A method described to purify pluripotent hemopoietic stem cells ( PHSC ) from adult mouse bone marrow. The method consists of three separation steps. First, bone marrow cells are centrifuged in a discontinuous metrizamide gradient and simultaneously labeled with wheat germ agglutinin-fluorescein isothiocyanate (WGA-FITC). Second, the low density cells are analyzed by a fluorescence-activated cell sorter (FACS) and the WGA-positive cells with medium forward and low perpendicular light scatter intensities are sorted. The WGA-FITC is removed from the cells by incubation with N-acetyl-D-glucosamine. Finally, the sorted cells are incubated with anti-H-2K-biotin and avidin-FITC and sorted a second time to enrich cells with high H-2K density. The sorted cells gave rise to 2 spleen colonies per 100 injected cells at 8 d and 6.6 colonies per 100 cells at 12 d after transplantation into lethally irradiated syngeneic recipients. The average enrichment factor for day 12 CFU-S (colony-forming unit/spleen) was 135 (range, 90--230; n = 15) and was similar to that for the cell type that provides radioprotection (180 +/- 70), indicating that these functional properties were copurified. Indirect evidence suggests that the spleen-seeding efficiency (f factor) of these cells is 0.10 and, therefore, the average purity of the sorted PHSC was 65% (range in 15 experiments, 35--110%). The sorted cells were all in the G1 or G0 phase of the cell cycle. They appeared to be undifferentiated blasts by morphological criteria. Electron microscopy revealed that the sorted cells consisted primarily of two cell types, possibly representing G0 and G1 cells. The FACS was used to deposit single selected cells into individual microwells of Terasaki trays. 32% of the sorted cells could be induced to form myeloid progeny in vitro. This procedure should be useful for direct studies on the regulation of hemopoietic cell differentiation.

Inactivation of protein kinase Cdelta (PKCdelta) is associated with resistance to terminal cell death in epidermal tumor cells, suggesting that activation of PKCdelta in normal epidermis may be a component of a cell death pathway. To test this hypothesis, we constructed an adenovirus vector carrying an epitope-tagged PKCdelta under a cytomegalovirus promoter to overexpress PKCdelta in normal and neoplastic keratinocytes. While PKCdelta overexpression was detected by immunoblotting in keratinocytes, the expression level of other PKC isozymes, including PKCalpha, PKCepsilon, PKCzeta, and PKCeta, did not change. Calcium-independent PKC-specific kinase activity increased after infection of keratinocytes with the PKCdelta adenovirus. Activation of PKCdelta by 12-O-tetradecanoylphorbol-13-acetate (TPA) at a nanomolar concentration was lethal to normal and neoplastic mouse and human keratinocytes overexpressing PKCdelta. Lethality was inhibited by PKC selective inhibitors, GF109203X and Ro-32-0432. TPA-induced cell death was apoptotic as evidenced by morphological criteria, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, DNA fragmentation, and increased caspase activity. Subcellular fractionation indicated that PKCdelta translocated to a mitochondrial enriched fraction after TPA activation, and this finding was confirmed by confocal microscopy of cells expressing a transfected PKCdelta-green fluorescent protein fusion protein. Furthermore, activation of PKCdelta in keratinocytes altered mitochondrial membrane potential, as indicated by rhodamine-123 fluorescence. Mitochondrial inhibitors, rotenone and antimycin A, reduced TPA-induced cell death in PKCdelta-overexpressing keratinocytes. These results indicate that PKCdelta can initiate a death pathway in keratinocytes that involves direct interaction with mitochondria and alterations of mitochondrial function.

