. grevillei possesed two nuclei. Regeneration efficiency of protoplasts was 0.4% forS. luteus and 0.05% forS. grevillei. The regeneration ofB. cavipes protoplasts was also confirmed. Optimal conditions for regeneration were determined. Addition of gellan gum instead of agar to the medium and activated charcoal treatment of agar medium increased the regeneration efficiency significantly.     

Phenotypic Characterization and Molecular Mapping of an acaulis2 Mutant of Arabidopsis thaliana with Flower Stalks of Much Reduced Length

An acaulis2-1 (acl2-1) mutant of Arabidopsis thaliana (L.) Heynh.was isolated and characterized. The mutant had inflorescencesof much reduced length. The defect was severer in lower internodesand was visible preferentially in type 2 metamers. The defectin the elongation of internodal cells of inflorescences wasattributable to a defect in the mechanism for elongation ofthese cells. The cortical microtubules (MTs) were analyzed inthe acl2 mutant, in a comparison with those of an acl1 mutantwhich was described earlier, but no changes were detected inamounts of MTs. In the type 2 and type 3 metamers, the defectin elongation in acl mutants was loosely correlated with a changein the orientation of MTs. Thus, the abnormal arrangement ofMTs appears to be one of the pleiotropic effects of acaulismutations. Genetic mapping was performed using molecular markers and theACL2 gene was located at 100 map units on chromosome 1. ¹Present address: Institute of Molecular and Cellular Biosciences,The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, 113 Japan ²Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan