Contribution of the endoplasmic reticulum to the glucose-induced [Ca(2+)](c) response in mouse pancreatic islets

Thapsigargin (TG), a blocker of Ca(2+) uptake by the endoplasmic reticulum (ER), was used to evaluate the contribution of the organelle to the oscillations of cytosolic Ca(2+) concentration ([Ca(2+)](c)) induced by repetitive Ca(2+) influx in mouse pancreatic beta-cells. Because TG depolarized the plasma membrane in the presence of glucose alone, extracellular K(+) was alternated between 10 and 30 mM in the presence of diazoxide to impose membrane potential (MP) oscillations. In control islets, pulses of K(+), mimicking regular MP oscillations elicited by 10 mM glucose, induced [Ca(2+)](c) oscillations whose nadir remained higher than basal [Ca(2+)](c). Increasing the depolarization phase of the pulses while keeping their frequency constant (to mimic the effects of a further rise of the glucose concentration on MP) caused an upward shift of the nadir of [Ca(2+)](c) oscillations that was reproduced by raising extracellular Ca(2+) (to increase Ca(2+) influx) without changing the pulse protocol. In TG-pretreated islets, the imposed [Ca(2+)](c) oscillations were of much larger amplitude than in control islets and occurred on basal levels. During intermittent trains of depolarizations, control islets displayed mixed [Ca(2+)](c) oscillations characterized by a summation of fast oscillations on top of slow ones, whereas no progressive summation of the fast oscillations was observed in TG-pretreated islets. In conclusion, the buffering capacity of the ER in pancreatic beta-cells limits the amplitude of [Ca(2+)](c) oscillations and may explain how the nadir between oscillations remains above baseline during regular oscillations or gradually increases during mixed [Ca(2+)](c) oscillations, two types of response observed during glucose stimulation.     

Evaluation of adolescent statural growth in health and disease: reliability of assessment from height measurement series and development of an automated algorithm

OBJECTIVES: The precise evaluation of adolescent growth spurt is necessary for numerous clinical research studies of growth disorders and treatments. The objectives of our study were: (1) to evaluate the reliability of clinicians' 'manual' evaluation of the adolescent growth spurt from a collected series of height data, and (2) to construct an automated algorithm to determine the duration of the two phases of growth in health and disease (spurt and final slow growth) independent of clinical pubertal stages. METHODS: One hundred and seventy-four growth curves of normally growing, GH-deficient and Turner's syndrome subjects were presented twice to 2 experienced clinicians. Disagreement between evaluations and clinicians were settled to obtain a 'consensual gold standard' evaluation versus which the algorithm was assessed. Kappa statistics and Bland-Altman analyses were used to evaluate the reliability and agreement of the evaluations. RESULTS: The reliability of 'manual' evaluation of adolescent growth spurt from collected series of height data appeared to be poor. Conversely, the developed algorithm is perfectly reliable and satisfactorily valid. Discrepancies with the clinical consensual gold standard were always fewer than the discrepancies between the expert clinicians, and were observed in similarly difficult curves. CONCLUSION: The developed algorithm may be useful for diverse clinical and biological research applications in children with growth disorders. This study also confirms the value of a comprehensive investigation of growth during adolescence independent of clinical staging.     

Direct evidence for membrane pore formation by the apoptotic protein Bax

Direct imaging of the interaction of the apoptotic protein, Bax, with membrane bilayers shows the presence of toroidal-shaped pores using atomic force microscopy. These pores are sufficiently large to allow passage of proteins from the intermitochondrial space. Both the perturbation of the membrane and the amount of protein bound to the bilayer are increased in the presence of calcium. The results from the imaging are consistent with leakage studies from liposomes of the same composition. The work shows that Bax by itself can form pores in membrane bilayers.     

