[Muscle pain in myositis in a young female patient: what is behind this?] / Muskelschmerzen bei Myositis bei einer jungen Patientin ­ was steckt dahinter?

Myositis is a connective tissue disease which is most frequently diagnosed in women aged 40-60 years. Due to a clear association with underlying malignant diseases, general tumor screening is recommended whenever it is diagnosed. Colorectal carcinoma (CRC) is a common malignant disease, and the typical at-risk group comprised, to date, patients older than 55 years. However, with the rising incidence of so-called early-onset colorectal carcinoma (EO-CRC), an increasingly important patient population is emerging in the 20- to 50-year age range. One reason for the rising incidence is suggested to be an increase in classic risk factors at younger ages. Here, the case of a 34-year-old female patient who presented with the leading paraneoplastic syndrome of myositis and was diagnosed with a sporadic form of CRC is reported. Monitoring of known risk factors as early on as in young adulthood and greater attention in the presence of symptoms such as gastrointestinal hemorrhage or paraneoplastic syndromes seem necessary to compensate for the time delay in diagnosis that currently still exists and the associated worse oncologic outcome.

Visual learning in individually assayed Drosophila larvae.

An understanding of associative learning is facilitated if it can be analyzed in a simple animal like the fruit fly Drosophila. Here, we introduce the first visual associative learning paradigm for larval Drosophila; this is remarkable as larvae have an order of magnitude fewer neurons than adult flies. Larvae were subjected to either of two reciprocal training regimes: Light+/Dark- or Light-/Dark+. Subsequently, all larvae were individually tested for their preference between Light versus Dark. The difference between training regimes was therefore exclusively which visual situation was associated with which reinforcer; differences observed during the test thus reflected exclusively associative learning. For positive reinforcement (+) we used fructose (FRU), and for negative reinforcement (-) either quinine or sodium chloride (QUI, NaCl). Under these conditions, associative learning could be reproducibly observed in both wild-type strains tested. We then compared the effectiveness of training using differential conditioning, with both positive and negative reinforcement, to that using only positive or only negative reinforcement. We found that FRU only, but neither QUI nor NaCl, was in itself effective as a reinforcer. This is the first demonstration of appetitive learning in larval Drosophila. It is now possible to investigate the behavioral and neuronal organization of appetitive visual learning in this simple and genetically easy-to-manipulate experimental system.

Interactions of the drug amphotericin B with phospholipid membranes containing or not ergosterol: new insight into the role of ergosterol.

Amphotericin B (AmB) is an amphipathic polyene antibiotic which permeabilizes ergosterol-containing membranes, supposedly by formation of pores. In water, AmB forms chiral aggregates, modelled as stacks of planar dimers in which the joined polyene chains in each dimer turn round, from one dimer to the following in these stacks, by forming a helical array. Studies of the binding of AmB with L-dipalmitoylphosphatidylcholine (L-DPPC) and L-dilauroylphosphatidylcholine (L-DLPC) bilayers disclose the main following results. (1) An inversion of the helicity of the L-DPPC-bound AmB aggregates, when the L-DPPC bilayers are in the gel phase, is inferred from the evolution of the circular dichroism spectra of AmB+L-DPPC mixtures. (2) An AmB-induced gel-to-subgel transformation of L-DPPC bilayers, in the previous mixtures, is revealed by a differential scanning calorimetry study. (3) The role played by ergosterol in the location of phospholipid-bound AmB aggregates with respect to a phospholipid bilayer is directly demonstrated from atomic force microscopy observations of mica-supported AmB+L-DLPC mixtures, in the presence or absence of ergosterol. While in the absence of ergosterol AmB aggregates remained at the surface of the bilayer, in the presence of ergosterol they appeared embedded within this bilayer and became hollow-centered. As such an embedding in the hydrophobic core of a bilayer requires a rearrangement of the aggregates with respect to their architecture in water, this rearrangement is held responsible for the hollowing of aggregates. The hollow-centered sublayer-embedded AmB aggregates are thought to be the precursors of the formation of AmB pores.

The formation of empty shells upon pressure induced decapsidation of turnip yellow mosaic virus.

