Ophioluxin,a convulxin-like C-type lectin from Ophiophagus hannah (King cobra) is a powerful platelet activator via glycoprotein VI

Ophioluxin, a potent platelet agonist, was purified from the venom of Ophiophagus hannah (King cobra). Under nonreducing conditions it has a mass of 85 kDa, similar to convulxin, and on reduction gives two subunits with masses of 16 and 17 kDa, slightly larger than those of convulxin. The N-terminal sequences of both subunits are very similar to those of convulxin and other C-type lectins. Ophioluxin induces a pattern of tyrosine-phosphorylated proteins in platelets like that caused by convulxin, when using appropriate concentrations based on aggregation response, because it is about 2-4 times more powerful as agonist than the latter. Ophioluxin and convulxin induce [Ca(2+)](i) elevation both in platelets and in Dami megakaryocytic cells, and each of these C-type lectins desensitizes responses to the other. Convulxin agglutinates fixed platelets at 2 microg/ml, whereas ophioluxin does not, even at 80 microg/ml. Ophioluxin resembles convulxin more than echicetin or alboaggregin B because polyclonal anti-ophioluxin antibodies recognize both ophioluxin and convulxin, but not echicetin, and platelets adhere to and spread on ophioluxin- or convulxin-precoated surfaces in the same way that is clearly different from their behavior on an alboaggregin B surface. Immobilized ophioluxin was used to isolate the glycoprotein VI-Fcgamma complex from resting platelets, which also contained Fyn, Lyn, Syk, LAT, and SLP76. Ophioluxin is the first multiheterodimeric, convulxin-like snake C-type lectin, as well as the first platelet agonist, to be described from the Elapidae snake family.     

Annexin V-CLIO: a nanoparticle for detecting apoptosis by MRI

Annexin V, which recognizes the phosphatidylserine of apoptotic cells, was conjugated to crosslinked iron oxide (CLIO) nanoparticles, a functionalized superparamagnetic preparation developed for target-specific magnetic resonance imaging (MRI). The resulting nanoparticle had an average of 2.7 annexin V proteins linked per CLIO nanoparticle through disulfide bonds. Using camptothecin to induce apoptosis, a mixture of Jurkat T cells (69% healthy and 31% apoptotic) was incubated with annexin V-CLIO and was applied to magnetic columns. The result was an almost complete removal of the apoptotic cells (> 99%). In a phantom MRI experiment, untreated control cells (12% apoptotic cells, 88% healthy cells) and camptothecin-treated cells (65% apoptotic cells, 35% healthy cells) were incubated with either annexin V-CLIO (1.0, 0.5, and 0.1 microgram Fe/mL) or with unlabeled CLIO. A significant signal decrease of camptothecin-treated cells relative to untreated cells was observed even at the lowest concentration tested. Unmodified CLIO failed to cause a significant signal change of apoptotic cells. Hence, annexin V-CLIO allowed the identification of cell suspensions containing apoptotic cells by MRI even at very low concentrations of magnetic substrate. Conjugation of annexin V to CLIO affords a strategy for the development of a MRI imaging probe for detecting apoptosis.     

Oligomerization of paramagnetic substrates result in signal amplification and can be used for MR imaging of molecular targets

Magnetic resonance imaging (MRI) has evolved into a sophisticated, noninvasive imaging modality capable of high-resolution anatomical and functional characterization of transgenic animals. To expand the capabilities MRI, we have developed a novel MR signal amplification (MRamp) strategy based on enzyme-mediated polymerization of paramagnetic substrates into oligomers of higher magnetic relaxtivity. The substrates consist of chelated gadolinium covalently bound to phenols, which then serve as electron donors during enzymatic hydrogen peroxide reduction by peroxidase. The converted monomers undergo rapid condensation into paramagnetic oligomers leading to a threefold increase in atomic relaxtivity (R1/Gd). The observed relaxtivity changes are largely due to an increase in the rotational correlation time tau r of the lanthanide. Three applications of the developed system are demonstrated: (1) imaging of nanomolar amounts of an oxidoreductase (peroxidase); (2) detection of a model ligand using an enzyme-linked immunoadsorbent assay format; and (3) imaging of E-selectin on the surface of endothelial cells probed for with an anti-E-selectin-peroxidase conjugate. The development of "enzyme sensing" probes is expected to have utility for a number of applications including in vivo detection of specific molecular targets. One particular advantage of the MRamp technique is that the same paramagnetic substrate can be potentially used to identify different molecular targets by attaching enzymes to various antibodies or other target-seeking molecules.     

