Ear identification: A multi-ethnic study sample.

The external human ear is considered to be highly variable among individuals. Hence, forensic applications could be explored for human identification. This research compares the usefulness of Cameriere's ear identification method, in samples originating from six different countries (Brazil, India, Japan, Russia, South Africa and Turkey) in order to examine possible differences in their accuracy values. A sample of 2,225 photographs of the external human ear (1,134 left and 1,091 right ears) from 1,411 individuals (633 females and 778 males) was collected. The samples included healthy subjects with no systemic disorders and without any craniofacial trauma, maxillofacial abnormalities, auricular anomalies, ear diseases or previous auricular surgery. Cameriere's ear identification method was applied and measurements were performed on the images of each ear, considering four anatomic regions: helix, antihelix, concha, and lobe. The quantified measurement values were converted into a proposed coded number system. A search for identical codes was accomplished to find out the distinctiveness of the morphology of the human ear. The combined codes of left and right ears of each of the 814 subjects were not repeated in this multi-ethnic study sample. Dirichlet's distribution and the inherent study equation showed that the probability of two different individuals having the same code (false-positive identification) was found to be <0.0007. Because of the distinctive metrics of the ratios of external human ears, studies with Cameriere's ear identification method may be valuable for human identification. Studying the differences between the left and right ears of the same individual and across different ethnic groups could contribute to the development of supplementary tools for human identification.

A full Bayesian calibration model for assessing age in adults by means of pulp/tooth area ratio in periapical radiography.

The Bayesian approach is being a fundamental tool in forensic and legal field where inferences and decisions are made. In this study, a full Bayesian calibration model was developed to make probabilistic inferences about age estimation in a reference sample of 891 periapical X-rays of upper and lower canines. These teeth belonged to both deceased and living adult subjects, aged between 20 and 86 years, coming from five different countries (Turkey, Italy, Portugal, Japan and Mexico). For this purpose, the narrowing of pulp chamber due to the apposition of secondary dentine was analysed by means of the pulp/tooth area ratio. To determine the agreement of the method, intra- and inter-observer differences for measuring process were calculated by means of the intraclass correlation coefficient (ICC) analysis. Observer error tests showed excellent agreement between observers and between repeated assessments. According to the results of the ANCOVA, neither nationality nor sex was associated to the secondary dentine apposition while it is associated with individual's age. The results of the present study indicated that the concept of probability is intrinsically linked to the assessment of age in a forensic context, and the Bayesian approach could be considered a robust tool to overtake the bias generated by traditional regression models, thus helping the decision-making process in a legal framework.

Comparison of the third molar maturity index (I3M) between left and right lower third molars to assess the age of majority: a multi-ethnic study sample.

The diagnostic accuracy of the I3M to assess the legal age of 18 years has already been tested in several specific-population samples. The left lower third molar has been extensively used for discriminating between minors and adults. This research aimed to compare the usefulness of lower third molar maturity indexes, from both left and right side (I3ML and I3MR), in samples originating from four distinct continents in order to examine possible differences in their accuracy values. For this purpose, a sample of 10,181 orthopantomograms (OPGs), from Europe, Africa, Asia and America, was analysed and previously scored in other studies. The samples included healthy subjects with no systemic disorders with both third molars and clear depicted root apices. Wilcoxon Signed Rank test for left and right asymmetry did not show any significant differences. Data about sensitivity, specificity, predictive values, likelihood ratio and accuracy were pooled together and showed similar results for I3ML and I3MR, respectively. In addition, all these quantities were high when only the I3MR was considered to discriminate between adults and minors. The present referable database was the first to pool third molar measurements using panoramic radiographs of subjects coming from different continents. The results highlighted that both I3ML and I3MR are reliable indicators for assessing the legal age of 18 years old in those jurisdictions where this legal threshold has been set as the age of majority.

Development of an application tool to support returnees in Fukushima.

To promote radiation protection and health promotion among returning residents (returnees) in coastal areas of Fukushima, eHealth principles were used to develop a new application tool (app) that can record radiation exposure and health status while providing comprehensive support to returnees. Intended users are returnees and health and welfare workers. After assessing their needs, a flowchart and prototype for operational logic were created using commercially available software tools. Professional developers will focus on improving the user interface and ensuring data security. The finished app will be compatible with mobile telephones and tablets. Utility and ease of use are paramount to serve returnees of all ages effectively.

Construction of a recombinant vaccinia virus expressing Babesia gibsoni thrombospondin-related anonymous protein and evaluation of its immunogenicity in mice.

