DNA survival and physical and histological properties of heat-induced alterations in burnt bones.

During forensic casework, it is vital to be able to obtain valuable information from burnt bone fragments to ascertain the identity of the victim. Here, we report the findings of an experimental study on burnt bovine compact bone segments. Compact bones were cut to size and heated in an electric furnace at a temperature range of 100­1,100 °C with 100 °C increments. Heat-induced alterations to the bone color,weight, volume, and density were monitored using gross morphology and micro-focus X-ray computed tomography.We found that the increase in temperature caused the color of the compact bones to change in order of yellow, brown, gray,and white. In contrast to the weight reduction that occurred immediately after burning, we measured no significant reduction in volume even at 600 °C; however, volume reduced drastically once the temperature reached 700 °C. Light microscopic histological observations of burnt bone revealed heat induced alterations such as cracking and separation of the osteons at higher temperatures. In addition to these findings,we sought to examine the survival of DNA in the burnt bones using polymerase chain reaction of mitochondrial DNA. No amplification was found in the specimens burnt at 250 °C or higher, indicating the likely difficulty in testing the DNA of burnt bones from forensic casework. The results of this study will enable an estimation of the burning temperatures of burnt bones found in forensic cases and will provide an important framework with which to interpret data obtained during anthropological testing and DNA typing.

Increased phosphatidylcholine concentration in saliva reduces surface tension and improves airway patency in obstructive sleep apnoea.

Surface tension may have important role for maintaining upper airway patency in patients with obstructive sleep apnoea. It has been demonstrated that elevated surface tension increases the pharyngeal pressures required to reopen the upper airway following collapse. The aim of the study was to evaluate the associations between the concentrations of endogenous surfactants in saliva with indices of upper airway patency in obstructive sleep apnoea. We studied 20 male patients with obstructive sleep apnoea (age: 60·3 ± 10·3 years; BMI: 25·9 ± 4·6 kg m(-2); AHI: 41·5 ± 18·6 events h(-1)). We obtained 100-µL samples of saliva prior to overnight polysomnographic sleep study. The surface tension was determined using the pull-off force technique. The concentration of phosphatidylcholine (PC) was evaluated by liquid chromatography-mass spectrometry (LC-MS/MS). Regression analysis between apnoea, hypopnoea and apnoea/hypopnoea indices and the ratio of hypopnoea time/total disordered breathing time (HT/DBT) with surface tension and PC were performed. P < 0·05 was considered significant. The mean saliva surface tension was 48·8 ± 8·0 mN m(-1) and PC concentration was 15·7 ± 11·1 nM. The surface tension was negatively correlated with the PC concentration (r = -0·48, P = 0·03). There was a significant positive correlation between surface tension with hypopnoea index (r = 0·50, P = 0·03) and HT/DBT (r = 0·6, P = 0·006), but not apnoea or apnoea/hypopnoea index (P > 0·11). Similarly, PC concentration negatively correlated with hypopnoea index (r = -0·45, P = 0·04) and HT/DBT (r = -0·6, P = 0·004), but not with apnoea index or AHI (P > 0·08). An increase in salivary PC concentration may increase upper airway patency in obstructive sleep apnoea through a reduction in surface tension.

Presenilin-1 mutations downregulate the signalling pathway of the unfolded-protein response.

Missense mutations in the human presenilin-1 (PS1) gene, which is found on chromosome 14, cause early-onset familial Alzheimer's disease (FAD). FAD-linked PS1 variants alter proteolytic processing of the amyloid precursor protein and cause an increase in vulnerability to apoptosis induced by various cell stresses. However, the mechanisms responsible for these phenomena are not clear. Here we report that mutations in PS1 affect the unfolded-protein response (UPR), which responds to the increased amount of unfolded proteins that accumulate in the endoplasmic reticulum (ER) under conditions that cause ER stress. PS1 mutations also lead to decreased expression of GRP78/Bip, a molecular chaperone, present in the ER, that can enable protein folding. Interestingly, GRP78 levels are reduced in the brains of Alzheimer's disease patients. The downregulation of UPR signalling by PS1 mutations is caused by disturbed function of IRE1, which is the proximal sensor of conditions in the ER lumen. Overexpression of GRP78 in neuroblastoma cells bearing PS1 mutants almost completely restores resistance to ER stress to the level of cells expressing wild-type PS1. These results show that mutations in PS1 may increase vulnerability to ER stress by altering the UPR signalling pathway.

Effect of endurance training supplemented with green tea extract on substrate metabolism during exercise in humans.