Fluorescence data analysis on gel-based biochips

A series of biochip readers developed for gel-based biochips includes three imaging models and a novel nonimaging biochip scanner. The imaging readers, ranging from a research-grade versatile reader to a simple portable one, use wide-field objectives and 12-bit digital large-coupled device cameras for parallel addressing of multiple array elements. This feature is valuable for monitoring the kinetics of sample interaction with immobilized probes. Depending on the model and the label used, the sensitivity of these readers approaches 0.3 amol of a labeled sample per gel element. In the selective scanner, both the spot size of the excitation laser beam and the detector field of view match the size of the biochip array elements so that the whole row of the array can be read in a single scan. The portable version reads 50-mm long, 150-element, one-dimensional arrays in 5 s. With a dynamic range of 4000:1, a sensitivity of 1-5 amol of a labeled sample per gel element, and a data format facilitating online processing, the scanner is an attractive, inexpensive solution for biomedical diagnostics. Fluorophores for sample labeling were compared experimentally in terms of detection sensitivity, influence on duplex stability, and suitability for multilabel analysis and thermodynamic studies. Texas Red and tetracarboxyphenylporphyn proved to be the best choice for two-wavelength analysis using the imaging readers.     

Muscle electrolyte measurements during and after hypokinesia in determining muscle electrolyte depletion during hypokinesia in the rat

Hypokinesia (diminished movement) induces muscle mineral depletion. However, the mechanism of muscle mineral depletion during hypokinesia (HK) remains unknown. Measuring electrolyte retention and electrolyte values in muscle, plasma, and urine during and after HK, the aim of this study was to discover if HK could depress mineral retention and lead to muscle mineral depletion. Studies were done on 204 13-wk-old male Wistar rats (370–390 g) during 10 d pre-HK period, 98 d HK period, and 15 d post-HK period. Rats were equally divided into two groups: vivarium control rats (VCR) and hypokinetic rats (HKR). All hypokinetic rats were kept for 98 d in small individual cages, which restricted their movements in all directions without hindering food and water intakes. All control rats were housed for 98 d in individual cages under vivarium control conditions. Both groups of rats were pair-fed. During the HK period skeletal muscle sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), and water content and electrolyte retention decreased significantly (p < 0.05), while urinary and plasma electrolyte levels increased significantly (p < 0.05) in HKR compared with their pre-HK values and their respective VCR. During the initial days of the post-HK period, mineral retention increased significantly (p < 0.05), plasma and urinary electrolyte level decreased significantly (p < 0.05), while muscle electrolyte and water content remained significantly (p < 0.05) depressed in HKR compared with VCR. Muscle mineral and water content, electrolyte retention, plasma, and urinary electrolyte values did not change in VCR compared with their pre-HK values. It was concluded that during HK decreased muscle mineral content may suggest muscle mineral depletion, while increased urinary electrolyte loss and muscle mineral depletion may demonstrate reduced mineral retention. Reduced electrolyte excretion and depressed muscle mineral content during post-HK may indicate skeletal muscle mineral depletion during HK. Dissociation between electrolyte retention and muscle mineral depletion may demonstrate the presence of decreased electrolyte retention as the mechanism of muscle electrolyte depletion during prolonged HK.     

