Atomic force microscopy measurement of leukocyte-endothelial interaction

Leukocyte adhesion to vascular endothelium is a key initiating step in the pathogenesis of many inflammatory diseases. In this study, we present real-time force measurements of the interaction between monocytic human promyelocytic leukemia cells (HL-60) cells and a monolayer of human umbilical vein endothelial cells (HUVECs) by using atomic force microscopy (AFM). The detachment of HL-60-HUVEC conjugates involved a series of rupture events with force transitions of 40-100 pN. The integrated force of these rupture events provided a quantitative measure of the adhesion strength on a whole cell level. The AFM measurements revealed that HL-60 adhesion is heightened in the borders formed by adjacent HUVECs. The average force and mechanical work required to detach a single HL-60 from the borders of a tumor necrosis factor-alpha-activated HUVEC layer were twice as high as those of the HUVEC bodies. HL-60 adhesion to the monolayer was significantly reduced by a monoclonal antibody against beta1-integrins and partially inhibited by antibodies against selectins ICAM-1 and VCAM-1 but was not affected by anti-alphaVbeta3. Interestingly, adhesion was also inhibited in a dose-dependent manner (IC50 approximately 100 nM) by a cyclic arginine-glycine-aspartic acid (cRGD) peptide. This effect was mediated via interfering with the VLA-4-VCAM-1 binding. In parallel measurements, transmigration of HL-60 cells across a confluent HUVEC monolayer was inhibited by the cRGD peptide and by both anti-beta1 and anti-alphaVbeta3 antibodies. In conclusion, these data demonstrate the role played by beta1-integrins in leukocyte-endothelial adhesion and transmigration and the role played by alphaVbeta3 in transmigration, thus underscoring the high efficacy of cRGD peptide in blocking both the adhesion and transmigration of monocytes.     

Simultaneous cytomegalovirus infection and Kaposi's sarcoma of the thyroid diagnosed by fine needle aspiration in an AIDS patient. A case report and first cytologic description of the two entities occurring together

BACKGROUND: Cytomegalovirus (CMV) infection and Kaposi's sarcoma (KS) are common in AIDS patients but rarely involve the thyroid, and coexistence of these two entities in that organ has not yet been described before. CASE: A 41-year-old female AIDS patient presented with a 2 x 1-cm, well-demarcated, rubbery mass in the right side of the thyroid. On fine needle aspiration (FNA), spindle cells were retrieved singly or in small, loose clusters; they had bland, fusiform to cigar-shaped nuclei; inconspicuous nucleoli; delicate cytoplasmic vacuoles; cytoplasmic hyaline drops; and hemosiderin granules. A single endothelial cell showed an enlarged nucleus with a basophilic intranuclear inclusion and periinclusional halo. CONCLUSION: This is the first reported case of an AIDS patient with KS and CMV infection simultaneously involving the thyroid diagnosed by FNA.     

Frequency and coverage of trinucleotide repeats in eukaryotes

In the aim to assess whether the tri-repeat shortage reported in vertebrates affects specific motifs, such as those causing neuromuscular diseases in man, we detected approximate di-, tri- and tetra-repeats (STR) longer than 25 bases in human chromosomes 21 and 22, and in some model organisms (M. musculus, D. melanogaster, C. elegans, A. thaliana and S. cerevisiae). We found that overall STR are more represented in mouse and in man than in the other organisms. However, tri-repeats are less represented than di- and tetra- in man and mouse, but show intermediate values between di- and tetra- in the other organisms. In man, ACG shows the lowest both frequency and coverage, ATC the highest coverage and AAT the highest frequency. In general, coverage and frequency of tri-repeats are linearly related, except for ACC, ATC, AAG, AGG motifs in man and AAG, AGG in mouse, which exhibit unexpectedly long repeats. Often their copy numbers exceed that found responsible for the dynamic mutations, set at around 40. The shortage in frequency and coverage of tri- vs. di- and tetra-repeats observed in man and mouse can be ascribed to a subset of the remaining tri-repeat motifs, but among them those recognized as dynamically mutable (AAG, AGC and CCG) are not the least represented. Possible constraints in tri-repeat expansion seem to be structural and conserved along the evolutionary scale: a motif-specific relaxation of the relevant controls may be responsible for the occasional expansions found in mouse and man.     