The highly reactive species, peroxynitrite, is produced in endothelial cells in pathological states in which the production of superoxide anion and NO is increased. Here, we show that peroxynitrite added exogenously or generated endogenously in response to exposure to an NO donor or oxidized low-density lipoproteins (oxLDL) increases p21ras activity in bovine aortic endothelial cells. The activation is not dependent on upstream elements but rather is due to direct targeting of p21ras by reversible S-glutathiolation of cysteine thiols as demonstrated by biotin-labeling techniques. The time course of p21ras S-glutathiolation following peroxynitrite corresponds to the increase in its Raf-1 binding activity and translocation to the membrane. Moreover, p21ras S-glutathiolation and activation can be reversed by dithiothreitol, confirming the importance of a disulfide bond. S-glutathiolation also promoted guanine nucleotide exchange of recombinant p21ras. In addition, the oxidant-induced activation of Mek/Erk and PI3 kinase/Akt was abrogated by dominant-negative and Cys-118 p21ras mutants, and the latter also prevented S-glutathiolation of p21ras. These results indicate that peroxynitrite arising from NO donors or pathological stimuli such as oxLDL triggers direct S-glutathiolation of p21ras Cys-118, which increases p21ras activity and mediates downstream signaling.

Epithelial cadherin (E-cadherin) is a Ca(2+)-dependent cell-cell adhesion molecule that connects cells via homotypic interactions. Its function is critical in the induction and maintenance of cell polarity and differentiation, and its loss of downregulation is associated with an invasive and poorly differentiated phenotype in colon and other tumors. We have used an avidin-biotin immunoperoxidase technique to localize E-cadherin in microwave-treated, paraffin-embedded sections from 36 patients with pancreatic adenocarcinomas. E-cadherin was expressed by normal ductal and acinar cells with typical membranous staining at the intercellular junctions. Loss of normal surface E-cadherin expression was found in 19/36 (53 per cent) tumours compared to the adjacent normal ductal cells. Abnormal E-cadherin expression was found more frequently in poorly differentiated (grade III) (6/7, 86 per cent) than in well-differentiated tumors (grade I) (4/14, 28 per cent) (P = 0.012). Membranous E-cadherin expression was also lost more frequently in primary tumours with lymph node (stage III) (14/23, 61 per cent) and distant metastasis (stage IV) (2/2, 100 per cent) compared with 3/11 (27 per cent) lymph node-negative tumours (stage I) (P = 0.043). In conclusions, our data indicate that loss of membranous E-cadherin expression is associated with high grade and advanced stage in pancreatic cancer.

Biotin-containing proteins are found in all forms of life, and they catalyze carboxylation, decarboxylation, or transcarboxylation reactions that are central to metabolism. In plants, five biotin-containing proteins have been characterized. Of these, four are catalysts, namely the two structurally distinct acetyl-CoA carboxylases (heteromeric and homomeric), 3-methylcrotonyl-CoA carboxylase and geranoyl-CoA carboxylase. In addition, plants contain a noncatalytic biotin protein that accumulates in seeds and is thought to play a role in storing biotin. Acetyl-CoA carboxylases generate two pools of malonyl-CoA, one in plastids that is the precursor for de novo fatty acid biosynthesis and the other in the cytosol that is the precursor for fatty acid elongation and a large number of secondary metabolites. 3-Methylcrotonyl-CoA carboxylase catalyzes a reaction in the mitochondrial pathway for leucine catabolism. The exact metabolic function of geranoyl-CoA carboxylase is as yet unknown, but it may be involved in isoprenoid metabolism. This minireview summarizes the recent developments in our understanding of the structure, regulation, and metabolic functions of these proteins in plants.

When human U937 cells are placed in agarose microbeads and treated with a detergent, the cytoplasmic membrane is lysed and the nuclear membrane is permeabilized. However, the nuclei remain intact and maintain both replication and transcription. Biotin labeled monoclonal antibodies against Z-DNA have been diffused into this system and used to measure the amount of Z-DNA present in the nuclei. It has previously been shown that the amount of Z-DNA present decreases due to relaxation by topoisomerase I and increases as the level of transcription increases. Here we measure the formation of Z-DNA in the c-myc gene by crosslinking the antibodies to DNA using laser radiation at 266 nm for 10 ns. The crosslinked DNA is isolated by restriction digestion, separation of antibody labeled fractions through the biotin residue, and subsequent proteolysis to remove the crosslinked antibody. Three AluI restriction fragments of the c-myc gene are shown to form Z-DNA when the cell is transcribing c-myc. The Z-DNA forming segments are near the promoter regions of the gene. However, when U937 cells start to differentiate and transcription of the c-myc gene is down-regulated, the Z-DNA content goes to undetectable levels within 30-60 min.