The stability of turnip yellow mosaic virus (TYMV) was investigated under pressure, using solution neutron small angle scattering. Dissociation products were characterized by analytical ultracentrifugation and electron microscopy. At pH 6.0, TYMV remained unaffected by pressure, up to 260 Megapascals (MPa), the highest pressure reached in these experiments. At pH 8.0, TYMV remained unaffected by pressure up to 160 MPa, but decapsidated irreversibly above 200 MPa, giving rise to more and more empty shells upon increasing pressure. The organization of these empty shells was similar to that of the capsid of native virions, apart from the presence of a hole corresponding to the loss of a group of 5-8 coat protein subunits, through which the RNA may have escaped. At variance with other small isometric viruses, the capsid of TYMV never dissociated under pressure into subunits or small aggregates of subunits. This exceptional behavior of TYMV is probably due to the importance of van der Waals contacts and hydrogen bonds in the stability of its capsid.

Binding of nystatin and amphotericin B with sterol-free L-dilauroylphosphatidylcholine bilayers resulting in the formation of dichroic lipid superstructures.

Interactions of multilamellar vesicles (MLV) of dilauroylphosphatidylcholine (DLPC) with the polyene antibiotics, amphotericin B (AmB) and nystatin (Ny), were followed by circular dichroism (CD). These interactions proceed with both antibiotics through a slow association with high [DLPC]/[antibiotic] stoichiometric molar ratios (> or = 130), at room temperature for which DLPC membranes are in a fluid state. Microscopic investigations of the spatial distributions of the antibiotic and the MLV in the mixtures revealed that MLV form clusters inside which the antibiotic is strongly concentrated and lipid superstructures appear. Concomitantly with the appearance of these superstructures a DLPC dichroic signal emerges. This observation indicates that the chiral properties of antibiotic oligomers can induce a chirality of the DLPC molecules which are bound to them. These results support the hypothesis of a recent molecular modeling of AmB oligomers which postulates that their chiral properties result from a chiral assemblage of antibiotic molecules (Millié et al., J. Phys. Chem. B, in press).

Polymorphism of turnip yellow mosaic virus empty shells and evidence for conformational changes occurring after release of the viral RNA. A differential scanning calorimetric study.

Turnip yellow mosaic virus (TYMV) is a small isometric plant virus which decapsidates by releasing its RNA through a hole in the capsid, leaving behind an empty shell [R. E. F. Matthews and J. Witz, (1985) Virology 144, 318-327]. Similar empty shells (artificial top component, ATC) can be obtained by submitting the virions to various treatments in vitro. We have used differential scanning calorimetry, analytical sedimentation, and electron microscopy to investigate the thermodenaturation of natural empty shells (NTC, natural top component) present in purified virus suspensions, and of several types of ATCs. ATCs divided in two major classes. Those obtained by alkaline titration, by the action of urea or butanol behaved as NTC: their thermograms contained only one peak corresponding to the irreversible dissociation of the shells and the denaturation of the coat protein. The temperature of this unique transition varied significantly with pH, from 71 degrees C at pH 4.5 to 84 degrees C at pH 8.5. The thermograms of ATCs obtained by freezing and thawing, or by the action of high pressure, contained two peaks: shells dissociated first into smaller protein aggregates at 57 degrees C (at pH 5.0) to 61 degrees C (at pH 8.5), which denatured at the temperature of the unique transition of NTC. Shells obtained by heating virions to 55 degrees C at pH 7.6, changed conformation after the release of the viral RNA, as upon continuous heating to 95 degrees C, their thermograms were similar to those of the shells obtained by freezing and thawing, whereas after purification they behaved like NTC. Structural implications of these observations are discussed.

Dynamics of Micelles of Polyethyleneoxide-Polypropyleneoxide-Polyethyleneoxide Block Copolymers in Aqueous Solutions.

