Biomedical subjects

T Ugarova

Publications and source records attributed to T Ugarova.

5 recordsLinked to original sources

PubMed2000

Modulating fibroblast adhesion, spreading, and proliferation using self-assembled monolayer films of alkylthiolates on gold.

Ultrathin, highly organized functionalized alkylthiol monolayers were applied as model substrates for cell growth and protein adsorption studies. The aim of this approach was to improve the understanding of molecular surface determinants required for adhesion-dependent cell growth and proliferation using well-controlled surface chemistry. Carboxyl- and methyl-terminated alkylthiol monolayers on gold were used to monitor Swiss 3T3 fibroblast adhesion, spreading, and growth. Stress fiber and focal contact formation were determined by immunostaining of actin filaments and paxillin. Fibronectin deposition and conformation on these surface chemistries in the presence and absence of competing proteins were also determined. The relative levels of adsorbed fibronectin were assessed using radiolabeled proteins. Exposure of the 10th type III cell integrin binding domain of fibronectin was assessed using a radiolabeled monoclonal antibody. Distinct alkylthiol substrate chemistry-dependent differences were observed in fibroblast adhesion, spreading, and growth. The formation of focal contacts and stress fibers was enhanced on the carboxyl-terminated surface relative to the methyl surface. Relative deposition and conformations of adsorbed fibronectin were shown to be dependent on surface chemistry in both the presence and absence of competing proteins. The results indicated that well-controlled culture surfaces modulate differential cell adhesion, spreading, and growth through modulations of the amounts and conformations of adsorbed extracellular matrix molecules (e.g., fibronectin).

3T3 Cells↗

PubMed2000

Alpha 4 integrin-dependent leukocyte recruitment does not require VCAM-1 in a chronic model of inflammation.

Rats immunized with Mycobacterium butyricum in Freund's adjuvant develop a chronic vasculitis, with large increases in leukocyte rolling and adhesion in mesenteric postcapillary venules that are significantly inhibited with an alpha 4 integrin Ab. Using intravital microscopy to visualize chronically inflamed microvessels, we demonstrated that alpha 4 integrin-dependent leukocyte rolling and adhesion was inhibited with a beta 1 integrin, but not a beta 7 integrin Ab. To date, VCAM-1 has been presumed to be the primary ligand for alpha 4 beta 1 integrin in the vasculature. However, alpha 4 beta 1 integrin-dependent interactions were not reduced by monoclonal or polyclonal VCAM-1 Abs or a VCAM-1 antisense oligonucleotide despite increased VCAM-1 expression in the mesenteric vasculature. To ensure that the VCAM-1 Abs were functional and used at saturating concentrations, blood from Ab-treated rats was perfused over monolayers of CHO cells transfected with rat VCAM-1. Sufficient alpha 4 integrin or VCAM-1 Ab was present to inhibit leukocyte interactions with rat VCAM-1 by 95-100%. Under in vitro flow conditions, only mononuclear leukocytes were recruited from blood of control rats onto purified VCAM-1. However, neutrophils were also recruited onto VCAM-1 from whole blood of adjuvant-immunized animals via alpha 4 integrin. Another ligand for alpha 4 beta 1 integrin is the connecting segment-1 (CS-1) region of fibronectin. An Ab to the CS-1 portion of fibronectin, which did not reduce rolling and adhesion in adjuvant arthritis animals, completely inhibited leukocyte adhesion to CS-1 under static conditions. These findings provide the first evidence that alpha 4 beta 1 integrin-dependent leukocyte rolling and adhesion can occur in vivo via a mechanism other than VCAM-1.

Adjuvants, Immunologic↗

PubMed1997

Domain structure and functional activity of the recombinant human fibrinogen gamma-module (gamma148-411).

Human fibrinogen gamma-module comprising residues gamma148-411 was expressed in Escherichia coli and refolded in vitro. Differential scanning calorimetry revealed that in addition to the two previously identified independently folded thermolabile domains, one in each half of the module, the gamma-module also contains one or two thermostable domains that melt above 65 degrees C. To localize the latter, an NH2-terminal 6-kDa fragment was prepared by limited proteolysis of the recombinant gamma-module. It melted at high temperature, indicating that this portion is folded into a compact structure that represents a thermostable domain, also identified in the proteolytic fibrinogen fragment D1 which contains the natural gamma-module. Thus the NH2-terminal half of the gamma-module forms two domains, a thermostable one and a thermolabile one, leaving the rest of the module to be responsible for the formation of the other one or two domains. The thermal stability of some domains was lower in the recombinant gamma-module than in its natural counterpart in D1, reflecting most probably the loss of interactions with neighboring domains; however, the major functional sites were essentially preserved. The module bound Ca2+ and was stabilized by it against denaturation and proteolysis. It inhibited fibrin polymerization and was efficiently cross-linked by factor XIIIa. The gamma-module supported adhesion of platelets via their GP IIbIIIa (alpha(IIb)beta3) receptor in the same manner as D1 fragment. It also supported the adhesion of alpha(M)beta2- (Mac-1-) transfected cells and in the fluid phase was more effective than D1 as an inhibitor of that adhesion, suggesting that the Mac-1 binding site is better exposed.

Calorimetry, Differential Scanning↗

PubMed1995

Conformational changes in adhesive proteins modulate their adhesive function.

A large family of glycoproteins mediates cell adhesion by binding to cellular receptors. As a group, these adhesive proteins are large in size, multi-domainal in composition, and are capable of self-association. A general property of the adhesive proteins is their susceptibility to structural modulation, and conformational change provides a mechanism for regulation of their adhesive functions. To illustrate this concept, conformational alterations of thrombospondin, fibrinogen and fibronectin are shown to modulate their adhesive potential. Thus, conformational status of the adhesive proteins contributes to the regulation of cell adhesion.

Cell Adhesion↗

PubMed1990

Electron microscope investigation of the early stages of fibrin assembly. Twisted protofibrils and fibers.

Structures formed during the early stages of clot formation have been produced in a controlled manner by polymerization of soluble fibrin monomers prepared from dissolved normal clots that had been formed upon addition of thrombin to fibrinogen. In agreement with other studies using different approaches, electron microscopy of negatively contrasted or rotary-shadowed specimens of these preparations reveal two-stranded protofibrils, as well as shorter oligomers and fibrin monomers. Individual fibrin molecules are similar in appearance to fibrinogen, suggesting that no large-scale changes in conformation occur on removal of the fibrinopeptides. Moreover, these micrographs show details of the protofibril structure not previously seen. The visualization of clear cross-over points of the filaments making up the protofibril indicate that these structures are twisted. Diffraction patterns of electron micrographs of both protofibrils and fibers and computer modeling of protofibrils also suggest that these structures are twisted but not precisely ordered.

Animals↗