Kartagener syndrome (KS) is a trilogy of symptoms (nasal polyps, bronchiectasis, and situs inversus totalis) that is associated with ultrastructural anomalies of cilia of epithelial cells covering the upper and lower respiratory tracts and spermatozoa flagellae. The axonemal dynein intermediate-chain gene 1 (DNAI1), which has been demonstrated to be responsible for a case of primary ciliary dyskinesia (PCD) without situs inversus, was screened for mutation in a series of 34 patients with KS. We identified compound heterozygous DNAI1 gene defects in three independent patients and in two of their siblings who presented with PCD and situs solitus (i.e., normal position of inner organs). Strikingly, these five patients share one mutant allele (splice defect), which is identical to one of the mutant DNAI1 alleles found in the patient with PCD, reported elsewhere. Finally, this study demonstrates a link between ciliary function and situs determination, since compound mutation heterozygosity in DNAI1 results in PCD with situs solitus or situs inversus (KS).

Our understanding of conformational conversion of proteins in diseases is essential for any diagnostic and therapeutic approach. Although not fully understood, misfolding of the prion protein (PrP) is implicated in the pathogenesis of prion diseases. Despite several efforts to produce the pathologically misfolded conformation in vitro from a recombinant PrP, no positive result has yet been obtained. Within the "protein-only hypothesis", the reason for this hindrance may be that the experimental conditions used did not allow selection of the pathway adopted in vivo resulting in conversion into the infectious form. Here, using a pressure perturbation approach, we show that recombinant PrP is converted to a novel misfolded conformer, which is prone to aggregate and ultimately form amyloid fibrils. A short incubation at high pressure (600 MPa) of the truncated form of hamster prion protein (SHaPrP(90-231)) resulted in the formation of pre-amyloid structures. The mostly globular aggregates were characterized by ThT and ANS binding, and by a beta-sheet-rich secondary structure. After overnight incubation at 600 MPa, amyloid fibrils were formed. In contrast to pre-amyloid structures, they showed birefringency of polarized light after Congo red staining and a strongly decreased ANS binding capacity, but enhanced ThT binding. Both aggregate types were resistant to digestion by PK, and can be considered as potential scrapie-like forms or precursors. These results may be useful for the search for compounds preventing pathogenic PrP misfolding and aggregation.

Voltage-gated Ca 2 ϩ channels contribute to the maintenance of contractile tone in vascular myocytes and are potential targets for vasodilating agents. There is no information available about their nature and regulation in human coronary arteries. We used the whole-cell voltage-clamp technique to characterize Ca 2 ϩ -channel currents immediately after enzymatic dissociation and after primary culture of coronary myocytes taken from heart transplant patients. We recorded a dihydropyridine-sensitive L-type current in both freshly isolated and primary cultured cells. A T-type current was recorded only in culture. The L-(but not the T-) type current was inhibited by permeable analogues of cGMP in a dose-dependent manner. This effect was mimicked by the nitric oxide-generating agents S -nitroso-N -acetylpenicillamine (SNAP) and 3-morpholinosydnonimine which increased intracellular cGMP. Methylene blue, known to inhibit guanylate cyclase, antagonized the effect of SNAP. Inhibitions by SNAP and cGMP were not additive and seemed to occur through a common pathway. We conclude that ( a ) L-type Ca 2 ϩ channels are the major pathway for voltage-gated Ca 2 ϩ entry in human coronary myocytes; ( b ) their inhibition by agents stimulating nitric oxide and/or intracellular cGMP production is expected to contribute to vasorelaxation and may be involved in the therapeutic effect of nitrovasodilators; and ( c ) the expression of T-type Ca 2 ϩ channels in culture may be triggered by cell proliferation. ( J. Clin. Invest. 1997. 99:185-193.)

Protein misfolding and formation of structured aggregates are considered to be the earliest events in the development of neurodegenerative diseases, but the mechanism of these biological phenomena remains to be elucidated. Here, we report a study of heat-and pressure-induced unfolding of human Q26 and murine Q6 ataxin-3 using spectroscopic methods. UV absorbance and fluorescence revealed that heat and pressure induced a structural transition of both proteins to a molten globule conformation. The unfolding pathway was partly irreversible and led to a protein conformation where tryptophans were more exposed to water. Furthermore, the use of fluorescent probes (8-anilino-1-naphthalenesulfonate and thioflavin T) allowed the identification of different intermediates during the process of pressure-induced unfolding. At high temperature and pressure, human Q26, but not murine Q6, underwent concentration-dependent aggregation. Fourier transform infrared and circular dichroism spectroscopy revealed that these aggregates are characterized by an increased ␤-sheet content. As revealed by electron microscopy, heat-and pressure-induced aggregates were different; high temperature treatment led to fibrillar microaggregates (8 -10-nm length), whereas high pressure induced oligomeric structures of globular shape (100 nm in diameter), which sometimes aligned to higher order suprastructures. Several intermediate structures were detected in this process. Two factors appear to govern ataxin unfolding and aggregation, the length of the polyglutamine tract and its protein context.

Cell dedifferentiation and proliferation in vivo abolish the expression of L-type Ca(2+) channels and dystrophin in neointimal cells. These changes may be critical in the regulation of Ca(2+) homeostasis and, thereby, contraction of the arterial SMCs during restenosis following angioplasty.