Endurance training and ingestion of green tea extract (GTE), composed mainly of tea catechins (TC), are well known to enhance fat metabolism. However, their synergistic effects remain to be fully elucidated. We tested the hypothesis that endurance training supplemented with GTE would further accelerate whole-body fat utilization during exercise, compared with training alone, in humans. Twelve healthy male subjects [peak oxygen consumption (VO2peak), 50.7 ± 1.3 (SEM) mL/kg/min] were divided into two groups: GTE and placebo (PLA) groups. Subjects in both groups performed a cycle ergometer exercise at 60% of VO2peak for 60 min/day, 3 days/week, and daily ingested 572.8 or 0 mg TC in GTE and PLA groups for 10 weeks, respectively. Before and after training, respiratory gas exchange was measured during 90-min exercise at pre-training ∼55% of VO2peak. After training, the average respiratory exchange ratio during exercise remained unchanged in the PLA group (post-training: 0.834 ± 0.008 vs pre-training: 0.841 ± 0.004), whereas it was lower in the GTE group (post-training: 0.816 ± 0.006 vs pre-training: 0.844 ± 0.005, P<0.05). These results suggest that habitual GTE ingestion, in combination with moderate-intense exercise, was beneficial to increase the proportion of whole-body fat utilization during exercise.

A molecular chaperone inducer protects neurons from ER stress.

The endoplasmic reticulum (ER) stress response is a defense system for dealing with the accumulation of unfolded proteins in the ER lumen. Recent reports have shown that ER stress is involved in the pathology of some neurodegenerative diseases and cerebral ischemia. In a screen for compounds that induce the ER-mediated chaperone BiP (immunoglobulin heavy-chain binding protein)/GRP78 (78 kDa glucose-regulated protein), we identified BiP inducer X (BIX). BIX preferentially induced BiP with slight inductions of GRP94 (94 kDa glucose-regulated protein), calreticulin, and C/EBP homologous protein. The induction of BiP mRNA by BIX was mediated by activation of ER stress response elements upstream of the BiP gene, through the ATF6 (activating transcription factor 6) pathway. Pretreatment of neuroblastoma cells with BIX reduced cell death induced by ER stress. Intracerebroventricular pretreatment with BIX reduced the area of infarction due to focal cerebral ischemia in mice. In the penumbra of BIX-treated mice, ER stress-induced apoptosis was suppressed, leading to a reduction in the number of apoptotic cells. Considering these results together, it appears that BIX induces BiP to prevent neuronal death by ER stress, suggesting that it may be a potential therapeutic agent for cerebral diseases caused by ER stress.

Involvement of endoplasmic reticulum stress in the neuronal death induced by transient forebrain ischemia in gerbil.

Endoplasmic reticulum (ER) stress, which is caused by an accumulation of unfolded proteins in the ER lumen, is associated with stroke and with neurodegenerative diseases such as Parkinson's and Alzheimer diseases. We assessed the expression patterns of immunoglobulin heavy chain binding protein (BiP)/glucose-regulated protein (GRP) 78 (an ER-resident molecular chaperone whose expression serves as a good marker of ER-stress), activating transcription factor (ATF)-4, and C/EBP homology protein (CHOP) by immunohistochemistry and/or Western blotting after transient forebrain ischemia in gerbils. Double-fluorescent staining involving CHOP immunohistochemistry and the terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) method was performed to clarify the involvement of CHOP in cell death. Immunohistochemical and Western blot analyses of the hippocampal Cornet d'Ammon (CA)1 subfield showed that BiP expression was increased at 12 h, peaked at 3 days, then decreased (versus the control group). A transient increase was detected in CA3 at 1 day after ischemia, but BiP expression was unchanged in dentate gyrus and cortex. Signals for ATF-4 and CHOP were increased at 1 day and 3 days in CA1, and at 12 h in CA3. Co-localization of CHOP immunoreactivity and DNA fragmentation was detected by the TUNEL method at 3 days after ischemia in CA1, but not at 12 h in CA3. These findings are consistent with ER stress playing a pivotal role in post-ischemic neuronal death in the gerbil hippocampal CA1 subfield.

Induction of BiP, an ER-resident protein, prevents the neuronal death induced by transient forebrain ischemia in gerbil.

Endoplasmic reticulum (ER) stress, which is caused by the accumulation of unfolded proteins in the ER lumen, is associated with stroke and neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. We evaluated the effect of a selective inducer of immunoglobulin heavy chain binding protein (BiP) (BiP inducer X; BIX) against both tunicamycin-induced cell death (in SH-SY5Y cells) and the effects of global transient forebrain ischemia (in gerbils). BIX significantly induced BiP expression both in vitro and in vivo. Pretreatment with BIX at 2 or 5 microM reduced the cell death induced by tunicamycin in SH-SY5Y cells. In gerbils subjected to forebrain ischemia, prior treatment with BIX (intracerebroventricular injection at 10 or 40 microg) protected against cell death and decreased TUNEL-positive cells in the hippocampal CA1 subfield. These findings indicate that this selective inducer of BiP could be used to prevent the neuronal damage both in vitro and in vivo.