Molecular cloning, gene localization, and structure of human cyclin B3

Here we describe the molecular cloning of human cyclin B3, its localization, and its structure. It is localized in the subcentromeric region of the X chromosome, still not completely sequenced by the Human Genome Project. This cyclin B3 is unusually large for a mitotic cyclin. Its mRNAs were found in all tissues and were particularly abundant in testis. At least three splice variants were found in the ORF and three variants in the 5'UTR.     

Multi-bead-and-spring model to interpret protein detachment studied by AFM force spectroscopy

This article deals with the detachment of molecules (fibrinogen) from a surface studied experimentally with an atomic force microscope. The detachment (or rupture) forces are measured as a function of the retraction velocity and exhibit a clear dependence on this parameter, even though the interaction between the molecules and the surface are nonspecific. To interpret these data, a mechanical multi-bead-and-spring model is developed. It consists of one to several parallel, "molecular" springs connected to an extra spring representing the cantilever that is moved at constant velocity. The free end of each molecular spring terminates with a particle that interacts with the surface through a Lennard-Jones potential. This Brownian dynamics model is used to analyze the experimental findings. In the framework of this model, it appears that the fibrinogen molecule must be ascribed a stiffness much smaller than that of the cantilever. In addition, several bonds between the molecule and the surface must be taken into account for the range of the molecule-surface interaction not to be unrealistically small. In future work, this model will be extended to more complex mechanisms such as the detachment of cells from a surface.     

Equilibrium and kinetic studies of the interactions of a porphyrin with low-density lipoproteins

Low-density lipoproteins (LDL) play a key role in the delivery of photosensitizers to tumor cells in photodynamic therapy. The interaction of deuteroporphyrin, an amphiphilic porphyrin, with LDL is examined at equilibrium and the kinetics of association/dissociation are determined by stopped-flow. Changes in apoprotein and porphyrin fluorescence suggest two classes of bound porphyrins. The first class, characterized by tryptophan fluorescence quenching, involves four well-defined sites. The affinity constant per site is 8.75 x 10(7) M(-1) (cumulative affinity 3.5 x 10(8) M(-1)). The second class corresponds to the incorporation of up to 50 molecules into the outer lipidic layer of LDL with an affinity constant of 2 x 10(8) M(-1). Stopped-flow experiments involving direct LDL porphyrin mixing or porphyrin transfer from preloaded LDL to albumin provide kinetic characterization of the two classes. The rate constants for dissociation of the first and second classes are 5.8 and 15 s(-1); the association rate constants are 5 x 10(8) M(-1) s(-1) per site and 3 x 10(9) M(-1) s(-1), respectively. Both fluorescence and kinetic analysis indicate that the first class involves regions at the boundary between lipids and the apoprotein. The kinetics of porphyrin-LDL interactions indicates that changes in the distribution of photosensitizers among various carriers could be very sensitive to the specific tumor microenvironment.     

Evidence of an odorant-binding protein in the human olfactory mucus: location,structural characterization,and odorant-binding properties

Odorant-binding proteins (OBPs) are small abundant extracellular proteins belonging to the lipocalin superfamily. They are thought to participate in perireceptor events of odor detection by carrying, deactivating, and/or selecting odorant molecules. Putative human OBP genes (hOBP) have recently been described [Lacazette et al. (2000) Hum. Mol. Genet. 9, 289-301], but the presence of the corresponding proteins remained to be established in the human olfactory mucus. This paper reports the first evidence of such expression in the mucus covering the olfactory cleft, where the sensory olfactory epithelium is located. On the contrary, hOBPs were not observed in the nasal mucus covering the septum and the lower turbinate. To demonstrate the odorant binding activity of these proteins, a corresponding recombinant protein variant, hOBP(IIa)(alpha), was secreted by the yeast Pichia pastoris and thoroughly characterized. It appears as a monomer with one disulfide bond located between C59 and C151, a conservative feature of all other vertebrate OBPs. By measuring the displacement of several fluorescent probes, we show that hOBP(IIa)(alpha) is able to bind numerous odorants of diverse chemical structures, with a higher affinity for aldehydes and large fatty acids. A computed 3D model of hOBP(IIa)(alpha) is proposed and reveals that two lysyl residues of the binding pocket may account for the increased affinity for aldehydes. The relatively limited specificity of hOBP(IIa)(alpha) suggests that other human OBPs are expected to take into account the large diversity of odorant molecules.     