Previously, we have identified a gene encoding thrombospondin-related anonymous protein of Babesia gibsoni (BgTRAP), and have shown that the antisera raised against recombinant BgTRAP expressed in Escherichia coli inhibited the growth of parasites. In the present study, a recombinant vaccinia virus expressing the BgTRAP (VV/BgTRAP) was constructed. A specific band with a molecular mass of 80 kDa, which is similar to that of native BgTRAP on the merozoites of B. gibsoni, was detected in the supernatant of VV/ BgTRAP-infected RK13 cells. Mice inoculated with VV/BgTRAP produced a specific antiBgTRAP response. The antiserum against VV/BgTRAP showed reactivity against the native BgTRAP on parasites. These results indicated that the recombinant vaccinia virus expressing BgTRAP might be a vaccine candidate against canine B. gibsoni infection.

Regulation of Op18 during spindle assembly in Xenopus egg extracts.

Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.

The Xenopus Chk1 protein kinase mediates a caffeine-sensitive pathway of checkpoint control in cell-free extracts.

We have analyzed the role of the protein kinase Chk1 in checkpoint control by using cell-free extracts from Xenopus eggs. Recombinant Xenopus Chk1 (Xchk1) phosphorylates the mitotic inducer Cdc25 in vitro on multiple sites including Ser-287. The Xchk1-catalyzed phosphorylation of Cdc25 on Ser-287 is sufficient to confer the binding of 14-3-3 proteins. Egg extracts from which Xchk1 has been removed by immunodepletion are strongly but not totally compromised in their ability to undergo a cell cycle delay in response to the presence of unreplicated DNA. Cdc25 in Xchk1-depleted extracts remains bound to 14-3-3 due to the action of a distinct Ser-287-specific kinase in addition to Xchk1. Xchk1 is highly phosphorylated in the presence of unreplicated or damaged DNA, and this phosphorylation is abolished by caffeine, an agent which attenuates checkpoint control. The checkpoint response to unreplicated DNA in this system involves both caffeine-sensitive and caffeine-insensitive steps. Our results indicate that caffeine disrupts the checkpoint pathway containing Xchk1.

Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts.

Cdc2, the cyclin-dependent kinase that controls mitosis, is negatively regulated by phosphorylation on its threonine-14 and tyrosine-15 residues. Cdc25, the phosphatase that dephosphorylates both of these residues, undergoes activation and phosphorylation by multiple kinases at mitosis. Plx1, a kinase that associates with and phosphorylates the amino-terminal domain of Cdc25, was purified extensively from Xenopus egg extracts. Cloning of its complementary DNA revealed that Plx1 is related to the Polo family of protein kinases. Recombinant Plx1 phosphorylated Cdc25 and stimulated its activity in a purified system. Cdc25 phosphorylated by Plx1 reacted strongly with MPM-2, a monoclonal antibody to mitotic phosphoproteins. These studies indicate that Plx1 may participate in control of mitotic progression.

Myt1: a membrane-associated inhibitory kinase that phosphorylates Cdc2 on both threonine-14 and tyrosine-15.

Cdc2 is the cyclin-dependent kinase that controls entry of cells into mitosis. Phosphorylation of Cdc2 on threonine-14 and tyrosine-15 inhibits the activity of the enzyme and prevents premature initiation of mitosis. Although Wee1 has been identified as the kinase that phosphorylates tyrosine-15 in various organisms, the threonine-14-specific kinase has not been isolated. A complementary DNA was cloned from Xenopus that encodes Myt1, a member of the Wee1 family that was discovered to phosphorylate Cdc2 efficiently on both threonine-14 and tyrosine-15. Myt1 is a membrane-associated protein that contains a putative transmembrane segment. Immunodepletion studies suggested that Myt1 is the predominant threonine-14-specific kinase in Xenopus egg extracts. Myt1 activity is highly regulated during the cell cycle, suggesting that this relative of Wee1 plays a role in mitotic control.

Positive regulation of Wee1 by Chk1 and 14-3-3 proteins.

