Inhomogeneities in sodium decylsulfate doped 1,2-dipalmitoylphosphatidylcholine bilayer.

Differential scanning calorimetry (DSC), dynamic light scattering (DLS), and turbidity measurements have been used to study the effect of sodium decylsulfate, C10OS, on 1,2-dipalmitoylphosphatidylcholine, DPPC, vesicles. DSC measurements, performed soon after addition of C10OS solutions to DPPC vesicular dispersion, at several C10OS/DPPC molar ratios, showed the contemporary presence of maxima ascribable both to transitions of an unmodified DPPC membrane and to transitions related to membrane domains in which C10OS is also present. In all the considered samples only unilamellar vesicles are present as shown by DLS measurements carried out on aqueous DPPC and C10OS-DPPC systems. Also the aging of the samples confirmed the presence of SUV in the time scale of DSC measurements. On the basis of DSC results the pseudo-phase diagram of the system was drawn. This diagram does not represent a system in equilibrium, from a thermodynamic point of view, but only kinetically probable states. In fact the prolonged aging of the solutions used both for DSC and DLS scans highlight the disappearance of the unmodified DPPC membrane and its conversion into a mixed membrane. Turbidity measurements at 25 degrees C, performed as a function of surfactant composition, showed that C10OS initially contributes to increase the vesicles dimensions and allowed to calculate the saturation ratio C10OS/DPPC in the membrane. In contrast, at 45 degrees C the presence of surfactant gives origin immediately to smaller structures in comparison to vesicles, probably gel-state micelles.

Analysis of thermal adaptation in the HSL enzyme family.

The recently solved three-dimensional (3D) structures of two thermostable members of the carboxylesterase/lipase HSL family, namely the Alicyclobacillus (formerly Bacillus) acidocaldarius and Archaeoglobus fulgidus carboxylesterases (EST2 and AFEST, respectively) were compared with that of the mesophilic homologous counterpart Brefeldine A esterase from Bacillus subtilis. Since the 3D homology models of other members of the HSL family were also available, we performed a structural alignment with all these sequences. The resulting alignment was used to assess the amino acid "traffic rule" in the HSL family. Quite surprisingly, the data were in very good agreement with those recently reported from two independent groups and based on the comparison of a huge number of homologous sequences from the genus Bacillus, Methanococcus and Deinococcus/Thermus. Taken as a whole, the data point to the statistical meaning of defined amino acid conversions going from psychrophilic to hyperthermophilic sequences. We identified and mapped several such changes onto the EST2 structure and observed that such mutations were localized mostly in loops regions or alpha-helices and were mostly excluded from the active site. A site-directed mutagenesis of two of the identified residues confirmed they were involved in thermal stability.

A 23Na NMR study of the effect of D(+) and L(-) arabitol on NaDNA in aqueous solution.

The 23Na NMR quadrupolar relaxation in NaDNA aqueous solutions has been investigated in the presence of D(+) and L(-) arabitol. Quite different results were produced by the enantiomers, i.e. the addition of D(+) arabitol produced a small increase of the 23Na NMR relaxation rates, while in the presence of L(-) arabitol a significant decrease was observed. These findings were analysed and discussed in terms of an effective interaction of L(-) arabitol with DNA.

Thermodynamic stability of the two isoforms of bovine seminal ribonuclease.

Bovine seminal ribonuclease (BS-RNase) is a dimeric protein with two identical subunits linked by two disulfide bridges, each subunit showing 80% of sequence identity with pancreatic RNase A. BS-RNase exists in two different quaternary conformations in solution: the MxM form, in which each subunit exchanges its alpha-helical N-terminal segment with its partner, and the M=M form with no exchange. By differential scanning microcalorimetry (DSC), the denaturation of the two dimeric forms of BS-RNase was found to be more complex than a simple two-state process. Monomeric derivatives of the dimeric protein follow instead a simple two-state mechanism, but are distinctly less stable than RNase A. The three-state N if I if D denaturation process of the two quaternary isoforms was interpreted by identifying in the dimers a central highly structured core, enclosing the covalently bonded subunit interface, which unfolds only after the periphery (mainly the N-terminal peptide) unfolds. Circular dichroism spectra of the two forms in the far-ultraviolet region show large differences between the secondary structure of the isoforms and that of the native BS-RNase mixture at equilibrium. This has been attributed to the presence in the equilibrium mixture of intermediate forms with displaced and disordered N-terminal alpha-helical segments.

