Discovery of a bone-like blood particle in the peripheral circulation of humans and rodents.

OBJECTIVE: To characterize ossified bone marrow blood vessels and confirm the presence of ossified particles (OSP) in humans and rodents. METHODS: Human bone marrow blood vessels were processed for scanning and transmission electron microscopy. Whole blood samples were collected from younger (26-39 years; n = 6) and older (55-63 years; n = 6) volunteers and male Fischer-344 rats (1 month, n = 7; 6 months, n = 7; 12 months, n = 7; 18-months, n = 6; 24 months, n = 8). OSP in the whole blood samples were sorted and imaged with microscopy to determine diameter, circularity, and solidity. Additionally, the chemical composition of OSP was determined via elemental analysis. RESULTS: SEM revealed two types of ossified bone marrow blood vessels: that is, "transitioning" and "ossified." OSP were adhered to the surface of transitioning vessels and theoretically gain access to and circulate within the blood. The majority of OSP were ≤15 µm in diameter, but many were of sufficient size to serve as emboli (ie, >15 µm).OSP were predominately oblong in shape and several had jagged tips and edges. CONCLUSIONS: We introduce a novel, bone-like blood particle that may be diagnostic of bone marrow blood vessel ossification. Further, OSP may associate with several disease states (eg, atherosclerosis).

Progressive ossification of the bone marrow vasculature with advancing age corresponds with reduced red blood cell count and percentage of circulating lymphocytes in male Fischer-344 rats.

OBJECTIVE: Assess the link between bone marrow blood vessel ossification, Tb. and cortical bone, and hematological parameters across the lifespan in rats. METHODS: Right femora and whole blood samples were taken from male Fischer-344 rats at 1, 6, 12, 18 and 24 months. Femora were scanned by micro-computed tomography (MicroCT) to determine bone marrow blood vessel ossification (ie, ossified vessel volume [OsVV], ossified vessel thickness (OsV.Th), ossified vessel density (OsV density), and structural model index [SMI]). Bone microarchitecture (ie, bone volume [BV/TV], trabecular thickness, trabecular number, and trabecular separation), density and SMI, and cortical bone parameters (ie, cortical shell thickness, porosity, and density) were also determined by MicroCT. Complete blood counts with differentials were conducted. RESULTS: Ossified vessel volume increased throughout the lifespan, coinciding with reduced trabecular BV/TV and cortical shell thickness at 24 months. Many of the hematological parameters were unchanged (ie, white blood cells, lymphocyte number) or increased (monocyte number, percent monocyte, granulocyte number, percent granulocyte, hemoglobin, hematocrit, mean corpuscular hemoglobin concentration, red blood cell distribution width, platelet, mean platelet volume) with advancing age; however, red blood cells (RBC) and percent lymphocytes (LY%) were reduced at 24 months. In addition, OsV density was similar to trabecular bone density. CONCLUSIONS: Declines in trabecular BV/TV, cortical shell thickness, RBC, and LY% with advanced age coincided with augmented ossification of bone marrow blood vessels.

Effect of the disintegrin eristostatin on melanoma-natural killer cell interactions.

Malignant melanoma is difficult to treat due to its resistance to chemotherapeutic regimens. Discovery of new pharmaceuticals with inhibitory potential can be helpful in the development of novel treatments. The snake venom disintegrin eristostatin, from the viper Eristicophis macmahoni, caused immunodeficient mice to be significantly protected from development of lung colonization when melanoma cells and the disintegrin were co-injected in vivo into the lateral tail vein compared to vehicle controls. Cytotoxicity assays suggested that eristostatin makes the melanoma cells a better target for lysis by human natural killer cells. Direct binding assays using atomic force microscopy showed eristostatin does specifically bind the surface of the six melanoma cell lines tested. Eristostatin binding was partially inhibited by the addition of soluble RGDS peptide, suggesting an integrin as one likely, but not the sole, binding partner. Studies done with melanoma cells on a culture dish and natural killer cells attached to a cantilever tip in atomic force microscopy showed four major populations of interactions which exhibited altered frequency and unbinding strength in the presence of eristostatin.

Research in clinical laboratory science: professionals' involvement.

