A mixture of lysophosphatidylcholines (lyso-PCs) are generated during blood storage and are etiologic in models of acute lung injury. We hypothesize that lyso-PCs stimulate polymorphonuclear neutrophils (PMNs) through Ca(2)(+)-dependent signaling. The lyso-PC mix (0.45-14.5 micro M) and the individual lyso-PCs primed formyl-Met-Leu-Phe (fMLP) activation of the oxidase (1.8- to 15.7-fold and 1.7- to 14.8-fold; P<0.05). Labeled lyso-PCs demonstrated a membrane association with PMNs and caused rapid increases in cytosolic Ca(2)(+). Receptor desensitization studies implicated a common receptor or a family of receptors for the observed lyso-PC-mediated changes in PMN priming, and cytosolic Ca(2)(+) functions were pertussis toxin-sensitive. Lyso-PCs caused rapid serine phosphorylation of a 68-kD protein but did not activate mitogen-activated protein kinases or cause changes in tyrosine phosphorylation. With respect to alterations in PMN function, lyso-PCs caused PMN adherence, increased expression of CD11b and the fMLP receptor, reduced chemotaxis, provoked changes in morphology, elicited degranulation, and augmented fMLP-induced azurophilic degranulation (P<0.05). Cytosolic Ca(2)(+) chelation inhibited lyso-PC-mediated priming of the oxidase, CD11b surface expression, changes in PMN morphology, and serine phosphorylation of the 68-kD protein. In conclusion, lyso-PCs affect multiple PMN functions in a Ca(2)(+)-dependent manner that involves the activation of a pertussis toxin-sensitive G-protein.

An 8 kDa ubiquitin-like peptide (ULP) was isolated by high performance liquid chromatography from the rabbit vitreous humor, and the N-terminal amino acid sequence of this peptide showed complete homology with ubiquitin. Western blot revealed the presence of free ULP in both the iris-ciliary (IC) complex and the aqueous humor extracts. In the IC complex, fluorescence and immunoelectron microscopy detected high concentrations of ULP in the posterior epithelial cells, suggesting this tissue as a possible source of ULP in the ocular fluids. Significantly, this is the first time that the presence of free ULP has been reported in mammalian extracellular fluids. Furthermore, we recently demonstrated that the 8 kDa fraction of vitreous humor containing ULP is a potent inhibitor of protein synthesis [Banerjee et al. (1992): J Cell Biochem 49:66-73]. These findings taken together suggest a novel biological role for ULP in the control of lens cell growth.

Calmodulin is believed to be the most important mediator of Ca2+-dependent signaling in eucarytic cells and is thought to play an essetial role in processes like cell proliferation and growth. Calmodulin is therefore a target for certain drugs and consequently a target of drug design experiments.The structures of calmodulin as well as its complexes with a number of substrates has received widespread attention recently. This is because calmodulin plays a regulatory role in a number of processes by transforming the value of the intracellular Ca2+ -ion concetration into a more structured information. If Ca2+ concentration increases than two hydrophobic binding pockets of calmodulin become exposed facilitating the binding of certain amphilphilic regulatory helices of at least 30 different proteins of high biological importance. Calmodulin mediated enzyme activation can be efficiently inhibited by a number of pharmacological agents (antipsychotics, antidepressants, muscle relaxants etc.). Crystal stmcture of TFP (a potent antipsychotic phenothiazine type drug, kind of a reference molecule on the area) with calmodulin is known from the literature!. This shows that due to the effect of the small molecular antagonist TFP. calmoduline undergoes a very similar conformational change to that it suffers when it binds regulatory oligopeptide pieces of proteins normally regulated by calmodulin.We have recently shown that drugs fairly different form TFP result in very similar calmodulin conformational changes. Fmthennore we have also demonstrated that at low, physiologically relevant TFP concentrations the binding of the second TFP occurs in the C-terminal domain of calmodulin, unlike it was proposed earlier. A quaterner complex of calmoduline with Ca2+ and two different type of drugs has also been investigated resulting in some new insights into the inactivation of calmodulin.Two proteins that represent promising drug design targets against human pathogens are the major ferric iron binding protein (FBP) from Neisseria and Haemophilus, and glutathione-Stransferase (GST) from Schistosoma. These proteins are both members of structural families for which several crystal structures are already known, pennitting rational design of inhibitors specific for particular family members.FBPs from two species of pathogenic bacte1ia have been crystallized. FBP crystals from Haemophilus inf/uen:::.ae (which causes many infections, including meningitis) diffract X-rays to 1.6 A resolution. Anomalous scattering from these crystals unambiguously reveals the position of the iron atom at the active site. Crystals of FBP from [!eisseria gonorrhoeae (which causes gononhea) diffract to 2.8 A. Crystallographic determination of these protein structures is underway.The crystal structure of GST from Schistosoma japonicum has previously been detem1ined, both in native fonn and in complex with praziquantel, the leading drug used to treat schistosomiasis (McTigue et al 1995). We are working to evaluate other potential inhibitors by X-ray crystallography.Diff...