Nitric oxide (NO) is a key redox-active, small molecule involved in various aspects of plant growth and development. Here, we report the identification of an NO accumulation mutant, nitric oxide excess1 (noe1), in rice (Oryza sativa), the isolation of the corresponding gene, and the analysis of its role in NO-mediated leaf cell death. Map-based cloning revealed that NOE1 encoded a rice catalase, OsCATC. Furthermore, noe1 resulted in an increase of hydrogen peroxide (H(2)O(2)) in the leaves, which consequently promoted NO production via the activation of nitrate reductase. The removal of excess NO reduced cell death in both leaves and suspension cultures derived from noe1 plants, implicating NO as an important endogenous mediator of H(2)O(2)-induced leaf cell death. Reduction of intracellular S-nitrosothiol (SNO) levels, generated by overexpression of rice S-nitrosoglutathione reductase gene (GSNOR1), which regulates global levels of protein S-nitrosylation, alleviated leaf cell death in noe1 plants. Thus, S-nitrosylation was also involved in light-dependent leaf cell death in noe1. Utilizing the biotin-switch assay, nanoliquid chromatography, and tandem mass spectrometry, S-nitrosylated proteins were identified in both wild-type and noe1 plants. NO targets identified only in noe1 plants included glyceraldehyde 3-phosphate dehydrogenase and thioredoxin, which have been reported to be involved in S-nitrosylation-regulated cell death in animals. Collectively, our data suggest that both NO and SNOs are important mediators in the process of H(2)O(2)-induced leaf cell death in rice.

The cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a common histopathological hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum (ALS/FTD). However, the composition of aggregates and their contribution to the disease process remain unknown. Here we used proximity-dependent biotin identification (BioID) to interrogate the interactome of detergent-insoluble TDP-43 aggregates and found them enriched for components of the nuclear pore complex and nucleocytoplasmic transport machinery. Aggregated and disease-linked mutant TDP-43 triggered the sequestration and/or mislocalization of nucleoporins and transport factors, and interfered with nuclear protein import and RNA export in mouse primary cortical neurons, human fibroblasts and induced pluripotent stem cell-derived neurons. Nuclear pore pathology is present in brain tissue in cases of sporadic ALS and those involving genetic mutations in TARDBP and C9orf72. Our data strongly implicate TDP-43-mediated nucleocytoplasmic transport defects as a common disease mechanism in ALS/FTD.

Redox-switches are critical cysteine thiols that are modified in response to changes in the cell's environment conferring a functional effect. S-nitrosylation (SNO) is emerging as an important modulator of these regulatory switches; however, much remains unknown about the nature of these specific cysteine residues and how oxidative signals are interpreted. Because of their labile nature, SNO-modifications are routinely detected using the biotin switch assay. Here, a new isotope coded cysteine thiol-reactive multiplex reagent, cysTMT(6), is used in place of biotin, for the specific detection of SNO-modifications and determination of individual protein thiol-reactivity. S-nitrosylation was measured in human pulmonary arterial endothelia cells in vitro and in vivo using the cysTMT(6) quantitative switch assay coupled with mass spectrometry. Cell lysates were treated with S-nitrosoglutathione and used to identify 220 SNO-modified cysteines on 179 proteins. Using this approach it was possible to discriminate potential artifacts including instances of reduced protein disulfide bonds (6) and S-glutathionylation (5) as well as diminished ambiguity in site assignment. Quantitative analysis over a range of NO-donor concentrations (2, 10, 20 μm; GSNO) revealed a continuum of reactivity to SNO-modification. Cysteine response was validated in living cells, demonstrating a greater number of less sensitive cysteine residues are modified with increasing oxidative stimuli. Of note, the majority of available cysteines were found to be unmodified in the current treatment suggesting significant additional capacity for oxidative modifications. These results indicate a possible mechanism for the cell to gauge the magnitude of oxidative stimuli through the progressive and specific accumulation of modified redox-switches.