Preliminary description of lesions in juvenile largemouth bass injected with largemouth bass virus

Juvenile largemouth bass Micropterus salmoides were intraperitoneally injected with largemouth bass virus (LMBV), a member of the genus Ranavirus, family Iridoviridae. Moribund fish which had been injected with 10(6.2) tissue culture infectious doses, 50% endpoint (TCID50), were sampled 4 d after injection; other largemouth bass injected with this dose died between 3 and 5 d after injection. Fish injected with 10(2.8) TCID50 of LMBV were also examined after 4 d and had lesions similar to those of fish injected with the high dose. Clinical signs included darker pigmentation, inflammation and necrosis at the site of injection, distended abdomen, corkscrew swimming, and lateral recumbency. Internally, fish had focally pale livers, bright red spleens and reddened intestinal ceca. Histologically acute fibrinous peritonitis affected the surface of all organs in the peritoneal cavity, but deeper portions of organs appeared normal. There was also necrosis of the gastrointestinal mucosa. Except for the injection site, lesions were confined to the peritoneal cavity.     

Acetabularia: A Unicellular Model for Understanding Subcellular Localization and Morphogenesis during Development

Acetabularia acetabulum is the organism that provided the first compelling experimental evidence both for the role of an organelle whose function was unknown, the nucleus, and for the existence of "morphogenetic substances," the behavior of which presaged the discovery of mRNA in other organisms. This giant unicell holds special appeal as a model system, because the contribution of its diploid nucleus to cellular processes can be assessed using simple amputation and grafting experiments and because it lends itself to a wide range of methods in cell, molecular, and developmental biology. It remains an excellent model system for understanding how body regions are functionally and structurally distinguished from each other without cellular compartmentation. Advances in genetics (that is, mutant selection and analysis, large-scale transformation) will greatly increase the power of this system to address fundamental questions in development and morphogenesis. We discuss strengths and weaknesses of the system and outline the body of knowledge that would make the system more powerful and broadly appealing.     

Size-exclusion chromatographic study of the reduction of recombinant hepatitis B surface antigen

The reduction of the P. pastoris-derived hepatitis B surface antigen (HBsAg) has been investigated by size exclusion chromatography performed in a detergent solution containing 0.3% sodium dodecyl sulfate (SDS) and 0.1 M Tris–HCl, pH 7.0. The HBsAg, reduced under different conditions and passed through the TSK G4000 SW column (600×7.5 mm I.D.) at 0.9 ml min⁻¹, was resolved into two peaks corresponding to the reduced, monomeric, and non-reduced forms, respectively. Under these conditions, the antigen fraction corresponding to the HBsAg dimer can be separated and completely reduced to monomers by repeated reductive treatment with simultaneous lipid removal. The efficiency of reduction was maximal after sample treatment with an equal volume of a solution containing 417 mM dithiothreitol, 4.2% (w/v) SDS and 16% (v/v) 2-mercaptoethanol. In conclusion, complete reduction of recombinant HBsAg to monomer subunits is possible and depends on the efficiency of lipid removal during the reductive treatment.     

Integrative structural modeling with small angle X-ray scattering profiles

Recent technological advances enabled high-throughput collection of Small Angle X-ray Scattering (SAXS) profiles of biological macromolecules. Thus, computational methods for integrating SAXS profiles into structural modeling are needed more than ever. Here, we review specifically the use of SAXS profiles for the structural modeling of proteins, nucleic acids, and their complexes. First, the approaches for computing theoretical SAXS profiles from structures are presented. Second, computational methods for predicting protein structures, dynamics of proteins in solution, and assembly structures are covered. Third, we discuss the use of SAXS profiles in integrative structure modeling approaches that depend simultaneously on several data types.     

Macromolecular docking restrained by a small angle X-ray scattering profile

While many structures of single protein components are becoming available, structural characterization of their complexes remains challenging. Methods for modeling assembly structures from individual components frequently suffer from large errors, due to protein flexibility and inaccurate scoring functions. However, when additional information is available, it may be possible to reduce the errors and compute near-native complex structures. One such type of information is a small angle X-ray scattering (SAXS) profile that can be collected in a high-throughput fashion from a small amount of sample in solution. Here, we present an efficient method for protein–protein docking with a SAXS profile (FoXSDock): generation of complex models by rigid global docking with PatchDock, filtering of the models based on the SAXS profile, clustering of the models, and refining the interface by flexible docking with FireDock. FoXSDock is benchmarked on 124 protein complexes with simulated SAXS profiles, as well as on 6 complexes with experimentally determined SAXS profiles. When induced fit is less than 1.5 Å interface C_α RMSD and the fraction residues of missing from the component structures is less than 3%, FoXSDock can find a model close to the native structure within the top 10 predictions in 77% of the cases; in comparison, docking alone succeeds in only 34% of the cases. Thus, the integrative approach significantly improves on molecular docking alone. The improvement arises from an increased resolution of rigid docking sampling and more accurate scoring.