Wee1 inactivates the Cdc2-cyclin B complex during interphase by phosphorylating Cdc2 on Tyr-15. The activity of Wee1 is highly regulated during the cell cycle. In frog egg extracts, it has been established previously that Xenopus Wee1 (Xwee1) is present in a hypophosphorylated, active form during interphase and undergoes down-regulation by extensive phosphorylation at M-phase. We report that Xwee1 is also regulated by association with 14-3-3 proteins. Binding of 14-3-3 to Xwee1 occurs during interphase, but not M-phase, and requires phosphorylation of Xwee1 on Ser-549. A mutant of Xwee1 (S549A) that cannot bind 14-3-3 is substantially less active than wild-type Xwee1 in its ability to phosphorylate Cdc2. This mutation also affects the intranuclear distribution of Xwee1. In cell-free kinase assays, Xchk1 phosphorylates Xwee1 on Ser-549. The results of experiments in which Xwee1, Xchk1, or both were immunodepleted from Xenopus egg extracts suggested that these two enzymes are involved in a common pathway in the DNA replication checkpoint response. Replacement of endogenous Xwee1 with recombinant Xwee1-S549A in egg extracts attenuated the cell cycle delay induced by addition of excess recombinant Xchk1. Taken together, these results suggest that Xchk1 and 14-3-3 proteins act together as positive regulators of Xwee1.

Control of the Cdc2/cyclin B complex in Xenopus egg extracts arrested at a G2/M checkpoint with DNA synthesis inhibitors.

Proliferating eukaryotic cells possess checkpoint mechanisms that block cell division in the presence of unreplicated or damaged DNA. Using cell-free extracts from Xenopus eggs, we have investigated the mechanisms underlying the inability of a recombinant Cdc2/cyclin B complex to induce mitosis in the presence of incompletely replicated DNA. We found that the activities of the kinases and phosphatases that regulate the major phosphorylation sites on Cdc2 (e.g., tyrosine 15, threonine 14, and threonine 161) are not altered significantly under conditions where Xenopus extracts remain stably arrested in interphase due to the presence of the replication inhibitor aphidicolin. However, at threshold concentrations, a Cdc2/cyclin B complex containing a mutant Cdc2 subunit that cannot be phosphorylated on either tyrosine 15 or threonine 14 displays a markedly reduced capacity to induce mitosis in the presence of aphidicolin. This observation indicates that the replication checkpoint in Xenopus egg extracts functions without the inhibitory tyrosine and threonine phosphorylation of Cdc2. We provide evidence that the checkpoint-dependent suppression of the Cdc2/cyclin B complex involves a titratable inhibitor that is regulated by the presence of unreplicated DNA.

14-3-3 proteins act as negative regulators of the mitotic inducer Cdc25 in Xenopus egg extracts.

Cdc25, the dual-specificity phosphatase that dephosphorylates the Cdc2-cyclin B complex at mitosis, is highly regulated during the cell cycle. In Xenopus egg extracts, Cdc25 is associated with two isoforms of the 14-3-3 protein. Cdc25 is complexed primarily with 14-3-3epsilon and to a lesser extent with 14-3-3zeta. The association of these 14-3-3 proteins with Cdc25 varies dramatically during the cell cycle: binding is high during interphase but virtually absent at mitosis. Interaction with 14-3-3 is mediated by phosphorylation of Xenopus Cdc25 at Ser-287, which resides in a consensus 14-3-3 binding site. Recombinant Cdc25 with a point mutation at this residue (Cdc25-S287A) is incapable of binding to 14-3-3. Addition of the Cdc25-S287A mutant to Xenopus egg extracts accelerates mitosis and overrides checkpoint-mediated arrests of mitotic entry due to the presence of unreplicated and damaged DNA. These findings indicate that 14-3-3 proteins act as negative regulators of Cdc25 in controlling the G2-M transition.

Synthesis and metabolism of arachidonyl- and eicosapentaenoyl-CoA in rat aorta.

In studies on the metabolism of polyunsaturated fatty acids, acyl-CoA synthetase for 5,8,11,14-20:4 (arachidonic acid) and 5,8,11,14,17-20:5 (eicosapentaenoic acid) and the incorporation of these fatty acids into complex lipids and their conversion to CO2 were investigated in rat aorta. The activity of acyl-CoA synthetase was 35.9 for arachidonic acid and 63.0 for eicosapentaenoic acid (nmol/mg protein per 10 min) and the apparent Km values were 45 microM for arachidonic acid and 56 microM for eicosapentaenoic acid. Inhibition of eicosapentaenoyl-CoA synthesis by arachidonic acid was stronger than that of arachidonyl-CoA synthesis by eicosapentaenoic acid. Arachidonic acid and eicosapentaenoic acid were mostly incorporated into phospholipids. The incorporation of these fatty acids into cholesterol ester and their conversion to CO2 were less than those of palmitic acid, but their incorporation into triacyglycerol was greater. The incorporation of these fatty acids into phosphatidylserine + phosphatidylinositol and phosphatidylethanolamine was also greater than that of palmitic acid. The patterns of incorporation of arachidonic acid and eicosapentaenoic acid were similar. The physiological roles of these polyunsaturated fatty acids and the interference of eicosapentaenoic acid in arachidonic acid metabolism are discussed on the basis of these results.