The dynamics of the micelles of five triblock poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) copolymers, the Pluronics P104 (EO27PO61EO27), P84 (EO19PO43EO19), P65 (EO18PO29EO18), P85 (EO26PO40EO26), and P103 (EO17PO60EO17), have been investigated using two chemical relaxation methods: the temperature-jump and the ultrasonic relaxation (absorption). In the frequency range investigated (0.5-50 MHz), the ultrasonic absorption spectra (absorption vs frequency plots) consisted in tails of relaxation curves, indicating characteristic times much longer than 0.3 µs for the exchange of copolymers between micelles and intermicellar solution. Absorption measurements at a fixed frequency yielded the critical micellization temperature of the solutions. The temperature-jump results obtained in this study together with those from a previous one for the copolymers L64 (EO13PO30EO13) and PF80 (EO73PO27EO73) (B. Michels et al., Langmuir 13, 3111, 1997) showed that the relaxation time associated with the formation/breakup of micelles becomes longer upon increasing copolymer molecular weight at constant composition. This time also increased when decreasing the length of the hydrophilic block at fixed hydrophobic block length or increasing the length of the hydrophobic block at fixed hydrophilic block length, similar to conventional surfactants. The dynamics of block copolymers micelles in aqueous solution are discussed. Copyright 1999 Academic Press.

Association of polyene antibiotics with sterol-free lipid membranes. II. Hydrophobic binding of nystatin to dilauroylphosphatidylcholine bilayers.

Interaction of nystatin A1 with multilamellar vesicles (MLV) of dilauroylphosphatidylcholine (DLPC), observed either by adding nystatin to preformed MLV (mixtures I) or by incorporating it during the formation of vesicles (mixtures II, inner lamellas of MLV in contact with nystatin) was investigated for 0.002 < or = nystatin/DLPC = R(A) < or = 0.20, by four complementary methods. The main results were: (i) Ultraviolet absorption and circular dichroism (CD) spectra of mixtures I revealed the occurrence of a saturable association with a stoichiometry (R(A) = 0.007 +/- 0.002) constant between 3 and 33 degrees C. (ii) By differential scanning calorimetry, thermograms of the two types of mixtures were similar only when water was in great excess. In the opposite (e.g., (H2O)/(DLPC) = R(W) < or = 300), mixture II thermograms displayed two features, upshifted by about 6.5 degrees C with respect to the sharp peak observed with mixture I, resembling those obtained for pure DLPC when the low-temperature phase was the subgel phase. For this R(W), the nystatin absolute concentrations were those for which nystatin form superaggregates as revealed by the nystatin CD spectra. It is proposed that these superaggregates are excluded from the interlamellar spacings of MLV and exert a pumping action on the interlamellar water. The subsequent dehydration of the inner lamellas is thought to convert them into the subgel state. (iii) 2H-NMR spectra of sn-2-perdeuterated DLPC MLV + nystatin mixtures II, confirmed such a temperature shift of the main transition. They showed, in addition, an ordering of the aliphatic chains immediately above the transition temperature, equivalent to a bilayer thickening of 2 A.

Heat-induced conversion of ovalbumin into a proteinase inhibitor.

Ovalbumin is a member of the serine proteinase inhibitor (serpin) family but is unable to inhibit proteinases. Here we show that heating transforms it into inhibitory ovalbumin (I-ovalbumin), a potent reversible competitive inhibitor of human neutrophil elastase (Ki = 5 nM) and cathepsin G (Ki = 60 nM) and bovine chymotrypsin (Ki = 30 nM). I-ovalbumin also inhibits bovine trypsin, porcine elastase and alpha-lytic proteinase with Ki values in the micromolar range. Thus, I-ovalbumin differs from active serpins by its inability to form irreversible complexes with proteinases. I-ovalbumin is unusually thermostable: it does not undergo any structural transition between 45 degrees C and 120 degrees C as tested by differential scanning calorimetry, and it retains full inhibitory capacity after heating at 120 degrees C. It has 8% less alpha-helices and 9% more beta-sheet structures than native ovalbumin, as shown by circular dichroism. Our results show that the primary sequence of ovalbumin contains the information required for enabling the first step of the serpin-proteinase interaction to occur, i.e. the formation of the Michaelis-like reversible complex, but does not contain the information needed for stabilizing this initial complex.