OBJECTIVES: To describe current qualitative and quantitative aspects of research engagement and other scholarly activities conducted by clinical laboratory science (CLS) professionals across a range of employment settings. DESIGN: A link to a 3-part online survey was sent by electronic mail to 7,572 members of the American Society for Clinical Laboratory Science and 500 program directors. SETTING: email message, on-line survey PARTICIPANTS: all ASCLS members and all directors of accredited clinical laboratory educational programs MAIN OUTCOME MEASURES: Quantitative and qualitative measures of professionals' engagement in research and other scholarly activities RESULTS: 556 of 7572 (7.3%) persons completed the survey. Thirty-two percent of survey respondents reported spending between 1 to > 40 work hours per week conducting research with 68% of respondents not participating in research activities. Conducting research is an employment requirement for 18% of survey participants. Twenty-nine percent of respondents have published at least one research article, and 47% of respondents who conduct research have published studies in the journal Clinical Laboratory Science. More than 57% of respondents participate in non-research scholarly activities as part of their employment. CLS professionals who conduct research are more likely to do applied, clinical, or educational research than other types of research. Fifty-seven percent of respondents who conduct research lack external funding for their work. Ninety-three percent of total research dollars is obtained by respondents who hold the Ph.D. degree. The perception of the importance of conducting research varies by employment position. Barriers to participation in research include lack of inclusion of research in the job description, time constraints, inadequate research funding, limited opportunity, and lack of space and equipment. CONCLUSIONS: CLS professionals participate in research in limited numbers, and are more likely to engage in non-research types of scholarly activities. Numerous barriers are identified which impose limits to conducting research. Over half of CLS's research efforts lack external funding. Although there was broad representation among participants across educational levels, employment settings, and job positions, the number of survey respondents was limited. Possible directions for future research include conducting this survey using members of additional professional organizations.

Research in clinical laboratory science: professionals' educational preparation.

OBJECTIVE: To describe the educational preparation of CLS professionals for conducting research. DESIGN: A link to 3-part online survey was sent by electronic mail to 7,572 members of the American Society for Clinical Laboratory Science and 500 program directors research project. Barriers to participation in research by undergraduates include time limitations within the curriculum, insufficient faculty time, and lack of funds, space, and equipment. Increased emphasis on developing research skills is found in educational programs at the master's degree level. CONCLUSIONS: The formal educational background of many CLS professionals may leave them unprepared or underprepared for conducting research. Although there was broad representation among participants across educational levels, employment settings, and job positions, the number of survey respondents was limited. Possible directions for future research include conducting this survey using members of additional professional organizations.

Disintegrins in health and disease.

Few of the proteins isolated and characterized from snake venom have proven to be more chemically diverse, exquisitely specific or promiscuously active than the family known as disintegrins. These small proteins have shown structural homology with hundreds of cell surface molecules from plants and animals other than snakes, and their precise mimicry of native receptor ligands speaks to evolutionary niches related to survival and geographic locale. Over 100 disintegrins have been named and studied, with the most recent efforts into molecular techniques providing significant clues to taxonomic relationships among four different snake families. Investigators have evaluated disintegrin applications in therapies for cancer, asthma, osteopenia and inappropriate angiogenesis. Crystal and NMR studies have confirmed hypotheses regarding ligand-receptor interactions while illuminating the complexities of structure-function evidence. Disintegrin chimeras with viruses, microbubbles and fluorescent labels have become useful tools in many investigations. While many disintegrin studies still involve platelets, previously unexplored interactions with glial cancer, T lymphocytes and the bacteria Yersinia have blazed new trails for this field. This review will summarize disintegrin investigations since 2003.

Inhibition of lung tumor colonization and cell migration with the disintegrin crotatroxin 2 isolated from the venom of Crotalus atrox.

Disintegrins are low molecular weight proteins (4-15 kDa) with an RGD binding region at their binding loop. Disintegrin and disintegrin-like proteins are found in the venom of four families of snakes: Atractaspididae, Elapidae, Viperidae, and Colubridae. This report describes the biological activity of a disintegrin, crotatroxin 2, isolated by a three-step chromatography procedure from the venom of the Western diamondback rattlesnake (Crotalus atrox). The intact molecular mass for crotatroxin 2 was 7.384 kDa and 71 amino acids. Crotatroxin 2 inhibited human whole blood platelet aggregation with an IC(50) of 17.5 nM, inhibited cell (66.3p) migration by 63%, and inhibited experimental lung tumor colonization in BALB/c mice at 1000 microg/kg. Our data suggest that while crotatroxin 2 inhibits platelet aggregation, cancer cell migration, and lung tumor colonization, it is done via different integrins.

Inhibition of melanoma cell motility by the snake venom disintegrin eristostatin.

