Humanin improves impaired metabolic activity and prolongs survival of serum-deprived human lymphocytes

Humanin (HN) has been reported to be an endogenous peptide that exerts highly selective neuroprotection against cell death induced by various types of Alzheimer's disease-related insults. We previously proposed the much broader cytoprotective potential of HN from the result that HN suppressed serum-deprivation-induced death of rat pheochromocytoma cells. In this study, we showed that HN also suppressed death of human lymphocytes cultured under serum-deprived condition. Further, we revealed, by assaying metabolic activity and survival rate, that HN was a potent factor capable of increasing the metabolic activity of individual serum-deprived lymphocytes. To our knowledge, there is no report described about a rescue factor that increases the metabolic activity of individual serum-deprived cells and prolongs their survival. This novel feature of HN may enable us to apply this peptide for the management of diseases involving poor metabolic activity, such as mitochondria-related disorders and brain ischemia.     

Association of yeast adenylyl cyclase with cyclase-associated protein CAP forms a second Ras-binding site which mediates its Ras-dependent activation

Posttranslational modification, in particular farnesylation, of Ras is crucial for activation of Saccharomyces cerevisiae adenylyl cyclase (CYR1). Based on the previous observation that association of CYR1 with cyclase-associated protein (CAP) is essential for its activation by posttranslationally modified Ras, we postulated that the associated CAP might contribute to the formation of a Ras-binding site of CYR1, which mediates CYR1 activation, other than the primary Ras-binding site, the leucine-rich repeat domain. Here, we observed a posttranslational modification-dependent association of Ras with a complex between CAP and CYR1 C-terminal region. When CAP mutants defective in Ras signaling but retaining the CYR1-binding activity were isolated by screening of a pool of randomly mutagenized CAP, CYR1 complexed with two of the obtained three mutants failed to be activated efficiently by modified Ras and exhibited a severely impaired ability to bind Ras, providing a genetic evidence for the importance of the physical association with Ras at the second Ras-binding site. On the other hand, CYR1, complexed with the other CAP mutant, failed to be activated by Ras but exhibited a greatly enhanced binding to Ras. Conversely, a Ras mutant E31K, which exhibits a greatly enhanced binding to the CYR1-CAP complex, failed to activate CYR1 efficiently. Thus, the strength of interaction at the second Ras-binding site appears to be a critical determinant of CYR1 regulation by Ras: too-weak and too-strong interactions are both detrimental to CYR1 activation. These results, taken together with those obtained with mammalian Raf, suggest the importance of the second Ras-binding site in effector regulation.     

Role of Raf-1 conserved region 2 in regulation of Ras-dependent Raf-1 activation

Full activation of Raf-1 requires the interaction of its CRD with Ras. The serine/threonine-rich region, CR2, of Raf-1 was implicated in Raf-1 regulation, but the underlying mechanism was unclear. Here we show that CRD loses its Ras-binding activity when expressed in connection with CR2, suggesting that CR2 masks CRD. This masking effect is abolished by substitution of Asp or Ala for Ser-259, a growth factor- and TPA-induced phosphorylation site in CR2. Treatment of COS-7 cells expressing Ha-Ras(Val-12) and Raf-1 with TPA enhances the Ha-Ras(Val-12)-dependent Raf-1 kinase activity. In contrast, the Ha-Ras(Val-12)-dependent activities of the Raf-1(S259D) and Raf-1(S259A) mutants are comparable to that of wild-type Raf-1 stimulated by both Ha-Ras(Val-12) and TPA and cannot be further stimulated by TPA treatment. These results suggest that the in vivo phosphorylation of Ser-259 may comprise a crucial step for Ras-dependent Raf-1 activation by unmasking CRD and promoting its association with Ras.     

Tension Force‐Induced ATP Promotes Osteogenesis Through P2X7 Receptor in Osteoblasts

Orthodontic tooth movement induces alveolar bone resorption and formation by mechanical stimuli. Force exerted on the traction side promotes bone formation. Adenosine triphosphate (ATP) is one of the key mediators that respond to bone cells by mechanical stimuli. However, the effect of tension force (TF)‐induced ATP on osteogenesis is inadequately understood. Accordingly, we investigated the effect of TF on ATP production and osteogenesis in MC3T3‐E1 cells. Cells were incubated in the presence or absence of P2X7 receptor antagonist A438079, and then stimulated with or without cyclic TF (6% or 18%) for a maximum of 24 h using Flexercell Strain Unit 3000. TF significantly increased extracellular ATP release compared to control. Six percent TF had maximum effect on ATP release compared to 18% TF and control. Six percent TF induced the expression of Runx2 and Osterix. Six percent TF also increased the expression of extracellular matrix proteins (ECMPs), ALP activity, and the calcium content in ECM. A438079 blocked the stimulatory effect of 6% TF on the expression of Runx2, Osterix and ECMPs, ALP activity, and calcium content in ECM. This study indicated that TF‐induced extracellular ATP is released in osteoblasts, suggesting that TF‐induced ATP promotes osteogenesis by autocrine action through P2X7 receptor in osteoblasts. J. Cell. Biochem. 116: 12–21, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.     