Biosynthesis of N-methyl-L-glucosamine from D-glucose by Streptomyces griseus.

Biosynthesis of N-methyl-L-glucosamine moiety of streptomycin from D-glucose by Streptomyces griseus was studied. A mixture of D-[1-(14) C] glucose and D-[6(-3)H]glucose was given to the culture of S. griseus. The 3H/14C ratio found in N-methyl-L-glucosamine further supports a mechanism that the conversion of D-glucose to L-hexose is carried out without scission of carbon skeleton. When D-[1-14C]glucose and D-[3-3H]glucose were used, the fall of 3H/14C ratio in N-metyl-L-glucosamine showed that the hydrogen atom at C-3 plays a rôle in such a transformation.

Effects of sulfonylureas on membrane-bound low Km cyclic AMP phosphodiesterase in rat fat cells.

The effects of sulfonylureas and a biguanide on membrane-bound low Km cyclic AMP phosphodiesterase and lipolysis were examined in rat fat cells. Pharmacologically active sulfonylureas, such as tolbutamide (10 mM), acetohexamide (10 mM) and glibenclamide (200 microM) activated the phosphodiesterase when incubated with fat cells and suppressed lipolysis induced by isoproterenol. However, neither of these actions was observed in the presence of a pharmacologically inactive sulfonylurea, carboxytolbutamide (10 mM) and a biguanide, buformin (500 microM). Tolbutamide (0.5-10 mM) activated the enzyme, concentration dependently, and this manner of activation appears to coincide with that of the suppressive effect on the lipolysis. The time course of the enzyme activation was similar to that seen with insulin. Km, optimal pH and sensitivity to temperature of the enzyme from tolbutamide-treated cells were the same as those of the enzyme from control and insulin-treated cells. Direct incubation of the enzyme from control cells with tolbutamide did not affect the activity, while as little as 10 microM 3-isobutyl-1-methylxanthine markedly inhibited the enzyme. Tolbutamide continued to activate the enzyme in cells in which insulin receptor had been destroyed by trypsin-pretreatment. These results are compatible with the idea that the enzyme activated by sulfonylurea and that activated by insulin may be the same species of phosphodiesterase and that the antilipolytic action of sulfonylurea may be mediated by the activation of the enzyme which does not occur through the insulin receptor.

Effects of dithiothreitol on insulin-sensitive phosphodiesterase in rat fat cells.

Dithiothreitol activates the low-Km membrane-bound cyclic AMP phosphodiesterase when incubated with the enzyme in a cell-free system. To investigate the mechanism of its activation, we studied the effect of protease inhibitors. Isolated fat cells obtained from Sprague-Dawley rats were incubated in Krebs-Henseleit Hepes buffer, pH 7.4, at 37 degrees C with and without insulin (2 nM, 10 min). A crude microsomal fraction prepared by differential centrifugation was suspended in 0.25 M sucrose containing 10 mM Tes buffer, pH 7.5, with and without 2 mM dithiothreitol and protease inhibitors at 4 degrees C for 48 h. Dithiothreitol stimulated the phosphodiesterase, in a time-dependent manner. As little as 0.02 mM dithiothreitol activated the enzyme, and the maximally effective dose was 2-10 mM. Among the various protease inhibitors tested, antipain, leupeptin, chymostatin and E-64 were the most effective in preventing activation of the enzyme by dithiothreitol. Antipain also inhibited release of the enzyme from the bound fraction. These results suggest that activation of the low-Km phosphodiesterase by dithiothreitol may be provoked by stimulation of an endogenous thiol protease.

Effect of dextran sulfate on the interaction between very low density lipoprotein and lipoprotein lipase.

The effect of dextran sulfate on the interaction between very low density lipoprotein (VLDL) and purified bovine milk lipoprotein was studied. Dextran sulfate increased VLDL-triacylglycerol hydrolysis by lipoprotein lipase about 2-fold, but did not alter the Km value for triacylglycerol in VLDL. Strong association of dextran sulfate with the VLDL-lipoprotein lipase complex was demonstrated by gel filtration on BioGel A-5m, although dextran sulfate did not bind to VLDL and only very slightly to lipoprotein lipase. These findings suggest that dextran sulfate increases triacylglycerol hydrolysis in VLDL by binding to the VLDL-lipoprotein lipase complex.

Differential glycosylation of the GLUT1 glucose transporter in brain capillaries and choroid plexus.