Eristostatin, an RGD-containing disintegrin isolated from the venom of Eristicophis macmahoni, inhibits lung or liver colonization of melanoma cells in a mouse model. In this study, transwell migration and in vitro wound closure assays were used to determine the effect of eristostatin on the migration of melanoma cells. Eristostatin significantly impaired the migration of five human melanoma cell lines. Furthermore, it specifically inhibited cell migration on fibronectin in a concentration-dependent manner, but not that on collagen IV or laminin. In contrast, eristostatin was found to have no effect on cell proliferation or angiogenesis. These results indicate that the interaction between eristostatin and melanoma cells may involve fibronectin-binding integrins that mediate cell migration. Mutations to alanine of seven residues within the RGD loop of eristostatin and four residues outside the RGD loop of eristostatin resulted in significantly less potency in both platelet aggregation and wound closure assays. For six of the mutations, however, decreased activity was found only in the latter assay. We conclude that a different mechanism and/or integrin is involved in these two cell activities.

Molecular engineering of an EGFP/disintegrin-based integrin marker.

Disintegrins are viper venom peptides, which bind integrins with high affinity (10(-8) M) and selectivity. Among them, eristostatin (Er) selectively binds and inhibits alphaIIbbeta3 integrin function. In this work we have engineered an enhanced green fluorescence protein (EGFP)-tagged Er as an alphaIIbbeta3 biomarker to be used in bioassays involving fluorescence detectors. For this, we have first constructed an EGFP bacterial expression vector, which resulted in a 6xHis tag-coding region followed by the EGFP gene and a 3' multiple cloning site (MCS) comprising nine restriction sites. This vector, termed pAZ, was used to clone the Er gene, resulting in a 32 kDa EGFP-Er fusion protein when expressed as characterized by SDS-PAGE and Western blot. Both EGFP-Er and EGFP (expressed from the empty pAZ vector) were purified by immobilized metal affinity chromatography (IMAC) and their fluorescence was measured showing similar values, thus suggesting that the Er portion is not affecting the EGFP activity. EGFP-Er, but not EGFP selectively bound to immobilized platelets as detected by confocal microscopy indicating the preservation of Er disintegrin activity and its potential use as a marker for alphaIIbbeta3 integrin. Our data suggest the use of the pAZ vector for expressing soluble EGFP-labeled proteins and the use of EGFP-fused disintegrins as markers for integrins.

Scratching below the surface: wound healing and alanine mutagenesis provide unique insights into interactions between eristostatin, platelets and melanoma cells.

To study the molecular mechanism of the disintegrin eristostatin, cellular functional studies were performed using ten recombinant alanine mutants. ADP-induced platelet aggregation revealed critical contributions of seven residues within the 'RGD loop' (R24, R27, G28, N31) and C-terminus (W47, N48, G49) of this disintegrin. Using an in vitro scratch wound healing assay, four human melanoma cell lines yielded similar results when exposed to wildtype eristostatin. All eristostatin-treated cells healed less of the wounded area than control conditions. This phenomenon was reproduced when using fibronectin as the matrix. C8161 cells showed significant delay in wound closure with the N-terminal mutant P4A but not with R24A or G28A. Evidence from our laboratory and others suggests neither alpha IIb, alpha 4 nor alpha 5 integrins are directly involved in eristostatin's interactions. Eristostatin did not affect the number of melanoma cells in culture after 24 h or the development of apoptosis. However, phosphorylation studies performed after these melanoma cells were exposed to eristostatin revealed changes in several tyrosine phosphorylated molecules.

Disintegrins.

The existence of disintegrins, non-enzymatic, small molecular weight proteins from viper venom, has been known for 2 decades, and their impact on cellular research has been substantial and far-reaching. Disintegrins have been the molecular scaffold used in the design of therapeutics for the prevention of thrombosis and cancer. Their sequencing has provided insights into the evolution of proteins over millennia. Production of recombinant disintegrin mutants and fusion proteins has allowed investigations into molecular mechanisms at work in cell-extracellular matrix interactions. Structural homologies with non-snake proteins have shown disintegrin-like molecules in species ranging from slime mold to humans. Intracellular signaling events have been elucidated through the use of venom disintegrins, including events related to programmed cell death, motility, cell proliferation and viral pathogenesis. Disintegrin sequences (protein or genes) have been placed in microbubbles and liposomes and been found to target neovascular endothelium and metastatic tumors in two mouse models. The purpose of this review is to highlight the members of this disintegrin family discovered since 1998 as well as the increased understanding of their usefulness in therapeutics and technical assays.

The disintegrin echistatin stabilizes integrin alphaIIbbeta3's open conformation and promotes its oligomerization.