Crucial role of conserved lysine 277 in the fidelity of tRNA aminoacylation by Escherichia coli valyl-tRNA synthetase

Valyl-tRNA synthetase (ValRS) from Escherichia coli undergoes covalent valylation by a donor valyl adenylate synthesized by the enzyme itself. ValRS could also be modified, although to a lesser extent, by the noncognate isosteric substrate L-threonine from a donor threonyl adenylate synthesized by the synthetase itself, or by the nonsubstrate methionine from methionyl adenylate produced by catalytic amounts of methionyl-tRNA synthetase. MALDI mass spectrometry analysis designated lysines 154, 162, 170, 533, 554, 593, 894, 930, and 940 of ValRS as the target residues for the attachment of valine. Following autothreonylation, lysines 162, 170, 178, 277, 291, 554, 580, 593, 861, 894, and 930 were found to be modified. Finally, L-Met-labeled residues were lysines 118, 162, 170, 178, 277, and 938. Alignment of the available ValRS amino acid sequences showed that lysines 277 and 554 are strictly conserved (with the exception concerning replacement of Lys-277 with a methionine or a tyrosine in archaebacteria), suggesting that these residues might be functionally significant. Indeed, lysine 554 of ValRS is the first lysine of the Lys-Met-Ser-Lys-Ser signature of the catalytic site of class I aminoacyl-tRNA synthetases. Lys-277 which is labeled by L-threonine or L-methionine, and not by L-valine, is located at or near the editing site, in the three-dimensional structure of ValRS. The role of lysine 277 was evaluated by site-directed mutagenesis. The Lys277Ala mutant (K277A) exhibited a posttransfer Thr-tRNA(Val) editing rate that was significantly lower than that observed for the wild-type enzyme. In addition, the K277A substitution altered amino acid discrimination in the editing site, resulting in hydrolysis of the correctly charged cognate Val-tRNA(Val). Finally, significant amounts of mischarged Thr-tRNA(Val) were produced by the K277A mutant, and not by wild-type ValRS. Altogether, our results designate Lys-277 as a likely candidate for nucleophilic attack of misacylated tRNA in the editing site of ValRS.     

Attaching histidine-tagged peptides and proteins to lipid-based carriers through use of metal-ion-chelating lipids

The therapeutic potential of selected peptides and proteins is enormous, with applications ranging from use as therapeutic vaccines, as modulators of intracellular signaling pathways and as highly selective agents capable of recognizing unique extracellular targets. We have been pursuing development of hybrid lipid-based carrier formulations designed to take advantage of the therapeutic benefits of peptides selected for their ability to act in a complementary fashion with the carrier system. In this regard, it is critical to have simple and versatile methods to promote and control the binding of diverse peptides to a broad range of carrier formulations. As demonstrated here, recombinant proteins and synthetic peptides containing poly-histidine residues (4 to 10) can be specifically bound to liposomes containing a metal-ion-chelating lipid, DOGS-NTA-Ni. The potential of this approach is demonstrated using two functional peptides, AntpHD-Cw3 (applications for vaccine production) and AHNP (specificity for Her-2 expressing cells).     

Optical tweezers in biology and medicine

Optical trapping techniques provide unique means to manipulate biological particles such as virus, living cells and subcellular organelles. Another area of interest is the measurement of mechanical (elastic) properties of cell membranes, long strands of single DNA molecule, and filamentous proteins. One of the most attractive applications is the study of single motor molecules. With optical tweezers traps, one can measure the forces generated by single motor molecules such as kinesin and myosin, in the piconewton range and, for the first time, resolve their detailed stepping motion.