Two cis-acting elements in the 3' untranslated region of alpha-CaMKII regulate its dendritic targeting.

Dendritic localization of the alpha subunit of Ca2+/calmodulin-dependent protein kinase II (alphaCaMKII) mRNA in CNS neurons requires its 3' untranslated region (3'UTR). We investigated this targeting mechanism by identifying two cis-acting elements in the 3'UTR. One is a 30-nucleotide element that mediated dendritic translocation. A homologous sequence in the 3'UTR of neurogranin, transcripts of which also reside in dendrites, also funtioned in cis to promote its dendritic transport. Other putative elements in the alphaCaMKII mRNA inhibit its transport in a resting state. This inhibition was removed in depolarized neurons, and such activity-dependent derepression was a primary requirement for their dendritic targeting.

Endoplasmic reticulum stress response in dendrites of cultured primary neurons.

The endoplasmic reticulum (ER) is an organelle in which secretory and transmembrane proteins are folded or processed, and is susceptible to various stresses that provoke the accumulation of unfolded proteins in the ER lumen. Recently, ER stress has been reported to be linked to neuronal death in various neurodegenerative diseases. Neurons contain the ER not only in the soma, but also in the dendrites, thus presenting a different case to non-neuronal cells. The ER in the dendrites has potential functions in local protein synthesis and sorting of synthesized proteins to postsynaptic membranes. It raises the possibility that ER stress could occur locally in the dendrites. Here we showed that ER stress sensors, inositol-requiring 1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) exist in the ER of both soma and dendrites in primary mouse neurons, and that under ER stress conditions, GRP78/BiP and phosphorylated eIF2alpha are induced. Furthermore, XBP1 mRNA was localized in the proximal dendrites where IRE1 was rapidly phosphorylated in response to ER stress. These results indicate that the ER in dendrites could respond to ER stress and retain the capacity of protein quality control.

Involvement of endoplasmic reticulum stress after middle cerebral artery occlusion in mice.

The endoplasmic reticulum (ER) plays an important role in ischemic neuronal cell death. ER stress-related markers [immunoglobulin binding protein (BiP)/glucose-regulated protein (GRP) 78, activating transcription factor-4 (ATF-4), and C/EBP-homologous protein (CHOP)] in the striatum and the cortex were investigated after permanent middle cerebral artery occlusion (MCAO) in mice. Using endoplasmic reticulum stress-activated indicator (ERAI) transgenic mice, which show splicing of X-box protein 1 (XBP-1) mRNA as green fluorescence, we monitored the regional changes in fluorescence after MCAO. BiP mRNA (by reverse-transcription polymerase chain reaction [RT-PCR] analysis) was increased in the cortex at 6 h. In immunohistochemical and/or Western blot analysis, the expressions of ER stress-related markers (BiP, ATF-4, and CHOP) were increased in the infarct region, more strongly in the cortex than in the striatum. ERAI fluorescence was observed in the ischemic area starting from 6 and 12 h, respectively, after MCAO, with the peaks at 1 day and the fluorescence co-localized with the 2,3,5-triphenyltetrazolium chloride (TTC)-visible extension of brain infarction. These findings suggest that permanent MCAO induces expression of ER-stress related genes mainly in the periphery of the MCA territory.

Branched-chain amino acids supplementation attenuates the accumulation of blood lactate dehydrogenase during distance running.

AIM: We investigated the effect of branched-chain amino acids (BCAA) supplementation on tissue damage during distance running. METHODS: Eight male distance runners (mean +/- standard deviation; age: 20.4+/-1.2 years, body weight: 58.4+/-4.2 kg) participated in a double blinded cross over designed study conducted during training camp. During each intervention period, the subjects were asked to participate in a 25-km run, and the blood BCAA and lactate dehydrogenase (LDH) level, an index of tissue damage, were measured pre- and post-run. Either a drink containing BCAA (0.4% BCAA in a 4% carbohydrate solution) or an iso-calorie placebo drink was provided to the subjects 5 times during the run without any restriction in the volume. RESULTS: The total volume of the drink consumed by the subjects did not differ substantially between the trials: 591+/-188 (2.36 g BCAA) vs 516+/-169 mL in BCAA and placebo trial, respectively. During the run, the blood BCAA concentration was maintained in the BCAA trial. However, the blood BCAA concentration level tended to decrease in the placebo trial (P<0.1). The extent of the blood LDH increase in the BCAA trial was significantly less than that of the placebo trail (48% vs 58%, P<0.05). CONCLUSION: Maintaining the blood BCAA level throughout a long distance run contributes to a reduction in the LDH release and, therefore, the effect of BCAA supplementation is suggested to reduce the degree of muscle damage.