Although biotin is an essential enzyme cofactor found in all three domains of life, our knowledge of its biosynthesis remains fragmentary. Most of the carbon atoms of biotin are derived from pimelic acid, a seven-carbon dicarboxylic acid, but the mechanism whereby this intermediate is assembled remains unknown. Genetic analysis in Escherichia coli identified only two genes of unknown function required for pimelate synthesis, bioC and bioH. We report in vivo and in vitro evidence that the pimeloyl moiety is synthesized by a modified fatty acid synthetic pathway in which the omega-carboxyl group of a malonyl-thioester is methylated by BioC, which allows recognition of this atypical substrate by the fatty acid synthetic enzymes. The malonyl-thioester methyl ester enters fatty acid synthesis as the primer and undergoes two reiterations of the fatty acid elongation cycle to give pimeloyl-acyl carrier protein (ACP) methyl ester, which is hydrolyzed to pimeloyl-ACP and methanol by BioH.

The immunodominant proteins and glycoproteins of Borrelia burgdorferi were analyzed by one-dimensional (1D) and 2D gel electrophoresis. More than 100 polypeptide species could be detected on silver-stained 2D gels. Separation of sonic extracts of the organism by differential centrifugation (100,000 X g) revealed several of the major proteins to reside predominantly within the pellet fraction. The antigenicity of the individual polypeptides was determined by Western (immuno-) blot analysis with sera from humans with chronic Lyme disease and from rabbits immunized with B. burgdorferi. Surface proteins of viable B. burgdorferi labeled with 125I or long-arm hydroxysuccinimide biotin were identified by gel analyses. Thirteen major surface proteins were apparent, including the highly immunogenic 41-kilodalton (kDa) endoflagellar antigen. Two of these proteins, with molecular masses of 22 and 41 kDa, were further characterized by electroblotting and microsequencing their amino termini. Significant (35%) homology between the first 20 amino acids of the 22-kDa protein and the deduced amino acid sequence of the 31-kDa (outer surface protein A) protein of B. burgdorferi may indicate that these proteins are processed similarly or are part of a gene family expressed at the surface of the organism. In addition, highly significant (88%) homology was found between the first nine amino acids of the 41-kDa protein of B. burgdorferi and the 33-kDa endoflagellar protein of Treponema pallidum, after which the sequences diverge. This observation provides in part a structural basis for the observed cross-reactivity between the two organisms and suggests alternative approaches to the development of specific immunodiagnostics.

OBJECTIVE

In 1961, Majno and Palade proposed that plasma leakage in acute inflammation caused by histamine, serotonin, or bradykinin results via gaps that form between endothelial cells of postcapillary venules. Now the relevance of endothelial gaps in plasma leakage is being questioned. The purpose of this review is to summarize experimental evidence from our studies showing that endothelial gaps participate in plasma leakage in inflammation.

METHODS

Using neurogenic inflammation as a model of plasma leakage in acute inflammation, we compared five methods to determine whether endothelial gaps form in the microvasculature of the rat trachea. 1) Endothelial cells borders and gaps were stained with silver nitrate and visualized by light, scanning, and transmission electron microscopy. 2) The luminal surface of endothelial cells was examined by scanning electron microscopy. 3) The luminal surface of endothelial cells was stained with a biotinylated lectin and avidin-biotin-peroxidase histochemistry, and then was examined by differential interference contrast microscopy. 4) Endothelial junctions were reconstructed from serial sections photographed by transmission electron microscopy. 5) Leakage was measured after perfusion of lectins or tracers through aldehyde-fixed vessels in situ.

RESULTS

The results from the five methods used in this system were consistent with the formation of gaps between endothelial cells. Endothelial gaps were rare or absent under baseline conditions, but appeared with the onset of plasma leakage and had a distribution that matched the distribution of leakage. Gaps had a complex morphology and were accompanied by fingerlike cell processes, which may anchor adjacent endothelial cells to one another and participate in gap closure. In contrast to normal vessels, vessels that were leaky in life continued to leak after aldehyde fixation, in evidence that, once formed, the leakage pathway did not require energy-dependent membrane movement or vesicle shuttling. Holes through endothelial cells were less than 1% as frequent as intercellular gaps.