The sodium-independent GLUT1 glucose transporter is expressed in high density in human erythrocytes and in tissues which serve a barrier function. In the polarized endothelial cells of the brain capillaries, which comprise the blood-brain barrier (BBB), GLUT1 is expressed on both apical and basolateral membranes; however, in the epithelium of the choroid plexus, GLUT1 expression is restricted to the basolateral surface. The present study examined whether these differences in subcellular localization of GLUT1 at the BBB and choroid plexus could be correlated with differential N-linked or O-linked glycosylation of the protein. Western blot analysis of solubilized brain capillaries (BC) and choroid plexus (CP) revealed that while the BC GLUT1 had an average molecular mass identical to that of the purified human erythrocyte transporter (54 kDa), the CP GLUT1 was of lower molecular mass (47 kDa). Treatment of brain capillaries and choroid plexus with N-glycanase resulted in a shift in the mobility of the GLUT1 of both samples to a lower molecular mass of 42 kDa; however, in contrast, treatment with O-glycanase produced no change in the mobility patterns of GLUT1, but did result in O-linked deglycosylation of another BBB marker, gamma-glutamyl transpeptidase. In conclusion, BBB and choroid plexus GLUT1 are subject to differential N-linked glycosylation with the protein having an N-linked carbohydrate side chain of higher molecular mass at the BBB in comparison to the choroid plexus.

Upregulation of blood-brain barrier GLUT1 glucose transporter protein and mRNA in experimental chronic hypoglycemia.

An in vivo model of chronic hypoglycemia was used to investigate changes in blood-brain barrier (BBB) glucose transport activity and changes in the expression of GLUT1 mRNA and protein in brain microvasculature occurring as an adaptive response to low circulating glucose levels. Chronic hypoglycemia was induced in rats by constant infusion of insulin via osmotic minipumps; control animals received infusions of saline. The criterion for chronic hypoglycemia was an average blood glucose concentration of < 2.3 mmol/l (42 mg/dl) after 5 days. The average blood glucose concentration at the end of the experimental period in the rats selected for study was 2.0 +/- 0.1 mmol/l (36 +/- 1 mg/dl) vs. 4.9 +/- 0.1 mmol/l (88 +/- 1 mg/dl) in the controls. Internal carotid artery perfusion studies demonstrated an increase in the BBB permeability-surface area (PS) product of 40% (P < 0.0005) in the chronically hypoglycemic animals as compared with controls. Western blotting of solubilized isolated brain capillaries demonstrated a 51% increase (P < 0.05) in immunoreactive BBB GLUT1 in the chronically hypoglycemic rats, and Northern blotting of whole-brain poly(A+) mRNA revealed a 50% increase in the GLUT1-to-actin ratio in the insulin-treated group (P < 0.05). Northern blotting analysis of microvessel-depleted total brain poly(A+) showed that the increase in GLUT1 mRNA in the chronically hypoglycemic rats was restricted to the BBB. The present study demonstrates increased expression of GLUT1 mRNA and protein at the BBB in chronic hypoglycemia and suggests that this increase is responsible for the compensatory increase in BBB glucose transport activity that occurs with chronically low circulating blood glucose levels.

Structure and dynamics of the taxocenoses of Pimplinae, Poemeniinae, Rhyssinae, Anomaloninae and Metopiinae in an urban secondary semideciduous montane forest.

Ichneumonidae (Hymenoptera) is one of the largest families of Insecta, but information on family diversity and distribution in Brazil is limited. The aim of the study was to assess the abundance, richness and seasonal distribution of Ichneumonidae in an urban secondary semideciduous montane forest. Insect specimens were captured in a Malaise trap placed within a restored sub-evergreen forest and sampling was performed every week during three non-consecutive 12-month periods. Of the 507 specimens collected, 338 were captured between May 1991 and May 1992, 95 between May 2000 and May 2001, and 74 between May 2007 and May 2008. Specimens were distributed among the subfamilies Pimplinae (n = 444), Anomaloninae (n = 42), Metopiinae (n = 16), Poemeniinae (n = 3) and Rhyssinae (n = 2). Species richness was highest in 1991-1992 with 33 rare and eight common species captured, followed by 2000-2001 with 31 rare and one common species captured, and 2007-2008 with 24 rare and one common species captured. The Shannon-Wiener diversity index (H') and Jackknife 1 species richness (S) values for the respective periods were 2.75/59.6, 3.15/35.8 and 2.83/35.8. In the 1991-1992 and 2000-2001 periods, parasitoid abundance was higher during the rainy season, while in 2007-2008 abundance was higher during the dry season. Colpotrochia mexicana (Cresson), Colpotrochia neblina Gauld & Sithole and Exochus izbus Gauld & Sithole were recorded for the first time in Brazil.