Association of polyene antibiotics with sterol-free lipid membranes: I. Hydrophobic binding of filipin to dimyristoylphosphatidylcholine bilayers.

The interaction of filipin III with multilamellar vesicles (MLV) of dimyristoylphosphatidylcholine (DMPC ) was studied by four complementary methods leading to the following results: (1) The modifications of the filipin dichroic spectrum, by adding preformed fluid DMPC MLV, provide evidence of a saturable association with the stoichiometry DMPC/filipin = 4.2 +/- 0.5, constant between 24 and 35 degrees Celsius. (2) Thermograms obtained by differential scanning calorimetry (DSC) on mixtures where filipin is incorporated during the formation of MLV exhibit a high-temperature tail the more marked the higher the filipin content and some structures at temperatures which depend on this content. The corresponding evolution with the temperature of the CD spectra reveals that the characteristic bound filipin spectrum appears at the temperature at which a structure emerges. (3) Titration calorimetry measurements reveal that the association process is exothermic in the temperature range of the DSC endotherms in agreement with the filipin-induced ordering of the lipid chains, previously established by 2H-NMR in the same temperature range (Milhaud et al.(1989) Eur. Biophys. J. 17, 151-158). A discussion of the relevancy of this exothermicity to the hydrophobic effect is developed by referring to the paper by Wimley and White ((1993) Biochemistry 32, 6307-6312).

The thermal stability and decapsidation mechanism of tymoviruses: a differential calorimetric study.

The thermal stability of virions present in purified suspensions of three tymoviruses, turnip yellow mosaic virus (TYMV), belladonna mottle virus (BelMV) and eggplant mosaic virus (EMV) was investigated by microcalorimetry. Virions are less stable than natural empty shells at 4.5 < or = pH < or = 8.5. Polyvalent cations present in TYMV stabilize the virions at pH < or = 5.0 only. Virions decapsidate in three steps: i) the release of the viral RNA, probably through a hole in the capsid; ii) the dissociation of the artificial empty shells thus formed; and iii) the denaturation of the dissociated components. An exothermic process accompanies the first step. Structural implications are discussed.

Long term use of captopril or nifedipine in normotensive microalbuminuric patients with insulin-dependent diabetes mellitus.

Several studies have suggested that ACE-inhibition may be effective in postponing the onset of nephropathy in insulin-dependent diabetic subjects. In contrast, other drugs might have opposing effects. To study the long term effects of either captopril or nifedipine in normotensive, microalbuminuric patients with insulin-dependent diabetes mellitus, eighteen subjects received either placebo (n = 5, P), 20 mg nifedipine daily (n = 7, N) or 50 mg captopril daily (n = 6, C) for one year. Baseline clinical and laboratory variables were comparable in the three groups. Glomerular filtration rate (GFR), effective renal plasma flow (ERPF) and blood pressure did not differ between groups before and after one years medication. UAER did not change in the captopril and the placebo group (C: -12.6% (-58.1 to 51.8%)' P: -17.3 (-55.9 to 99.3%), medians and ranges. In contrast, in the patients that received nifedipine, UAER rose by 43.1% (-8.5 to 261.8%), (p < 0.05 Baseline vs one year, and one year nifedipine vs captopril and placebo). We therefore conclude, that long-term use of nifedipine increases UAER in normotensive microalbuminuric insulin-dependent subjects, in contrast to captopril or placebo. Whether this enhancement of microalbuminuria exerts an adverse effect on renal function in the long-term is yet unknown, but caution seems warranted.

Scanning calorimetric studies of the stability of tobacco mosaic virus and aggregates of its coat protein.

The thermal denaturation of the common strain of a rod-shaped plant virus, tobacco mosaic virus, has been investigated by differential scanning calorimetry, and compared to that of various aggregation states of its coat protein and to that of three other TMV strains. The state of the virions was monitored by electron microscopy and analytical ultracentrifugation. The observed endotherms could be analysed in terms of a step-wise dissociation of the virions. The transition temperatures of the three successive structural changes increased with decreasing pH, from pH = 8.0 to pH = 5.0, although the corresponding enthalpy changes did not vary appreciably with pH. TMV-HR showed a stronger pH dependence of the transition temperatures than the other strains, probably reflecting the importance of the changes in affecting the charged amino acids of its coat protein. The first step of the dissociation, which correlates with the breaking up of the virions into three or four shorter rods, implies a conformational change of the particle that may be related to the first step of the in situ decapsidation of TMV.