CONCLUSIONS

Taken together, the results show that endothelial gaps are a consistent feature of leaky vessels in the model system we studied, and are not an artifact of a particular method. The morphological complexity of the openings and accompanying fingerlike cell processes and overlapping endothelial cell borders make gaps difficult to distinguish from transcellular holes in thin sections viewed by transmission electron microscopy. However, scanning electron microscopic observations show that most of the openings in leaky venules are intercellular gaps, not transcellular holes. The formation and closure of gaps are likely to be energy-dependent, but the process of plasma leakage is not, provided there is adequate driving force for extravasation. The cellular mechanisms of gap opening and closure remain to be elucidated.

The trypanosomes are a family of parasitic protists of which the African trypanosome, Trypanosoma brucei, is the best characterized. The complex and highly ordered cytoskeleton of T. brucei has been shown to play vital roles in its biology but remains difficult to study, in large part owing to the intractability of its constituent proteins. Existing methods of protein identification, such as bioinformatic analysis, generation of monoclonal antibody panels, proteomics, affinity purification, and yeast two-hybrid screens, all have drawbacks. Such deficiencies-troublesome proteins and technical limitations-are common not only to T. brucei but also to many other protists, many of which are even less well studied. Proximity-dependent biotin identification (BioID) is a recently developed technique that allows forward screens for interaction partners and near neighbors in a native environment with no requirement for solubility in nonionic detergent. As such, it is extremely well suited to the exploration of the cytoskeleton. In this project, BioID was adapted for use in T. brucei. The trypanosome bilobe, a discrete cytoskeletal structure with few known protein components, represented an excellent test subject. Use of the bilobe protein TbMORN1 as a probe resulted in the identification of seven new bilobe constituents and two new flagellum attachment zone proteins. This constitutes the first usage of BioID on a largely uncharacterized structure, and demonstrates its utility in identifying new components of such a structure. This remarkable success validates BioID as a new tool for the study of unicellular eukaryotes in particular and the eukaryotic cytoskeleton in general.

The endocytosis of the T cell differentiation antigen CD4 has been investigated in CD4-transfected HeLa cells, the promyelocytic HL-60 cell line, and in a number of leukemia- or lymphoma-derived T cell lines. CD4 internalization was followed using radioiodinated antibodies in an acid-elution endocytosis assay, or by covalently modifying cell surface proteins with biotin and analyzing CD4 distributions by immunoprecipitation; both approaches gave equivalent results. The assays demonstrated that in transfected HeLa cells and in HL-60 cells CD4 was constitutively internalized and recycled in the absence of ligand. Immunogold labeling and electron microscopy demonstrated that CD4 enters cells through coated pits. In contrast to the nonlymphocytic cells, T cell lines showed very little endocytosis of CD4. Measurements of fluid phase endocytosis and morphometric analysis of the endosome compartment indicated that the endocytic capacities of HeLa and lymphoid cells are equivalent and suggested that the low level of CD4 uptake in lymphocytic cells is due to exclusion of CD4 from coated pits. This conclusion was supported by experiments using truncated CD4 molecules, lacking the bulk of the cytoplasmic domain, which were internalized equally efficiently in both transfected lymphocytes and HeLa cells. Together, these results indicate that the cytoplasmic domain of CD4 mediates the different interactions with the endocytic apparatus in lymphoid and nonlymphoid cells. We suggest that the CD4-associated lymphocyte-specific protein tyrosine kinase p56lck may be involved in preventing CD4 endocytosis in T cells.

