Selective expression of a splice variant of decay-accelerating factor in c-erbB-2-positive mammary carcinoma cells showing increased transendothelial invasiveness

By differential-display-PCR a subclone of the SK-BR-3 cell line with high in vitro transendothelial invasiveness was identified to express increased levels of a new alternative splice variant of decay-accelerating factor (DAF). DAF seems to play an important role in some malignant tumours since on the one hand the expression of complement inhibitors on the surface of tumour cells prevents the accumulation of complement factors and in consequence cell lysis. On the other hand, DAF has been identified as a ligand for the CD97 surface receptor which induces cell migration. Immunofluorescence procedures, Western blot analyses, and cDNA clone sequencing were employed to confirm the expression of DAF restricted to invasive tumour cells. Using a radioactive RNA-in situ hybridisation on freshly frozen tissue microarrays and RT-PCR on native tumour tissue, the expression of alternative spliced DAF mRNA was demonstrated in invasive breast cancer. Due to the fact that it could thereby not be detected in normal mammary tissues, it has to be confirmed in larger studies that the DAF splice variant might be a specific tumour marker for invasive breast cancer.     

Poly(azolyl) chelate chemistry 14.[1] κ-S,S′ vs κ-H,S,S′- H2B(mt)2 (mt=methimazolyl)borate coordination in the complex [W(CO){H2B(mt)2}2]

The reactions of [M(CC₆H₂Me₃-2,4,6)X(CO)₂(L)₂] (M = Mo, W; X = Cl, Br; L = pyridine, 3,5-dimethylpyrazole) with Na[H₂B(mt)₂] (mt = methimazolyl) are metal dependent, providing either the alkylidyne complex [Mo(CC₆H₂Me₃-2,4, 6)(CO)₂{κ³-H,S,S′-H₂B(mt)₂}] or the bis(chelate) complex [W(CO){κ²-S,S′-H₂B(mt)₂}{κ³-H,S,S′-H₂B(mt)₂}], the latter featuring both bi and tridentate coordination modes for the H₂B(mt)₂ ligand.     

Transition state stabilization by six arginines clustered in the active site of creatine kinase

Six fully conserved arginine residues (R129, R131, R235, R291, R319, and R340) closely grouped in the nucleotide binding site of rabbit muscle creatine kinase (rmCK) were mutated; four to alanine and all six to lysine. Kinetic analyses in the direction of phosphocreatine formation showed that all four alanine mutants led to substantial losses of activity with three (R129A, R131A, and R235A) having no detectable activity. All six lysine mutants retained variable degrees of reduced enzymatic activity. Static quenching of intrinsic tryptophan fluorescence was used to measure the binding constants for MgADP and MgATP. Nucleotide binding was at most only modestly affected by mutation of the arginine residues. Thus, the cluster of arginines seem to be primarily responsible for transition state stabilization which is further supported by the observation that none of the inactive mutants demonstrated the ability to form a transition analogue complex of MgADP.nitrate.creatine as determined by fluorescence quenching assays. As a whole, the results suggest that the most important role these residues play is to properly align the substrates for stabilization of the phosphoryl transfer reaction.     

CD8 kinetically promotes ligand binding to the T-cell antigen receptor

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.     

CD8+ cytotoxic T lymphocyte activation by soluble major histocompatibility complex-peptide dimers

CD8+ cytotoxic T lymphocyte (CTL) can recognize and kill target cells that express only a few cognate major histocompatibility complex class I-peptide (pMHC) complexes. To better understand the molecular basis of this sensitive recognition process, we studied dimeric pMHC complexes containing linkers of different lengths. Although dimers containing short (10-30-A) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers containing long linkers (> or = 80 A) did not. Based on this and on fluorescence resonance energy transfer experiments, we describe a dimeric binding mode in which two T cell receptors engage in an anti-parallel fashion two pMHC complexes facing each other with their constant domains. This binding mode allows integration of diverse low affinity interactions, which increases the overall binding and, hence, the sensitivity of antigen recognition. In proof of this, we demonstrated that pMHC dimers containing one agonist and one null ligand efficiently activate CTL, corroborating the importance of endogenous pMHC complexes in antigen recognition.     

Rapid exchange of pancreatic lipase between triacylglycerol droplets

Two types of experiments were performed to study the reversibility of interfacial adsorption of pancreatic lipase (PL) to fat droplets during lipolysis. Lipolysis was measured in olive oil/gum arabic emulsions containing radiolabeled triolein in the presence of bile salts and lecithin at rate-limiting concentrations of porcine PL (PPL) or human PL (HPL). The lipolysis rate in a labeled emulsion, i.e. release of [(14)C]oleic acid, was immediately reduced by around 50% upon dilution with an equal amount of an unlabeled emulsion. Further, lipolysis was rapidly and completely suppressed when a non-exchanging lipase inhibitor was present in the second emulsion. These results indicate hopping of lipase between emulsion droplets. Alternative explanations were excluded. Hopping of PL between triolein droplets stabilized with gum arabic at supramicellar bile salt concentrations was observed only in the presence, not in the absence, of lecithin. Displacement from a trioctanoin-water interface of active HPL by an inactive mutant (S152G) was studied in the presence of bile salts by measuring HPL distribution between the water phase and the oil-water interface. Colipase was limiting for HPL binding to the oil-water interface (colipase to lipase molar ratio: 0.5) and, thus, for lipolysis. Upon adding S152G, which has the same affinity for colipase, inactive and active HPL were found to compete for binding at the oil-water interface. When equal amounts of HPL and HPL S152G were used, the lipolysis rate dropped to half the maximum rate recorded with HPL alone, suggesting that half the active HPL was rapidly desorbed from the oil-water interface. Therefore, under various conditions, PL does not remain irreversibly adsorbed to the oil-water interface, but can exchange rapidly between oil droplets, via an equilibrium between soluble and lipid-bound PL.