A nonradioisotope, enzymatic microplate assay for in vivo evaluation of 2-deoxyglucose uptake in muscle tissue

To determine 2-deoxy-D-glucose (2DG) and 2-deoxy-D-glucose 6-phosphate (DG6P) in mouse tissue after injection of 2DG, we have developed a novel assay. This assay is a simple procedure involving incubation of samples with four independent, single reaction mixtures followed by measurement of fluorescence. From differences between the values obtained with the four reactions, each of glucose, glucose 6-phosphate, 2DG and DG6P were able to be quantified in a sensitive manner. Using this assay system, glucose and 2DG in blood and DG6P-accumulation in muscle were easily determined. Therefore, this assay may be useful for measuring in vivo glucose uptake without the use of radioisotopes.     

Relevance of the N-terminal NLS-like sequence of the prion protein for membrane perturbation effects

We investigated the nuclear localization-like sequence KKRPKP, corresponding to the residues 23-28 in the mouse prion protein (mPrP), for its membrane perturbation activity, by comparing effects of two mPrP-derived peptides, corresponding to residues 1-28 (mPrPp(1-28)) and 23-50 (mPrPp(23-50)), respectively. In erythrocytes, mPrPp(1-28) induced ∼ 60% haemoglobin leakage after 30 min, whereas mPrPp(23-50) had negligible effects. In calcein-entrapping, large unilamellar vesicles (LUVs), similar results were obtained. Cytotoxicity estimated by lactate dehydrogenase leakage from HeLa cells, was found to be ∼ 12% for 50 μM mPrPp(1-28), and ∼ 1% for 50 μM mPrPp(23-50). Circular dichroism spectra showed structure induction of mPrPp(1-28) in the presence of POPC:POPG (4:1) and POPC LUVs, while mPrPp(23-50) remained a random coil. Membrane translocation studies on live HeLa cells showed mPrPp(1-28) co-localizing with dextran, suggesting fluid-phase endocytosis, whereas mPrPp(23-50) hardly translocated at all. We conclude that the KKRPKP-sequence is not sufficient to cause membrane perturbation or translocation but needs a hydrophobic counterpart.     

Regulation of adaptor protein GIT1 in platelets, leading to the interaction between GIT1 and integrin alpha(IIb)beta3

GIT1 is an adaptor protein, which links signaling proteins to focal adhesion, thereby regulating cytoskeletal reorganization. Platelets undergo dynamic cytoskeletal reorganization during platelet activation, for which a large number of adaptor proteins are required. However, there has been no report of GIT1 in platelets. We found that GIT1 was abundantly expressed in platelets and underwent tyrosine phosphorylation downstream of integrin α_IIbβ₃, which was inhibited by the Src kinase inhibitor PP2. Furthermore, GIT1 constitutively associated with βPIX, a guanine nucleotide exchange factor (GEF) for Rac. The GIT1/βPIX complex associated with α_IIbβ₃, concomitantly with GIT1 tyrosine phosphorylation. Moreover, both GIT1 and α_IIbβ₃ rapidly translocated to the cytoskeletal fraction during platelet aggregation, which was not observed in the absence of aggregation. These results suggest that tyrosine phosphorylation of GIT1 by Src kinases may regulate cytoskeletal reorganization downstream of α_IIbβ₃ by bringing the Rac GEF βPIX to the vicinity of the integrin.     

Molecular dynamics simulations of Factor Xa: insight into conformational transition of its binding subsites

Protein flexibility and conformational diversity is well known to be a key characteristic of the function of many proteins. Human blood coagulation proteins have multiple substrates, and various protein-protein interactions are required for the smooth functioning of the coagulation cascade to maintain blood hemostasis. To address how a protein may cope with multiple interactions with its structurally diverse substrates and the accompanied structural changes that may drive these changes, we studied human Factor X. We employed 20 ns of molecular dynamics (MD) and steered molecular dynamics (SMD) simulations on two different conformational forms of Factor X, open and closed, and observed an interchangeable conformational transition from one to another. This work also demonstrates the roles of various aromatic residues involved in aromatic-aromatic interactions, which make this dynamic transition possible.     

Genomic Evidence for a Simpler Clotting Scheme in Jawless Vertebrates

Mammalian blood clotting involves numerous components, most of which are the result of gene duplications that occurred early in vertebrate evolution and after the divergence of protochordates. As such, the genomes of the jawless fish (hagfish and lamprey) offer the best possibility for finding systems that might have a reduced set of the many clotting factors observed in higher vertebrates. The most straightforward way of inventorying these factors may be through whole genome sequencing. In this regard, the NCBI Trace database ( http://www.ncbi.nlm.nih.gov/Traces/trace.cgi ) for the lamprey (Petromyzon marinus) contains more than 18 million raw DNA sequences determined by whole-genome shotgun methodology. The data are estimated to be about sixfold redundant, indicating that coverage is sufficiently complete to permit judgments about the presence or absence of particular genes. A search for 20 proteins whose sequences were determined prior to the trace database study found all 20. A subsequent search for specified coagulation factors revealed a lamprey system with a smaller number of components than is found in other vertebrates in that factors V and VIII seem to be represented by a single gene, and factor IX, which is ordinarily a cofactor of factor VIII, is not present. Fortuitously, after the completion of the survey of the Trace database, a draft assembly based on the same database was posted. The draft assembly allowed many of the identified Trace fragments to be linked into longer sequences that fully support the conclusion that lampreys have a simpler clotting scheme compared with other vertebrates. The data are also consistent with the hypothesis that a whole-genome duplication or other large scale block duplication occurred after the divergence of jawless fish from other vertebrates and allowed the simultaneous appearance of a second set of two functionally paired proteins in the vertebrate clotting scheme.     