Microglia were demonstrated in paraffin-embedded human nervous tissues with an avidin-biotin peroxidase method and Ricinus communis agglutinin-1 (RCA-1). Specific staining was observed in cell bodies and processes of microglia. Although endothelial cells and blood cells reacted with RCA-1, they were easily distinguished morphologically from microglia. Astrocytes, oligodendrocytes, and neurons did not react with RCA-1. These results suggest that RCA-1 can be used as a new histochemical marker for microglia in normal human brain.

Acetyl-CoA carboxylase is found in all animals, plants, and bacteria and catalyzes the first committed step in fatty acid synthesis. It is a multicomponent enzyme containing a biotin carboxylase activity, a biotin carboxyl carrier protein, and a carboxyltransferase functionality. Here we report the X-ray structure of the biotin carboxylase component from Escherichia coli determined to 2.4-A resolution. The structure was solved by a combination of multiple isomorphous replacement and electron density modification procedures. The overall fold of the molecule may be described in terms of three structural domains. The N-terminal region, formed by Met 1-Ile 103, adopts a dinucleotide binding motif with five strands of parallel beta-sheet flanked on either side by alpha-helices. The "B-domain" extends from the main body of the subunit where it folds into two alpha-helical regions and three strands of beta-sheet. Following the excursion into the B-domain, the polypeptide chain folds back into the body of the protein where it forms an eight-stranded antiparallel beta-sheet. In addition to this major secondary structural element, the C-terminal domain also contains a smaller three-stranded antiparallel beta-sheet and seven alpha-helices. The active site of the enzyme has been identified tentatively by a difference Fourier map calculated between X-ray data from the native crystals and from crystals soaked in a Ag+/biotin complex. Those amino acid residues believed to form part of the active site pocket include His 209-Glu 211, His 236-Glu 241, Glu 276, Ile 287-Glu 296, and Arg 338.2+ represents the first X-ray model of a biotin-dependent carboxylase.

The promoters of cell adhesion are ligands, which are often attached to flexible tethers that bind to surface receptors on adjacent cells. Using a combination of Monte Carlo simulations, diffusion reaction theory, and direct experiments (surface force measurements) of the biotin-streptavidin system, we have quantified polymer chain dynamics and the kinetics and spatial range of tethered ligand-receptor binding. The results show that the efficiency of strong binding does not depend solely on the molecular architecture or binding energy of the receptor-ligand pair, nor on the equilibrium configuration of the polymer tether, but rather on its "rare" extended conformations.

Farnesol, a precursor in the isoprenoid/sterol pathway, was recently identified as a quorum-sensing molecule produced by the fungal pathogen Candida albicans. Farnesol is involved in the inhibition of germination and biofilm formation by C. albicans and can be cytotoxic at certain concentrations. In addition, we have shown that farnesol can trigger apoptosis in mammalian cells via the classical apoptotic pathways. In order to elucidate the mechanism behind farnesol cytotoxicity in C. albicans, the response to farnesol was investigated, using proteomic analysis. Global protein expression profiles demonstrated significant changes in protein expression resulting from farnesol exposure. Among the downregulated proteins were those involved in metabolism, glycolysis, protein synthesis, and mitochondrial electron transport and the respiratory chain, whereas proteins involved in folding, protection against environmental and oxidative stress, actin cytoskeleton reorganization, and apoptosis were upregulated. Cellular changes that accompany apoptosis (regulated cell death) were further analyzed using fluorescent microscopy and gene expression analysis. The results indicated reactive oxygen species accumulation, mitochondrial degradation, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) in the farnesol-exposed cells concurrent with increased expression of antioxidant-encoding and drug response genes. More importantly, the results demonstrated farnesol-induced upregulation of the caspase gene MCA1 and the intracellular presence of activated caspases. In conclusion, this study demonstrated that farnesol promotes apoptosis in C. albicans through caspase activation, implying an important physiological role for farnesol in the fungal cell life cycle with important implications for adaptation and survival.