The effects of polyols on the thermal stability of calf thymus DNA.

The effects on thermal denaturation of calf thymus DNA (ct-DNA) and its conformational changes induced by the presence in solution of different polyols, namely glycerol, i-erytritol, L( -- ) and D( + ) arabitol, D-mannitol, D-sorbitol and myo-inositol, have been investigated by means of differential scanning calorimetry (DSC) and circular dichroism (CD). By increasing the concentration of these additives a decrease in both the denaturation enthalpy (deltadH) and temperature of the maximum of the denaturation peak (Tmax) of DNA is observed. The values of these thermodynamic parameters depend on both the nature and concentration of the solute. The overall destabilization of DNA molecule has been related to the different capability of polyhydric alcohols to interact with the polynucleotide solvation sites replacing water and to the modification of the electrostatic interactions between the polynucleotide and its surrounding atmosphere of counterions. The particular behaviour of L( -- ) arabitol, which showed a much greater destabilizing ability compared to the other polyols, was further investigated and attributed to a direct more effective interaction with the double helix of DNA. CD spectra showed only a slight alteration of DNA-B structure in the presence of all the molecules here studied, except for L( -- ) arabitol where the DNA molecule seems to undergo a meaningful conformational change. The salt concentration dependence of DNA thermal stability in the presence of L( -- ) arabitol indicates a conformational change of polynucleotide towards a more extended conformation.

A phase I/II trial of neoadjuvant chemotherapy with cisplatin and vinorelbine followed by accelerated irradiation for patients with inoperable nonsmall cell lung carcinoma.

BACKGROUND: Both locoregional and distant disease control remains poor in the treatment of Stage III nonsmall cell lung carcinoma (NSCLC). This trial was conducted to evaluate the tolerance and responses of patients with NSCLC given a neoadjuvant regimen of cisplatin and vinorelbine chemotherapy followed by accelerated thoracic radiotherapy. METHODS: Forty-two patients with Stage IIIA and IIIB NSCLC were entered into the study. Treatment consisted of cisplatin 100 mg/m2 given on Days 1 and 29 and vinorelbine 30 mg/m2 given weekly for 5 weeks, with a planned 50% dose reduction to 15 mg/m2 planned for Week 2. This was followed by thoracic irradiation of 60 gray (Gy) in 30 fractions of 2 Gy over 4 weeks (once daily during Weeks 1 and 2 and twice daily during Weeks 3 and 4). RESULTS: With a median follow-up time of 12.2 months (27-65 months for survivors), the median survival was 12.2 months (16.6 months for patients with no prior weight loss and 7.8 months for those with prior weight loss). The response rate after induction chemotherapy was 46.1%, increasing to 74.4% after radiation therapy (8 complete responses and 21 partial responses). The rate of progression was 13 of 18 (72%) for those who responded to chemotherapy (4 distant, 9 local) and 18 of 21 (86%) for those who did not respond to chemotherapy (14 distant, 7 local). The most frequent acute Grade 3 toxicity was nausea (21.4%). CONCLUSIONS: Accelerated thoracic irradiation after induction chemotherapy is well tolerated and yields therapeutic results that compare favorably with those reported for other regimens of chemotherapy and standard fractionated radiotherapy. The data from this study suggest that the responses of patients with clinically apparent disease to induction chemotherapy might indicate a likelihood of controlling microscopic distant metastases.

Is it arthritis? Beware the mimics.

BACKGROUND: The term arthritis is not specific enough to be regarded as a diagnosis. Many patients believe any joint pain is due to 'arthritis' but their understanding of the term can be very vague. OBJECTIVE: It is important to have a working protocol when a patient presents with aching joints. This article draws attention to the variety of possibilities that may underly the patient's symptoms. DISCUSSION: A variety of local and systemic causes may explain pain in one or more joints. Differentiating these requires a thorough and purposeful history. Eliciting certain features such as the presence of stiffness, the occurrence of night pain or associated features such as fever or weight loss help differentiate underlying pathology. In older people it is important to bear in mind the possibility of polymyalgia rheumatica. By considering the range of possibilities, the practitioner is less likely to be fooled by the mimics.

Temperature-induced denaturation of beta-glycosidase from the archaeon Sulfolobus solfataricus.

