Reconsideration of bone postmortem interval estimation by UV-induced autofluorescence.

Assessing the UV-fluorescence of a freshly cut cross section of the compact parts of a bone is often recommended as a first step to estimate the postmortem interval (PMI) of skeletal remains. Opinions differ concerning the cause of fluorescence and on how to categorize fluorescent properties as well as the significance of fluorescent characteristics in correlation with the PMI. In this study we evaluated the UV-fluorescence of over 200 bones with known PMI to reassess the diagnostic value of this method for differentiating between historical and recent skeletal remains. It could be shown that there is a correlation between the PMI and fluorescence colour, but not with fluorescence intensity. Furthermore, the quality of two UV-fluorescence test possibilities based on fluorescence colour was assessed by calculating the individual test efficiency, sensitivity and specificity. The results showed that blue bone fluorescence, as well as blue fluorescence combined with other colours (mainly yellow) does not allow the observer to draw any conclusions about sample age. Only overall yellow fluorescence may indicate a historical specimen. But still, 2% of all forensically relevant samples were falsely excluded, making bone fluorescent properties inappropriate as the sole criterion for deciding whether a specimen is included or excluded for further forensic investigation.

[A Roman orbital implant?]. / Römisches Orbitaimplantat?

During an excavation in Regensburg/Germany the skeleton of an approximately 20-year-old Roman man was found who was buried in the 3rd/4th century after Christ. A "stone" was found which fitted into the left orbit precisely. After a thorough investigation of the "stone" and with the ophthalmohistorical literature in mind an orbital "implant" as well as a petrified medical paste ("Kollyrium") could be ruled out almost with certainty. Possibly the "stone" served another medical purpose or was used for protection of the eye.

Synthetic analogs of FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] differentially regulate pulmonary vascular permeability in vivo and in vitro.

Novel therapies are needed to address the vascular endothelial cell (EC) barrier disruption that occurs in inflammatory diseases such as acute lung injury (ALI). We previously demonstrated the potent barrier-enhancing effects of both sphingosine 1-phosphate (S1P) and the structurally similar compound FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] in inflammatory lung injury. In this study, we examined the therapeutic potential of several novel FTY720 analogs to reduce vascular leak. Similar to S1P and FTY720, the (R)- and (S)-enantiomers of FTY720 phosphonate and enephosphonate analogs produce sustained EC barrier enhancement in vitro, as seen by increases in transendothelial electrical resistance (TER). In contrast, the (R)- and (S)-enantiomers of FTY720-regioisomeric analogs disrupt EC barrier integrity in a dose-dependent manner. Barrier-enhancing FTY720 analogs demonstrate a wider protective concentration range in vitro (1-50 microM) and greater potency than either S1P or FTY720. In contrast to FTY720-induced EC barrier enhancement, S1P and the FTY720 analogs dramatically increase TER within minutes in association with cortical actin ring formation. Unlike S1P, these FTY720 analogs exhibit differential phosphorylation effects without altering the intracellular calcium level. Inhibitor studies indicate that barrier enhancement by these analogs involves signaling via G(i)-coupled receptors, tyrosine kinases, and lipid rafts. Consistent with these in vitro responses, the (S)-phosphonate analog of FTY720 significantly reduces multiple indices of alveolar and vascular permeability in a lipopolysaccharide-mediated murine model of ALI (without significant alterations in leukocyte counts). These results demonstrate the capacity for FTY720 analogs to significantly decrease pulmonary vascular leakage and inflammation in vitro and in vivo.

Tyrosine dephosphorylation and deactivation of insulin receptor substrate-1 by protein-tyrosine phosphatase 1B. Possible facilitation by the formation of a ternary complex with the Grb2 adaptor protein.