The B vitamins are water-soluble vitamins required as coenzymes for enzymes essential for cell function. This review focuses on their essential role in maintaining mitochondrial function and on how mitochondria are compromised by a deficiency of any B vitamin. Thiamin (B1) is essential for the oxidative decarboxylation of the multienzyme branched-chain ketoacid dehydrogenase complexes of the citric acid cycle. Riboflavin (B2) is required for the flavoenzymes of the respiratory chain, while NADH is synthesized from niacin (B3) and is required to supply protons for oxidative phosphorylation. Pantothenic acid (B5) is required for coenzyme A formation and is also essential for alpha-ketoglutarate and pyruvate dehydrogenase complexes as well as fatty acid oxidation. Biotin (B7) is the coenzyme of decarboxylases required for gluconeogenesis and fatty acid oxidation. Pyridoxal (B6), folate and cobalamin (B12) properties are reviewed elsewhere in this issue. The experimental animal and clinical evidence that vitamin B therapy alleviates B deficiency symptoms and prevents mitochondrial toxicity is also reviewed. The effectiveness of B vitamins as antioxidants preventing oxidative stress toxicity is also reviewed.

Targeted ultrasound contrast materials (gas-filled microbubbles carrying ligands to endothelial selectins or integrins) have been investigated as potential molecular imaging agents. Such microbubbles normally exhibit good targeting capability at the slower flow conditions. However, in the conditions of vigorous flow, binding may be limited. Here, we describe a microbubble capable of efficient binding to targets both in slow and fast flow (exceeding 4 dyne/cm(2) wall shear stress) using a clustered polymeric form of the fast-binding selectin ligand sialyl Lewis(X). Microbubbles were prepared from decafluorobutane gas and stabilized with a monolayer of phosphatidylcholine, PEG stearate and biotin-PEG-lipid. Biotinylated PSLe(x) (sialyl Lewis(X) polyacrylamide) or biotinylated anti-P-selectin antibody (RB40.34) was attached to microbubbles via a streptavidin bridge. In a parallel plate flow chamber targeted adhesion model, PSLe(x) bubbles demonstrated specific adhesion, retention and slow rolling on P-selectin-coated plates. Efficiency of firm targeted adhesion to a P-selectin surface (140 molecules/microm(2)) was comparable for antibody-carrying bubbles and PSLe(x)-targeted bubbles at 0.68 dyne/cm(2) shear stress. At fast flow (4.45 dyne/cm(2)), PSLe(x)-targeted bubbles maintained their ability to bind, while antibody-mediated targeting dropped more than 20-fold. At lower surface density of P-selectin (7 molecules/microm(2)), targeting via PSLe(x) was more efficient than via antibody under all the flow conditions tested. Negative control casein-coated plates did not retain bubbles in the range of flow conditions studied. To confirm echogenicity, targeted PSLe(x)-bubbles were visualized on P-selectin-coated polystyrene plates by ultrasound imaging with a clinical scanner operated in pulse inversion mode; control plates lacking targeted bubbles did not show significant acoustic backscatter. In vivo, in a murine model of inflammation in the femoral vein setting, targeting efficacy of intravenously administered PSLe(x)-microbubbles was comparable with targeting mediated by anti-P-selectin antibody, and significantly exceeded the accumulation of non-targeted control bubbles. In the inflamed femoral artery setting, PSLe(x)-mediated microbubble targeting was superior to antibody-mediated targeting.

Osteocyte apoptosis appears to play a key role in the mechanism by which osteoclastic resorption activity targets bone for removal, because osteocyte apoptosis occurs in highly specific association with microdamage and subsequent remodeling after fatigue. However, beyond terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) assay, little is known about the mechanisms controlling osteocyte apoptosis in vivo. In the current studies, expression of Bax, a proapoptotic gene product, and Bcl-2, an antiapoptotic gene product, was determined in osteocytes of fatigued rat bone using immunocytochemical staining and compared with TUNEL staining patterns. Bax and Bcl-2 were evident in osteocytes by 6 h after loading. Moreover, Bax and Bcl-2 in osteocytes were expressed differently as a function of distance from microdamage sites. The peak of Bax expression and TUNEL+ staining in osteocytes was observed immediately at the microcrack locus, which is where bone resorption occurs in this system; in contrast, Bcl-2 expression, the antiapoptotic signal, reached its greatest level at some distance (1-2 mm) from microcracks. These data suggest that near sites of microinjury in bone, those osteocytes that do not undergo apoptosis are prevented from doing so by active protection mechanisms. Moreover, the zone of apoptotic osteocytes around microcracks was effectively "walled in" by a surrounding halo of surviving osteocytes actively expressing Bc1-2. Thus, the expression pattern of apoptosis-inhibiting gene products by osteocytes surrounding the apoptotic osteocyte at microdamage sites also may provide important signals in the guidance of resorption processes that occur in association with osteocyte apoptosis after fatigue.