MEPROCS framework for Craniofacial Superimposition: Validation study.

Craniofacial Superimposition (CFS) involves the process of overlaying a skull with a number of ante-mortem images of an individual and the analysis of their morphological correspondence. The lack of unified working protocols and the absence of commonly accepted standards, led to contradictory consensus regarding its reliability. One of the more important aims of 'New Methodologies and Protocols of Forensic Identification by Craniofacial Superimposition (MEPROCS)' project was to propose a common framework for CFS, what can be considered the first international standard in the field. The framework aimed to serve as a roadmap for avoiding particular assumptions that could bias the process. At the same time, it provides some empirical support to certain practices, technological means, and morphological criteria expected to facilitate the application of the CFS task and to improve its reliability. In order to confirm the utility and potential benefits of the framework use, there is a need to empirically evaluate it in CFS identification scenarios as close as possible to the reality. Thus, the purpose of this study is to validate the CFS framework developed. For that aim 12 participants were asked to report about a variable number of CFS following all the recommendations of the framework. The results are analysed and discussed according to the framework understanding and fulfilment, the participants' performance, and the correlation between expected decisions and those given by the participants. In view of the quantitative results and qualitative examination criteria we can conclude that those who follow the MEPROCS recommendations improve their performance.

Interchangeable binding of Bcl10 to TRAF2 and cIAPs regulates apoptosis signaling.

Bcl10 was identified as a candidate gene responsible for low grade B cell lymphomas of mucosa-associated lymphoid tissue. Overexpression of Bcl10 in cultured cells was reported to promote apoptosis, however, the mechanism of regulation of apoptosis mediated by Bcl10 has not been demonstrated. In the present study, we analysed the apoptosis signaling pathway mediated by Bcl10, focusing on phosphorylation of Bcl10 and the dynamic interaction with its binding partners during apoptosis. Previously, we have demonstrated that Bcl10 potentially interacts with the other apoptosis regulator, TNF receptor associated factor-2 (TRAF2) and inhibitor of apoptosis proteins (cIAPs). The present results showed that the complex formation of these molecules was regulated by phosphorylation of Bcl10, that is, phosphorylation of Bcl10 resulted in binding of Bcl10 to cIAPs and the dissociation of it from TRAF2. Moreover, hyperphosphorylation of Bcl10 enhanced apoptosis, suggesting that changes in the binding partners of Bcl10 were correlated to the promotion of apoptosis as mediated by Bcl10. Indeed, the mutant which was deleted from the binding site of Bcl10 for cIAPs, could not induce apoptosis. These findings indicate that Bcl10 is a mediator of apoptosis signaling, by switching over binding to cIAPs from TRAF2 through the events of Bcl10 phosphorylation.

Molecular cloning of a novel membrane glycoprotein, pal, specifically expressed in photoreceptor cells of the retina and containing leucine-rich repeat.

We have isolated a novel retina-specific gene in a screen for genes of which expression is not apparent neonatally in rat retina but is abundant postnatally on day 14 (P14). This gene, named Pal, encodes a putative type I transmembrane protein containing five leucine-rich repeats (LRRs), a single C2-type Ig-like domain, and a single fibronectin type III domain and is considered to be a new member of the LRR and Ig superfamily. No expression of Pal was found in rat retina at P1, but it was detected at P7 and markedly increased with subsequent development. These expression patterns of Pal appeared to be correlated with the development of the photoreceptor outer segments, because in the adult rat retina it was specifically localized in these segments. Ultrastructually, Pal immunoreactivity was distributed diffusely on the disk membrane in the lamellar regions. On the basis of its structural features and localization pattern, Pal may act as a receptor for a certain trophic factor or for an adhesion molecule participating in morphogenesis. The human homolog of Pal was mapped to chromosome 10q23.2-23.3 using fluorescence in situ hybridization.

Absorption and transport of base moieties of phosphatidylcholine and phosphatidylethanolamine in rats.