The microtubule-associated protein TOGp, which belongs to a widely distributed protein family from yeasts to humans, is highly expressed in human tumors and brain tissue. From purified components we have determined the effect of TOGp on thermally induced tubulin association in vitro in the presence of 1 mM GTP and 3.4 M glycerol. Physicochemical parameters describing the mechanism of tubulin polymerization were deduced from the kinetic curves by application of the classical theoretical models of tubulin assembly. We have calculated from the polymerization time curves a range of parameters characteristic of nucleation, elongation, or steady state phase. In addition, the tubulin subunits turnover at microtubule ends was deduced from tubulin GTPase activity. For comparison, parallel experiments were conducted with colchicine and taxol, two drugs active on microtubules and with tau, a structural microtubule-associated protein from brain tissue. TOGp, which decreases the nucleus size and the tenth time of the reaction (the time required to produce 10% of the final amount of polymer), shortens the nucleation phase of microtubule assembly. In addition, TOGp favors microtubule formation by increasing the apparent first order rate constant of elongation. Moreover, TOGp increases the total amount of polymer by decreasing the tubulin critical concentration and by inhibiting depolymerization during the steady state of the reaction.Microtubules are highly dynamic structures that switch between growing and shrinking phases both in vivo and in vitro. These cytoskeleton polymers are necessary for many functions within the cell including intracellular transport, motility, morphogenesis, and cell division. The intrinsic dynamic instability of microtubules is further modified in the cell by numerous protein factors that favor alternatively elongation, shortening, or anchoring of these polymers. Because the mitotic spindle plays a crucial role in cell division, it has been used for decades as an important target in cancer chemotherapy. Many tubulin poisons have been identified, some of them, taxanes and vinca alkaloids, have demonstrated therapeutic value. However, all tubulin poisons are not of clinical utility. This has led to extensive efforts to explore other targets that could affect spindle integrity. A promising approach is to identify the protein regulators that modulate tubulin polymerization and to investigate their mechanism of action.The dynamic instability of microtubules is controlled in vivo by several classes of cellular factors including depolymerizing kinesins (MCAK/XKCM1) (1, 2), stathmins (3), and microtubule-associated proteins (MAPs).2 This last group is composed of structural MAPs (MAP2, tau) that were first identified in brain tissue and of a group of XMAP215-related proteins whose generic member was first characterized in Xenopus eggs (4). TOGp (HUGO gene CKAP5), which is highly expressed in tumors and brain (5), is the human homolog of XMAP215. TOGp promotes microtubule assembly both in solution and from n...

Brevin, an actin-severing protein present in serum from numerous mammals, has been purified to homogeneity from bovine serum, using hydrophobic chromatography as the last purification step. The physicochemical parameters of brevin have been established and some of them studied in the absence and presence of Ca2+. Brevin exhibits an apparent Stokes radius, R,, of 3.4 nm, an intrinsic sedimentation coefficient s&,,, of 4.8 S and 4.4 S in the absence and presence of Ca2 + respectively, indicative of calcium-induced conformational change. The native molecular mass of brevin was found to be 68 kDa and the hydrodynamic data suggest that the protein is an asymmetric molecule. Sedimentation equilibrium studies demonstrated that Ca2 ' affects the shape (asymmetry) of brevin without altering its molecular mass. Limited tryptic and chymotryptic digestion of brevin distinguishes the Ca2 -induced conformation from the EGTA one. No change in the electrophoretic migration of brevin was seen upon CaZ + addition. Several isoforms were detected by two-dimensional gel electrophoresis. Brevin increases the rate of nucleation of actin but decreases the rate of elongation of the filaments and the steady-state viscosity of F-actin in substo'ichiometric amounts, as measured by viscometric assays under high shear conditions. Electron microscopic examination documents these effects. Brevin produces shorter actin filaments and binds to the 'barbed' end of filaments to which monomers add preferentially during elongation, as demonstrated by indirect immunogold staining of antibodies against brevin. Filament elongation occurs only at the slowly growing end. An enzyme-linked immunosorbent assay was developed and used to detect and quantify brevin and related proteins in extracts of different bovine cells and tissues. Liver and smooth muscles were found to contain the highest amounts of the severing protein.Recent years have seen rapid progress in studies on the composition of various filaments within cells. Their spatial organization and possible functions have focused much interest on the cytoskeleton, and particularly on the microfilament system.Actin is a contractile and structural protein widely distributed in all eukaryotic cells [l, 21. Besides its structural role and maintenance of cell shape, in non-muscle cells actin is constantly being reorganized according to the physiological state of the cell [3] and in response to functional demands. These dynamic events 11, 41 are related to a spatial and a temporal control of actin assembly and diassembly.The study of the molecular mechanisms underlying these events has led to the discovery within various cells of numerous proteins which modulate the assembly state of actin. Three major classes of actin binding proteins have been identified [5] : (a) actin cross-linking proteins, (b) severing and capping proteins, (c) G-actin stabilizing proteins.Several proteins which interact with F-actin in a calciumregulated way have been identified in a wide variety of cell types: gelsolin from rabbit mac...

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