Increase of sensitivity of sputum cytology using high-resolution image cytometry: field study results

Lung cancer remains the leading cause of cancer deaths in the developed world. There is no widely accepted method to screen for this cancer. The most commonly used method remains conventional sputum cytology, but this method is hampered by low sensitivity. We tested the hypothesis that sensitivity of sputum cytology for early lung cancer can be greatly improved by using image analysis of sputum cells, at a modest reduction of specificity.The study was double-blinded and used sputum samples from subjects with well-characterized clinical diagnoses. There were 177 cancers, 98 dysplasias, and 558 normals. The study samples were separated into two independent sets: training set and test set. Sputum samples were collected prospectively from subjects with a high probability of having lung cancer. Seven institutions from five countries participated in the study. All subjects had complete clinical diagnoses which included, as a minimum, negative chest x-rays for all negative cancers, while all cancers had confirmed tissue pathology. Samples were prepared according to the Saccomanno method. For conventional cytology, slides were stained using Papanicolaou stain. For image analysis, slides were stained using a DNA-specific (Feulgen-Thionin) stain. An automated, high-resolution image cytometer was used for measurements.At 90% specificity, sensitivity of 60% can be achieved for adenocarcinoma, compared to only 14% sensitivity of conventional cytology (at 99% specificity). Similarly, 45% sensitivity at 90% specificity can be reached for stages 0 and I lung cancer, compared to only 14% (at 99% specificity) using conventional cytology.Cytometry combined with conventional cytology shows an increase in sensitivity to early-stage cancer and to adenocarcinomas compared to conventional cytology alone. While the results are encouraging, the sensitivity to detect early lung cancer should be further improved to 70-80% at 90-95% specificity before this test can be considered for screening of high-risk individuals for lung cancer. Cytometry (Clin. Cytometry) 50:168-176, 2002.     

Redundancy in tumor necrosis factor (TNF) and lymphotoxin (LT) signaling in vivo: mice with inactivation of the entire TNF/LT locus versus single-knockout mice

Homologous genes and gene products often have redundant physiological functions. Members of the tumor necrosis factor (TNF) family of cytokines can signal activation, proliferation, differentiation, costimulation, inhibition, or cell death, depending on the type and status of the target cell. TNF, lymphotoxin alpha (LTalpha), and LTbeta form a subfamily of a larger family of TNF-related ligands with their genes being linked within a compact 12-kb cluster inside the major histocompatibility complex locus. Singly TNF-, LTalpha-, and LTbeta-deficient mice share several phenotypic features, suggesting that TNF/LT signaling pathways may regulate overlapping sets of target genes. In order to directly address the issue of redundancy of TNF/LT signaling, we used the Cre-loxP recombination system to create mice with a deletion of the entire TNF/LT locus. Mice with a triple LTbeta/TNF/LTalpha deficiency essentially manifest a combination of LT and TNF single-knockout phenotypes, except for microarchitecture of the spleen, where the disorder of lymphoid cell positioning and functional T- and B-cell compartmentalization is severer than that found in TNF or LT single-knockout mice. Thus, our data support the notion that TNF and LT have largely nonredundant functions in vivo.     

The role of lymphotoxin in development and maintenance of secondary lymphoid tissues

Secondary lymphoid organs provide the necessary microenvironment for the cooperation of antigen-specific T- and B-lymphocytes and antigen-presenting cells in order to initiate an efficient immune response. Remarkable progress in understanding of the mechanisms of lymphoid organogenesis was achieved due to the analysis of various gene-targeted mice. This review primarily focuses on the role of lymphotoxin (LT) in development, maturation and maintenance of secondary lymphoid organs.     

Bioinformatics classification and functional analysis of PhoH homologs

PhoH protein is a putative ATPase belonging to the phosphate regulon in Escherichia coli. EC-PhoH homologs are present in different organisms, but it is not clear if they are functionally related, besides nothing is known about their regulation. To distinguish true functional orthologs of EC-PhoH in different classes of bacteria and to identify their functional role in bacterial metabolic network we performed phylogenetic analysis of these proteins and comparative study of position and regulation of the related genes. Three groups of proteins were identified. Proteins of the first group (BS-PhoH orthologs) are present in most of bacteria and are proposed to be functionally linked to phospholipid metabolism and RNA modification. Proteins of the second group (BS-YlaK orthologs) are present in most of aerobes and Actinobacterial YlaK orthologs are shown to be members of a fatty acid beta-oxidation regulons. EC-PhoH orthologs are classified in a third group, specific for Enterobacteria. Functional role of PhoH homologs in the lipid and RNA metabolism and proposed interrelation of PhoH paralogs in one organism are discussed.