Hippocampal Subfield Volumes in First Episode and Chronic Schizophrenia

BackgroundReduced hippocampal volume in schizophrenia is a well-replicated finding. New imaging techniques allow delineation of hippocampal subfield volumes. Studies including predominantly chronic patients demonstrate differences between subfields in sensitivity to illness, and in associations with clinical features. We carried out a cross-sectional and longitudinal study of first episode, sub-chronic, and chronic patients, using an imaging strategy that allows for the assessment of multiple hippocampal subfields.MethodsHippocampal subfield volumes were measured in 34 patients with schizophrenia (19 first episode, 6 sub-chronic, 9 chronic) and 15 healthy comparison participants. A subset of 10 first episode and 12 healthy participants were rescanned after six months.ResultsTotal left hippocampal volume was smaller in sub-chronic (p = 0.04, effect size 1.12) and chronic (p = 0.009, effect size 1.42) patients compared with healthy volunteers. The CA2-3 subfield volume of chronic patients was significantly decreased (p = 0.009, effect size 1.42) compared to healthy volunteers. The CA4-DG volume was significantly reduced in all three patient groups compared to healthy group (all p < 0.005). The two affected subfield volumes were inversely correlated with severity of negative symptoms (p < 0.05). There was a small, but statistically significant decline in left CA4-DG volume over the first six months of illness (p = 0.01).ConclusionsImaging strategies defining the subfields of the hippocampus may be informative in linking symptoms and structural abnormalities, and in understanding more about progression during the early phases of illness in schizophrenia.     

A role for CFTR in the elevation of glutathione levels in the lung by oral glutathione administration

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is the only known apical glutathione (GSH) transporter in the lung. The purpose of these studies was to determine whether oral GSH or glutathione disulfide (GSSG) treatment could increase lung epithelial lining fluid (ELF) GSH levels and whether CFTR plays a role in this process. The pharmacokinetic profile of an oral bolus dose of GSH (300 mg/kg) was determined in mice. Plasma, ELF, bronchoalveolar lavage (BAL) cells, and lung tissue were analyzed for GSH content. There was a rapid elevation in the GSH levels that peaked at 30 min in the plasma and 60 min in the lung, ELF, and BAL cells after oral GSH dosing. Oral GSH treatment produced a selective increase in the reduced and active form of GSH in all lung compartments examined. Oral GSSG treatment (300 mg/kg) resulted in a smaller increase of GSH levels. To evaluate the role of CFTR in this process, Cftr knockout (KO) mice and gut-corrected Cftr KO-transgenic (Tg) mice were given an oral bolus dose of GSH (300 mg/kg) and compared with wild-type mice for changes in GSH levels in plasma, lung, ELF, and BAL cells. There was a twofold increase in plasma, a twofold increase in lung, a fivefold increase in ELF, and a threefold increase in BAL cell GSH levels at 60 min in wild-type mice; however, GSH levels only increased by 40% in the plasma, 60% in the lung, 50% in the ELF, and twofold in the BAL cells within the gut-corrected Cftr KO-Tg mice. No change in GSH levels was observed in the uncorrected Cftr KO mice. These studies suggest that CFTR plays an important role in GSH uptake from the diet and transport processes in the lung.     

Comparison of age-related peripheral nerve changes in mice housed in either plastic cages with sawdust-covered solid flooring or wire-mesh-floor cages.

A comparative histologic survey was conducted on the dorsal root, sciatic, tibial and medial plantar nerves of 90- and 110-week-old B6C3F1 female mice reared in either solid-floor cages covered in sawdust or wire-mesh-floor cages. Age-related peripheral nerve lesions, characterized by axonal degeneration and remyelination, were present in all nerves surveyed, and were especially prominent in the sciatic and medial plantar nerves at 110 weeks of age but, there were no differences associated with the type of cage floor in clinical signs, grasping power of the fore- and hind-limbs, motor nerve conduction velocity or histopathologic findings at these ages.     

Occurrence of chondroitin sulfate E in glycosaminoglycan isolated from the body wall of sea cucumber Stichopus japonicus

Glycosaminoglycan was isolated from the body wall of sea cucumber Stichopus japonicus by a method consisting of enzymatic digestion, gel filtration, and ion-exchange chromatography. One gram of sea cucumber glycosaminoglycan was composed of 2.50 mmol of sulfate, 0.47 mmol of N-acetylgalactosamine (GalNAc), 0.53 mmol of glucuronic acid (GlcA), 1.73 mmol of fucose, and a small amount of peptide. When mildly hydrolyzed with 0.1 N H2SO4, this glycosaminoglycan released two products, one consisting of fucose plus sulfate and the other of fucose only. Partially hydrolyzed glycosaminoglycan thus obtained was composed of sulfate, GalNAc, GlcA, and fucose at a molar ratio of 3:2:2:1. Partially hydrolyzed glycosaminoglycan was easily digested with chondroitinase AC II. In ion-exchange chromatography, the digest exhibited four sharp peaks whose retention times agreed with those of unsaturated 0-(delta Di-0S), mono-(delta Di-4S and delta Di-6S), and di-(delta Di-SE) sulfated disaccharide, respectively. The disaccharide unit of sea cucumber glycosaminoglycan was composed of 22.4% chondroitin sulfate E, 11.2% chondroitin, 10.4% chondroitin 4-sulfate, and 56.0% chondroitin 6-sulfate.