We have employed echistatin, a 5.4 kDa snake venom disintegrin, as a model protein to investigate the paradox that small ligand-mimetics can bind to the resting alphaIIbbeta3 integrin while adhesive macromolecules cannot. We characterized the interactions between purified human alphaIIbbeta3 and two recombinant echistatin variants: rEch (1-49) M28L, chosen for its selectivity toward beta3-integrins, and rEch (1-40) M28L, a carboxy-terminal truncation mutant. While both contain an RGD integrin targeting sequence, only rEch (1-49) M28L was an effective inhibitor of alphaIIbbeta3 function. Electron microscopy of rotary shadowed specimens yielded a variety of alphaIIbbeta3 conformers ranging from compact, spherical particles (maximum dimension 22 nm) to the classical "head with two tails" forms (32 nm). The population of larger particles (42-56 nm) increased from 17% to 28% in the presence of rEch (1-49) M28L, indicative of ligand-induced oligomerization. Sedimentation velocity measurements demonstrated that both full length and truncated echistatin perturbed alphaIIbbeta3's solution structure, yielding slower-sedimenting open conformers. Dynamic light scattering showed that rEch (1-49) M28L protected alphaIIbbeta3 from thermal aggregation, raising its transition mid-point from 46 degrees C to 69 degrees C; a smaller shift resulted with rEch (1-40) M28L. Sedimentation equilibrium demonstrated that both echistatin ligands induced substantial alphaIIbbeta3 dimerization. van't Hoff analysis revealed a pattern of entropy/enthalpy compensation similar to tirofiban, a small RGD ligand-mimetic that binds tightly to alphaIIbbeta3, but yields smaller conformational perturbations than echistatin. We propose that echistatin may serve as a paradigm for understanding multidomain adhesive macromolecules because its ability to modulate alphaIIbbeta3's structure resides on an RGD loop, while full disintegrin activity requires an auxiliary site that includes the carboxy-terminal nine amino acid residues.

Cloning, expression, and characterization of a bi-functional disintegrin/alkaline phosphatase hybrid protein.

Integrins are transmembrane heterodimeric glycoproteins responsible for cellular communication; therefore, they play an essential role in many physiological events. Viper snake venoms contain integrin antagonists called disintegrins which bind and inhibit integrin function. They present a loop containing an RGD motif responsible for integrin binding. The engineering of disintegrins fused to a reporter enzyme will be an interesting approach to build integrin markers. Even more, the disintegrin scaffold could be used to present other protein binding motifs. In this work, we have obtained alkaline phosphatase (APv) tagged eristostatin (Er) by cloning and expressing eristostatin DNA into the pLIP6-GN vector. Eristostatin, a 49 residue disintegrin, binds selectively to alphaIIbbeta3 integrin, inhibiting its binding to fibrinogen. The resulting fusion protein Er/APv was identified by SDS-PAGE and by Western blotting using both anti-Er and anti-AP antibodies. This fusion protein showed enzymatic AP activity similar to that of wild APv and its potential use for an alphaIIbbeta3 integrin assay was tested in a one-step dot blot using immobilized cells incubated with the marker and developed by AP substrate. Er/APv showed selectivity towards platelets and alphaIIbbeta3 integrin transfected cells and reacted with the same region as unlabeled Er, as analyzed in competition assays. Our data present a novel tool, Er/APv, with potential use as molecular marker in processes where the alphaIIbbeta3 integrin is involved.

Integrin antagonists affect growth and pathfinding of ventral motor nerves in the trunk of embryonic zebrafish.

Integrins are thought to be important receptors for extracellular matrix (ECM) components on growing axons. Ventral motor axons in the trunk of embryonic zebrafish grow in a midsegmental pathway through an environment rich in ECM components. To test the role of integrins in this process, integrin antagonists (the disintegrin echistatin in native and recombinant form, as well as the Arg-Gly-Asp-Ser peptide) were injected into embryos just prior to axon outgrowth at 14-16 h postfertilization (hpf). All integrin antagonists affected growth of ventral motor nerves in a similar way and native echistatin was most effective. At 24 hpf, when only the three primary motor axons per trunk hemisegment had grown out, 80% (16 of 20) of the embryos analyzed had abnormal motor nerves after injection of native echistatin, corresponding to 19% (91 of 480) of all nerves. At 33 hpf, when secondary motor axons were present in the pathway, 100% of the embryos were affected (24 of 24), with 20% of all nerves analyzed (196 of 960) being abnormal. Phenotypes comprised abnormal branching (64% of all abnormal nerves) and truncations (36% of all abnormal nerves) of ventral motor nerves at 24 hpf and mostly branching of the nerves at 33 hpf (94% of all abnormal nerves). Caudal branches were at least twice as frequent as rostral branches. Surrounding trunk tissue and a number of other axon fascicles were apparently not affected by the injections. Thus integrin function contributes to both growth and pathfinding of axons in ventral motor nerves in the trunk of zebrafish in vivo.