We have developed a two-site ELISA for measurement in serum of bone-related degradation products derived from C-terminal telopeptides of type I collagen. The assay is based on the application of two highly specific monoclonal antibodies against the amino acid sequence of AHD-beta-GGR, where the aspartic acid residue (D) is beta-isomerized. In a one-step incubation procedure, the degradation products containing cross-linked diisomerized EKAHD-beta-GGR peptides are captured by a biotinylated antibody and a peroxidase-conjugated antibody. The generated complex is then bound to the streptavidin surface via the biotin conjugate. Desalted urinary antigens are used for standardization, and parallelism is observed with serum samples. Results are obtained in <2.5 h, and both inter- and intraassay imprecision are <8%. The serum CrossLaps concentration was 1748+/-740 pmol/L (mean +/- SD) in premenopausal women (n = 65) and 2952+/-1325 pmol/L in a group of healthy postmenopausal women (n = 169). The Serum CrossLaps One Step ELISA was capable of detecting a highly significant (P <0.001) effect of hormone replacement therapy in a retrospective study involving 22 postmenopausal women.

A cDNA encoding a P450 monooxygenase was amplified from reverse transcribed rat heart and liver total RNA by polymerase chain reaction using primers based on the 5'- and 3'-end sequences of two rat pseudogenes, CYP2J3P1 and CYP2J3P2. Sequence analysis revealed that this 1,778-base pair cDNA contained an open reading frame and encoded a new 502 amino acid protein designated CYP2J3. Based on the deduced amino acid sequence, CYP2J3 was approximately 70% homologous to both human CYP2J2 and rabbit CYP2J1. Recombinant CYP2J3 protein was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 insect cells using a baculovirus expression system. Microsomal fractions of CYP2J3/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolized arachidonic acid to 14,15-, 11,12-, and 8, 9-epoxyeicosatrienoic acids and 19-hydroxyeicosatetraenoic acid as the principal reaction products (catalytic turnover, 0.2 nmol of product/nmol of cytochrome P450/min at 37 degrees C). Immunoblotting of microsomal fractions prepared from rat tissues using a polyclonal antibody raised against recombinant CYP2J2 that cross-reacted with CYP2J3 but not with other known rat P450s demonstrated abundant expression of CYP2J3 protein in heart and liver. Immunohistochemical staining of formalin-fixed paraffin-embedded rat heart tissue sections using the anti-CYP2J2 IgG and avidin-biotin-peroxidase detection localized expression of CYP2J3 primarily to atrial and ventricular myocytes. In an isolated-perfused rat heart model, 20 min of global ischemia followed by 40 min of reflow resulted in recovery of only 44 +/- 6% of base-line contractile function. The addition of 5 microM 11, 12-epoxyeicosatrienoic acid to the perfusate prior to global ischemia resulted in a significant 1.6-fold improvement in recovery of cardiac contractility (69 +/- 5% of base line, p = 0.01 versus vehicle alone). Importantly, neither 14,15-epoxyeicosatrienoic acid nor 19-hydroxyeicosatetraenoic acid significantly improved functional recovery following global ischemia, demonstrating the specificity of the biological effect for the 11, 12-epoxyeicosatrienoic acid regioisomer. Based on these data, we conclude that (a) CYP2J3 is one of the predominant enzymes responsible for the oxidation of endogenous arachidonic acid pools in rat heart myocytes and (b) 11,12-epoxyeicosatrienoic acid may play an important functional role in the response of the heart to ischemia.