Frequency and Pattern of Heteroplasmy in the Control Region of Human Mitochondrial DNA

In this work, we present the results of the screening of human mitochondrial DNA (mtDNA) heteroplasmy in the control region of mtDNA from 210 unrelated Spanish individuals. Both hypervariable regions of mtDNA were amplified and sequenced in order to identify and quantify point and length heteroplasmy. Of the 210 individuals analyzed, 30% were fully homoplasmic and the remaining presented point and/or length heteroplasmy. The prevalent form of heteroplasmy was length heteroplasmy in the poly(C) tract of the hypervariable region II (HVRII), followed by length heteroplasmy in the poly(C) tract of hypervariable region I (HVRI) and, finally, point heteroplasmy, which was found in 3.81% of the individuals analyzed. Moreover, no significant differences were found in the proportions of the different kinds of heteroplasmy in the population when blood and buccal cell samples were compared. The pattern of heteroplasmy in HVRI and HVRII presents important differences. Moreover, the mutational profile in heteroplasmy seems to be different from the mutational pattern detected in population. The results suggest that a considerable number of mutations and, particularly, transitions that appear in heteroplasmy are probably eliminated by drift and/or by selection acting at different mtDNA levels of organization. Taking as a whole the results reported in this work, it is mandatory to perform a broad-scale screening of heteroplasmy to better establish the heteroplasmy profile which would be important for medical, evolutionary, and forensic proposes.     

Emergent vascular network inhomogeneities and resulting blood flow patterns in a growing tumor

Tumors acquire sufficient oxygen and nutrient supply by coopting host vessels and neovasculature created via angiogenesis, thereby transforming a highly ordered network into chaotic heterogeneous tumor specific vasculature. Vessel regression inside the tumor leads to large regions of necrotic tissue interspersed with isolated surviving vessels. We extend our recently introduced model to incorporate Fahraeus-Lindqvist- and phase separation effects, refined tissue oxygen level computation and drug flow computations. We find, unexpectedly, that collapse and regression accelerates rather than diminishes the perfusion and that a tracer substance flowing through the remodeled network reaches all parts of the tumor vasculature very well. The reason for decreased drug delivery well known in tumors should therefore be different from collapse and vessel regression. Implications for drug delivery in real tumors are discussed.     

XRASGRP2 expression during early development of Xenopus embryos

Previously, we described the DNA microarray screening of vascular endothelial cells that were formed by treatment of aggregates prepared from Xenopus animal cap cells with activin and angiopoietin-2. One of the genes identified in this screening showed homology to human RASGRP2 which plays a role in the regulation of GTP-GDP exchange of the Ras and Rap proteins, and was named XRASGRP2. In the present study, we analyzed the expression pattern of xrasgrp2 during Xenopus embryogenesis. The xrasgrp2 mRNA was expressed after stage 24, as assessed by stage PCR analysis. Whole-mount in situ hybridization showed that xrasgrp2 mRNA was located in the vascular region of the embryo. Loss-of-function analysis revealed that the formation of blood and endothelial cells in the explants transplanted into Xenopus embryos was inhibited by antisense morpholino oligonucleotides that block xrasgrp2 translation. These results suggest that XRASGRP2 plays a role in angiogenesis in Xenopus embryos.     

Isolation of Bothrops jararaca Snake Antithrombin from the Supernatant of Fibrinogen Purification

A novel method of antithrombin (AT) purification from Bothrops jararaca snake plasma was developed to obtain this protein using a waste supernatant from B. jararaca fibrinogen purification. The AT purification was achieved by affinity chromatography on HiTrap Heparin HP. The results showed an efficient purification process yielding pure AT (purity 65-fold and specific activity 368.91). In conclusion, we showed a feasible purification method of AT from B. jararaca plasma using a discarded material. This feature is important, considering the limitation of material, such as snake plasma, and could also be useful to obtain pure plasma proteins from other animals, including human plasma.     

Blood volume measurement: The comparison of pulse dye densitometry and Dill and Costill's methods

In many clinical situations, it is crucial to determine circulating blood volume (BV) easily and to repeat this measurement. The Dye DensitoGram Analyzer® (DDG, Nihon Kohden Corp) measures semi-automatically BV, using an injection of IndoCyanine Green (ICG, 10 mg), and avoiding intermittent blood samples. The DDG was used during a 90-day microgravity simulation by Head-Down-Tilt bed rest (HDT) to measure BV and compared with the calculation of the plasma volume (PV) variations according to Dill and Costill's formula (DC). Seventeen healthy volunteers were included: 8 control subjects (Co) and 9 subjects submitted to a resistive exercise counter-measure (CM). Measurements were performed, one day before HDT, on days 3 and 90 of HDT and on day 9 after HDT. A double measurement of the BV was performed to assess the repeatability of this method. On the last day of HDT a significant decrease (p < 0.05) in the PV was noted with the DDG (Co: − 12.3 ± 5.7%, CM: − 9.0 ± 5.3%) and DC; (Co: − 4.7 ± 1.8%, CM: − 6.8 ± 2.5%). A good repeatability of the technique was shown with a low intrasubjects coefficient of variation (4.95 ± 0.95%) and an acceptable intersubjects coefficient of variation (15.30 ± 1.13%). No correlation was noted between DDG and DC (r² = 0.27). The DDG gives a good repeatability, not affected by the microgravity exposure. Thanks to its capacity to measure accurately the BV within 7-10 min, this device presents major advantages for clinical use and research purpose.