Regulation of the steady-state tyrosine phosphorylation of the insulin receptor and its postreceptor substrates are essential determinants of insulin signal transduction. However, little is known regarding the molecular interactions that influence the balance of these processes, especially the phosphorylation state of postinsulin receptor substrates, such as insulin receptor substrate-1 (IRS-1). The specific activity of four candidate protein-tyrosine phosphatases (protein-tyrosine phosphatase 1B (PTP1B), SH2 domain-containing PTPase-2 (SHP-2), leukocyte common antigen-related (LAR), and leukocyte antigen-related phosphatase) (LRP) toward IRS-1 dephosphorylation was studied using recombinant proteins in vitro. PTP1B exhibited the highest specific activity (percentage dephosphorylated per microg per min), and the enzyme activities varied over a range of 5.5 x 10(3). When evaluated as a ratio of activity versus IRS-1 to that versus p-nitrophenyl phosphate, PTP1B remained significantly more active by 3.1-293-fold, respectively. Overlay blots with recombinant Src homology 2 domains of IRS-1 adaptor proteins showed that the loss of IRS-1 binding of Crk, GRB2, SHP-2, and the p85 subunit of phosphatidylinositol 3'-kinase paralleled the rate of overall IRS-1 dephosphorylation. Further studies revealed that the adaptor protein GRB2 strongly promoted the formation of a stable protein complex between tyrosine-phosphorylated IRS-1 and catalytically inactive PTP1B, increasing their co-immunoprecipitation from an equimolar solution by 13.5 +/- 3.3-fold (n = 7; p < 0.01). Inclusion of GRB2 in a reaction mixture of IRS-1 and active PTP1B also increased the overall rate of IRS-1 tyrosine dephosphorylation by 2.7-3.9-fold (p < 0.01). These results provide new insight into novel molecular interactions involving PTP1B and GRB2 that may influence the steady-state capacity of IRS-1 to function as a phosphotyrosine scaffold and possibly affect the balance of postreceptor insulin signaling.

Expression of insulin receptor mRNA and insulin receptor substrate 1 in pancreatic islet beta-cells.

The expression of insulin receptor mRNA was examined in rat pancreatic islet cells by single-cell reverse transcriptase (RT)-polymerase chain reaction (PCR). Single cells from disaggregated islets were individually isolated in a microcapillary pipet, and the beta-cells were identified by amplification of the mRNA for insulin. We found that in single beta-cells, the mRNA for the insulin receptor was also expressed. The fraction of single islet cells expressing both insulin receptor and insulin mRNAs corresponds closely to the fraction of beta-cells in the disaggregated islet cell preparation. These results indicate that normal beta-cells have the potential to express authentic insulin receptors. Immunohistochemical analysis was insufficiently sensitive for assaying insulin receptor protein; however, insulin receptor substrate 1 (IRS-1) was readily immunolocalized in islet beta-cells. Since IRS-1 links several cell surface receptors, including those for insulin and IGF-I, to distal signal transduction pathways, our observations indicate that hormonal regulation of islet beta-cells potentially involves the same signal transduction pathway that mediates insulin and growth factor signaling in peripheral insulin target tissue cell types.

Purification and identification of bovine liver gamma-carboxylase.

The microsomal gamma-carboxylase catalyzes modification of a limited set of glutamyl residues to gamma-carboxyglutamyl residues in a vitamin K-dependent reaction that also utilizes O2 and CO2. We report the purification to apparent homogeneity of the bovine liver microsomal carboxylase. Affinity chromatography exploiting the association of the carboxylase with prothrombin precursor and carboxylase binding to the propeptide sequence were combined with ion-exchange chromatography and fractionation using immobilized lectins. A 3.5 x 10(5)-fold purification was obtained, which is the highest purification, by a factor of 35, yet reported for this enzyme. A single 98-kDa protein is obtained from this isolation. Carboxylase activity is associated with this protein by two different criteria. Antibodies prepared against the carboxylase detected the 98-kDa protein when used in Western analysis. In addition, the single 98-kDa protein was shown to comigrate with activity when electrophoresed in a nondenaturing gel system. The availability of purified preparations of carboxylase will facilitate an increased understanding of the complex biochemical reaction carried out by this protein.

Vitamin K-dependent carboxylase: partial purification of the enzyme by antibody affinity techniques.

The vitamin K-dependent carboxylase activity of bovine liver microsomes has been purified 500-fold by adsorption to an antiprothrombin column and elution with a dodeca peptide which competes with a prothrombin precursor enzyme recognition site. The purified enzyme is devoid of bound precursors, and has the same ratio of vitamin K epoxidase activity to carboxylase activity as the crude microsomal preparation.