The beta-glycosidase isolated from the extreme thermophilic archaeon Sulfolobus solfataricus, grown at 87 degrees C, is a tetrameric protein with a molecular mass of 240 kDa. This enzyme is barely active at 30 degrees C and has optimal activity, over 95 degrees C, at pH 6.5. Its thermal stability was investigated at pH 10.1 and 10.6 by means of functional studies, circular dichroism and differential scanning calorimetry. There was no evidence of thermal activation of the enzyme and the temperature-induced denaturation was irreversible and not well represented by the two-state transition model. A more complex process occurred, involving the dissociation and unfolding of subunits, and subsequent nonspecific association and/or aggregation. Denaturation temperature was around 85 degrees C, depending on protein concentration. The denaturation enthalpy change was between 7,500 and 9,800 kJ.mol-1, depending on the pH. The collapse of the native structure around 85 degrees C was confirmed by circular dichroism measurements and time-dependent activity studies. Finally, preliminary investigations were performed on the recombinant enzyme expressed in Escherichia coli.

Involvement of Ca2+ in the H2O2-induced increase in endothelial permeability.

We studied the role of Ca2+ in mediating the hydrogen peroxide (H2O2)-induced increase in endothelial permeability to 125I-labeled albumin using bovine pulmonary microvessel endothelial cells (BMVEC). Changes in cytosolic-free Ca2+ ([Ca2+]i) were monitored in BMVEC monolayers loaded with the Ca(2+)-sensitive membrane permeant fluorescent dye fura 2-AM. H2O2 (100 microM) produced a rise in [Ca2+]i within 10 s that was reduced by the addition of EGTA to the medium. Uptake of 45Ca2+ from the extracellular medium increased in the presence of H2O2 (100 microM) compared with control monolayers, suggesting that the H2O2-induced rise in [Ca2+]i is partly the result of extracellular Ca2+ influx. The effects of [Ca2+]i on endothelial permeability were addressed by pretreatment of BMVEC monolayers with BAPTA-AM (3-5 microM), a membrane permeant Ca2+ chelator, before the H2O2 exposure. BAPTA-AM produced an approximately 50% decrease in the H2O2-induced increase in endothelial permeability compared with endothelial cell monolayers exposed to H2O2 alone. The increase in endothelial permeability was independent of Ca2+ influx, since LaCl3 (0-100 microM), which displaces Ca2+ from binding sites on the cell surface, did not modify the permeability response. These results indicate that the rise in [Ca2+]i produced by H2O2 is a critical determinant of the increase in endothelial permeability.

The liquid amide transfer model and the unfolding thermodynamics of small globular proteins.

In this paper, the solid cyclic dipeptide model developed by Murphy and Gill is analysed in order to point out that, apart from general thermodynamic features shown by well-characterized small globular proteins, only the polar and apolar contributions to the net denaturation heat capacity change are necessary to calculate the so-called protein stability curve, delta dGzero versus temperature. We propose that these specific heat capacity contributions can be determined in a reliable manner by a group additivity analysis of the transfer process of liquid amides from pure liquid phase into water. This suggests that the unfolding process, thought of as the transfer of amino acid residues from the protein 'core' to contact with water molecules, can be modelled based on the transfer process of organic amides. The reliability of the model is tested in comparison with literature data.

Evidence for cellular heterogeneity in primary cultures of human orbital fibroblasts.

Orbital fibroblasts in culture display phenotypic attributes that distinguish them from fibroblasts derived from other anatomical regions. The current studies were conducted to define potential cellular heterogeneity among orbital fibroblasts with regard to 1) differential expression of Thy-1, a 25-kilodalton glycoprotein associated with cell signaling; 2) cells undergoing a change in shape in response to prostaglandin E2 (PGE2); and 3) differences in morphology and Thy-1 expression between single cell-derived clonal fibroblast strains. On the basis of flow cytometric analysis using an anti-Thy-1 monoclonal antibody, 65% of intact orbital fibroblasts expressed surface Thy-1 (n = 5; range, 54-71%). In contrast, greater than 95% of the fibroblasts present in the five dermal strains tested were Thy-1 positive. A total of six strains of orbital fibroblasts were assessed for their shape change response to a 4-h treatment with PGE2 (100 nmol/L). A mean of 37% of the fibroblasts present in each culture responded to PGE2 (range, 22-50%). In contrast, only 1% of dermal fibroblasts exhibited any change in morphology. Three separate clones were generated from a single parent strain of Graves' orbital fibroblasts. These clones consisted of homogeneous appearing cells; however, substantial clone to clone differences in morphology were stably expressed for several population doublings. Thy-1 was expressed uniformly in cells of two clones, whereas the third was Thy-1 negative. Factor VIII and smooth muscle-specific alpha-actin were undetectable in any of the orbital or dermal cultures examined. Thus, Thy-1 expression is uniform in fibroblasts from certain anatomical regions such as the skin and heterogeneous in cells derived from human lung and orbit. These findings suggest that human orbital connective tissue may have a complexity not previously appreciated.