Enhanced fluctuations in small phospholipid bilayer vesicles containing cholesterol.

Ultrasonic and calorimetric studies of small homogeneously-sized DMPC unilamellar vesicles showed two thermal transitions at temperatures Tc1 and Tc2 (Tc2 greater than or equal to Tc1); Tc2 is close to the phase transition temperature, Tc, of large vesicles. The process at Tc2 is not a fusion of vesicles and is interpreted as characterizing an order-disorder transition essentially similar to that of large vesicles. The temperatures Tc1 and Tc2 become increasingly similar as the cholesterol content is increased, while the clusters at Tc2 (congruent to 85 lipid molecules in pure DMPC) increase in size up to approximately 180 lipid molecules at 12 mol% cholesterol. Incorporation of cholesterol thus brings about enhanced fluctuations in this model system of a membrane.

Ultrasonic absorption in tobacco mosaic virus and its protein aggregates.

The structural fluctuations specific to self-assembled biological systems have been investigated further with ultrasonic techniques by using two strains of tobacco mosaic virus (TMV), as well as the helical aggregate of the common strain protein and subassemblies of it. We confirmed our earlier conclusion that protein assemblies exhibit specific structural fluctuations detected in ultrasonic experiments. As in spherical viruses, the fluctuations exhibited by the protein aggregates having a quaternary structure similar to that of the virion were modified in the virus by interaction with the RNA strand. It is unlikely that the origin for the observed effect is due either to: (1) the difference in local mobility of the segment 89 to 113 of the polypeptide chain in TMV and in the helical aggregate on the one hand, and in smaller aggregates, on the other hand; or (2) a local fluctuation associated with proton transfer reactions or ion-pair interactions. The most remarkable feature in the TMV system is the fact that the two-ring disk showed no excess of ultrasonic absorption with respect to the A-protein oligomer, while a large increase of ultrasonic absorption was observed in the rod-like aggregate that had undergone the disk-helix transition.

Ultrasonic absorption evidence for structural fluctuations in frog virus 3 and its subparticles.

The structural fluctuations specific to self-assemblies of biological molecules have been investigated further with ultrasonic techniques by using frog virus 3 (FV3). We compared the ultrasonic properties of complete FV3 virions and of several subparticles that may be obtained from this DNA virus: (i) the central nucleoprotein core versus its component DNA and proteins in a dissociated state; (ii) the core versus the capsidless subparticle, consisting of the core surrounded by the lipid membrane; and (iii) the complete virus versus the capsidless subparticle. The ultrasonic absorption by the core particle was quite large compared with the absorption by other nucleoprotein assemblies, suggesting that the core contains some organized structure. Both the core and the complete virus absorbed ultrasound more than did the capsidless subparticle. The difference spectrum for the virion relative to the capsidless subparticle may represent a single relaxation and is analyzed, by using a recent model, in terms of volume fluctuations due to radial movements in the virion. These fluctuations are much smaller than can be detected in virus crystals with present-day x-ray techniques.

Ultrasonic absorption evidence of structural fluctuations in viral capsids.

When the coat protein of the small icosahedral virus, brome mosaic virus, reassembles into capsids, the ultrasonic absorption of the solution greatly increases. Submitting the solution to an ultrasonic field thus appears to reveal spontaneous molecular motions within a protein assembly. Confirmatory evidence of a dynamics of a protein shell comes from measurements on brome mosaic virus at various degrees of swelling and on tomato bushy stunt virus treated with the crosslinking agent glutaraldehyde. The detected fluctuations may be related either with cooperative deformational motion in the capsid or with more localized structural changes. Such structural changes may help liberate the RNA at an early stage of viral infection.