The absorption and transport of the base moieties of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) which were fed to rats were compared. The major absorption site of ethanolamine-labeled PE was proximal jejunum while choline-labeled PC was absorbed almost equally throughout the jejunum. Lysophospholipids, glycerophosphoryl bases and constituent bases were the main digested products in intestinal content. This shows that base-labeled phospholipids were hydrolyzed to water-soluble products as well as lysophospholipids before absorption. The radioactivities from both phospholipids existed mainly in their parent phospholipids and water-soluble products in the intestinal mucosa. The amounts of lymphatic transport of the radioactivities from choline-labeled PC and ethanolamine-labeled PE were 17% and 8%, respectively, at 8 h after administration. The liver in lymph-cannulated rats contained 23% and 48% radioactivity from PC and PE, respectively, suggesting that base moieties of phospholipids, especially PE, were transported mainly via a non-lymphatic route, probably the portal vein, to the liver, as water-soluble products. The radioactivity from both base-labeled phospholipids in the liver was distributed in the parent phospholipids and water-soluble fractions. Ethanolamine-labeled PE was also incorporated into PC in the liver. These results indicate that intestinal absorption and transport of the base moiety of dietary PC and PE are similar; however, their intestinal absorption site and the extent of their separation during transport between the lymphatic and portal systems differ markedly.

Mechanism of autocatalytic oxidation of oxyhemoglobin by nitrite. An intermediate detected by electron spin resonance.

Oxidation of oxyhemoglobin by nitrite is characterized by the presence of a lag phase followed by the autocatalysis. Just before the autocatalysis begins, an asymmetric ESR signal is detected which is similar to that of the methemoglobin radical generated from methemoglobin and H2O2 in shape, g value (2.005), peak-to-peak width (18 G) and other properties, except the difference in the dependence on temperature. Generation of H2O2 is indicated by the prolongation of the lag phase by the addition of catalase. On the other hand, the oxidation is modified by neither superoxide dismutase nor Nitroblue tetrazolium. The oxidation is prolonged in the presence of KCN. The present results indicate a free-radical mechanism for the oxidation in which the asymmetric radical catalyzes the formation of NO2 from NO2- by a peroxidase action and NO2 oxidizes oxyhemoglobin in the autocatalytic phase.

Radioimmunoassay of arginine-rich apolipoprotein of rat serum.

A double-antibody radioimmunoassay was developed for quantification of rat arginine-rich apolipoprotein in sodium decyl sulfate. Arginine-rich protein, labeled with 125I by the chloramine-T method, had the same chromatographic characteristics on Sephadex G-200 as unlabeled arginine-rich protein and up to 70% of 125I-labeled arginine-rich protein was precipitated by antisera to arginine-rich protein in rabbits. The assay is sensitive at the level of 1-10 ng and has intraassay and interassay coefficients of variation of 5.4 and 6.8%, respectively. The specificity of the assay was established by competitive displacement of 125I-labeled arginine-rich protein from its antiserum by arginine-rich protein and lipoproteins containing this protein, but not by rat albumin or other purified apolipoproteins. Immunoreactivity of rat serum and lipoproteins was complete as demonstrated by comparison with their delipidated form. The accuracy of the immunoassay was further substantiated by comparison with the amount of arginine-rich protein in chromatographic fractions of total apoprotein of very low and high density lipoproteins, and by recovery experiments in ultracentrifugally separated fractions of serum. In contrast to an immunoassay reported previously for rat apo A-I, sodium decyl sulfate was not required for complete immunoreactivity of serum and lipoproteins. The inclusion of sodium decyl sulfate (9 mM final concentration) was necessary, however, for stability of labeled and unlabeled preparations of arginine-rich protein. Content (weight %, means values +/- S.D.), of immunoassayable arginine-rich protein in isolated lipoproteins was 15 +/- 1.5% in very density lipoproteins; 6.8% in low density lipoproteins (1.02 less than d less than 1.04 g/m); 7.1 +/- 0.3% in high density lipoproteins; and 4.8 +/- 0.5% in lymph chylomicrons. Concentration in whole serum was 18.1 +/- 1.4 and 20.4 +/- 2.3 mg/dl for male and female rats, respectively. Only about 55% of arginine-rich protein was recovered in the major lipoprotein classes and about 40% was in "lipoprotein-free" serum (d greater than 1.25 g/ml). Among the lipoproteins, the high density lipoprotein fraction contained twice the amount of arginine-rich protein recovered in very low or low density lipoproteins (26.6 vs. 13.5 and 13.4%, respectively). The significance of the large amount of arginine-rich protein in the 1.25 g/ml infranatant fraction is not apparent. Although repetitive centrifugation did not alter the amount recovered in this fraction, the possibility of an artifact induced by centrifugation and high salt concentration cannot be excluded.