Extracellular matrix hyaluronan is a determinant of the endothelial barrier.

We measured the hydraulic conductivity (Lp) of the extracellular matrix (ECM) obtained after detaching bovine pulmonary microvascular endothelial (BPMVEC) and bovine pulmonary arterial endothelial cell (BPAEC) monolayers from the ECM at different days postseeding. From day 1 to day 5 in culture, the total Lp (i.e., of cell monolayer + ECM) decreased from basal values of 17.1 +/- 4.0 to 8.5 +/- 1.6 x 10(-6) cm.s-1.cmH2O-1 in BPAEC (P < 0.05) and 7.6 +/- 1.1 to 3.7 +/- 0.8 in BPMVEC (P < 0.05), respectively, and on day 5 the total Lp values were lower in BPMVEC than in BPAEC (P < 0.05). On the 5th day, ECM Lp was 55.0 +/- 8.3 in BPAEC and 10.7 +/- 0.9 cm.s-1.cmH2O-1 in BPMVEC (P < 0.05), indicating that the contribution of ECM to the total Lp was greater in BPMVEC than in BPAEC. Treatment of [3H]acetate-labeled ECM with Streptomyces hyaluronidase (HAse; 6 U/ml for 10 min) released sixfold greater radioactivity in BPMVEC compared with untreated BPMVEC controls; a similar treatment of BPAEC did not release detectable radioactivity indicative of a higher hyaluronan content in the BPMVEC ECM. HAse treatment reduced the differences in total Lp between BPMVEC and BPAEC at different days postseeding. Moreover, on the 5th day after seeding, the ECM Lp of BPMVEC increased to a greater extent after HAse treatment than the ECM of BPAEC. These data indicate that the hyaluronan component of the ECM is an important determinant of the endothelial liquid-exchange barrier.

Shoulder pain in a community-based rheumatology clinic.

The objective of this study was to assess the prevalence of different shoulder disorders likely to be experienced by a rheumatologist in a community-based rheumatology clinic. We assessed patients with shoulder pain presenting to a large general practice at a community-based rheumatology clinic. It was found that the more common conditions seen were rotator cuff lesions (65%), pericapsular soft tissue pain (11%), acromioclavicular joint pain (10%) and referred pain from cervical spine (5%). In conclusion this study has established the spectrum of shoulder disorders referred from general practice to a highly accessible community-based rheumatology clinic. The diagnostic processes to distinguish the different conditions rely chiefly on an accurate history and directed examination.

Growth characteristics of pulmonary artery smooth muscle cells from fawn-hooded rats.

We compared the proliferative rates of vascular smooth muscle cells (VSMC) from pulmonary arteries of pulmonary hypertensive fawn-hooded rats (FHR) with VSMC from normotensive Sprague-Dawley rats (SDR). VSMC from FHR grew at increased rates and reached higher densities at all serum concentrations studied (5-20%) than the VSMC from SDR. The VSMC from FHR also responded to epidermal growth factor (EGF) at low serum concentrations, as evidenced by significantly greater DNA synthetic rates, than the control VSMC. The increased growth in these cells could be due to increased number and/or affinity of EGF receptors because of the higher specific binding of 125I-EGF to the VSMC from FHR. The VSMC from FHR and SDR were equally sensitive to the antiproliferative effects of heparin, suggesting that the heparin-sensitive pathways are not altered in the VSMC from FHR. These results suggest that the development of pulmonary hypertension in FHR may be related to the higher proliferative capacity of the pulmonary VSMC, which may be coupled to increased activity of the EGF receptors on these cells.