Lactose permease of Escherichia coli: properties of mutants defective in substrate translocation.

Mutants of lactose permease of Escherichia coli with amino acid changes (Gly-24----Glu; Gly-24----Arg; Pro-28---Ser; Gly-24, Pro-28----Glu-Ser and Gly-24, Pro-28----Arg-Ser) within a putative membrane-spanning alpha-helix (Phe-Gly-Leu-Phe-Phe-Phe-Phe-Tyr-Phe-Phe-Ile-Met-Gly- Ala-Tyr-Phe-Pro-Phe-Phe-Pro-Ile) are incorporated into the cytoplasmic membrane. The mutant proteins retain the ability to bind galactosides, and the affinity for several substrates is actually increased. However, the rate of active transport is decreased to 0.01% of the wild-type rate in the mutants carrying Arg-24 or Arg-24, Ser-28. Kinetic analysis demonstrates that the two mutants require 10 min to cause occupied binding sites for galactoside and H+ to change their exposure from the periplasm to the cytoplasm as compared to 50 ms in the wild type. The effect is less pronounced when these sites are unoccupied.     

Effects of short-term exposure to fine and ultrafine particles from indoor sources on arterial stiffness – A randomized sham-controlled exposure study

ObjectivesParticulate air pollution is linked to adverse cardiovascular effects, including arterial stiffness. The aim of the study was to investigate the effect of short-term exposure to indoor fine and ultrafine particles on augmentation index (AIx), augmentation pressure (AP), and pulse wave velocity (PWV), early signs of vascular damage.MethodsWe analyzed the association of particle emissions from typical indoor sources (candle burning - CB, toasting bread - TB, and frying sausages - FS) with changes in pulse wave analysis indices in 55 healthy adults in a randomized cross-over controlled exposure study. Particle mass concentration (PMC), size-specific particle number concentration (PNC) and lung-deposited particle surface area concentration (PSC) were measured during the 2 h exposure. AIx and AP were measured before, directly, 2, 4 and 24 h after exposure. PWV was measured directly and 24 h after exposure. We performed multiple mixed linear regression analyses of different particle metrics and AIx, AP and PWV.ResultsThe highest mean PMC was observed during FS reaching a maximum of 210 μg/m³ PM₁₀. The maximal PNC for UFP <100 nm was reached during CB with 2.3 million particles/cm³. PSC was similar across all three exposures (about 3000 μm²/cm³). Strongest associations between different particles metrics and arterial stiffness indices could be observed for UFP from CB and FS and for PMC from TB. The highest mean increase could be observed for the UFP fraction <10 nm, measured during CB, and AIx with an increase of 9.5%-points (95%-CI: 3.1; 15.9). PSC seemed to follow the pattern of PNC. PM₁₀ and PM_2.5 from TB led to clear changes in AIx with biggest increases for PM₁₀ of 5.8%-points (95%-CI: 3.2; 8.4) 2 h after exposure and for PM_2.5 of 8.1%-points (95%-CI: 2.5; 13.7) directly after exposure.ConclusionsOur study indicates effects of indoor exposure to fine and ultrafine particles on systemic arterial stiffness indices that depend on the indoor source as well as on particle metric. Differences in size-specific physical characteristics of source-specific particles might account for these differential effects. We did not observe clear and stable associations of indoor particle exposure and PWV.     

A Gene Expression Profile of the Myocardial Response to Clenbuterol

Clenbuterol is currently being used as part of a clinical trial into a novel therapeutic approach for the treatment of end-stage heart failure. The purpose of this study was to determine the global pattern of myocardial gene expression in response to clenbuterol and to identify novel targets and pathways involved. Rats were treated with clenbuterol (n = 6) or saline (n = 6) for periods of 1, 3, 9, or 28 days. Rats treated for 28 days were also subject to continuous electrocardiogram analysis using implantable telemetry. RNA was extracted from rats at days 1 and 28 and used from microarray analysis, and further samples from rats at days 1, 3, 9, and 28 were used for analysis by real-time polymerase chain reaction. Clenbuterol treatment induced rapid development of cardiac hypertrophy with increased muscle mass at day 1 and elevated heart rate and QT interval throughout the 28-day period. Microarray analysis revealed a marked but largely transitory change in gene expression with 1,423 genes up-regulated and 964 genes down-regulated at day 1. Up-regulated genes revealed an unexpected association with angiogenesis and integrin-mediated cell adhesion and signaling. Moreover, direct treatment of endothelial cells cultured in vitro resulted in increased cell proliferation and tube formation. Our data show that clenbuterol treatment is associated with rapid cardiac hypertrophy and identify angiogenesis and integrin signaling as novel pathways of clenbuterol action. The data have implications both for our understanding of the physiologic hypertrophy induced by clenbuterol and for treatment of heart failure. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of cell-specific soluble mediators and cellular targets during cell therapy for the treatment of heart failure

Cell therapy, the transplantation of progenitor cells into the myocardium, has been proposed as a possible treatment strategy for heart failure. Despite the lack of repopulation of the heart with progenitor cells, cell therapy induces a modest but well-documented functional improvement in patients. It is thought that paracrine mechanisms may account for the observed changes in heart function. However, there is little evidence that directly supports this hypothesis. We discuss the current views in the literature and present some preliminary data proposing that adult progenitor cells influence contractility and Ca2+ handling in neighboring failing cardiomyocytes by soluble mediators. This can be tested using a co-culture system. Our results suggest that soluble mediators from adult progenitor cells can enhance failing cardiomyocyte function, supporting the paracrine hypothesis. This co-culture strategy can be employed to identify cell-specific soluble mediators and their cellular targets during cell therapy for the treatment of heart disease.     

Spatial changes in the water quality of Itajaí-Açú Fluvial-Estuarine System, Santa Catarina, Brazil

This study was carried out with the aim of evaluating the spatial variation of the water quality in the Itajaí-A?ú River estuary. Seven stations along the estuary were monitored on a weekly basis, from October 2003 to December 2004, plus two stations in tributaries (Itajaí-Mirim River, the main tributary, and one reference station). This monitoring included measurements of salinity, pH, dissolved oxygen, temperature, nutrients(NH+4,NO3-2,NO-3,PO3-4,H4SiO4) Biochemical Oxygen Demand (BOD), total phosphorous and dissolved organic phosphorus (TP and DOP), particulate organic carbon (POC), suspended particulate matter (SPM) and chlorophyll-a (Cla). Multivariate analyses demonstrated the compartmentalization of the system based on the deterioration in water quality and marine influence. Urban development was the main factor responsible for the spatial variation of the monitored variables, resulting in increases in the indicators for organic matter and a progressive decrease in O2. Despite the effect of dilution by marine influence, there was an increase in ammonium, attributed to the influence of the municipal districts of Itajaí and Navegantes, close to the river mouth.     

Spiral CT angiography and 3D reconstruction in patients with aortic coarctation

The objective of this study was to assess the reliability of spiral CT angiography (CTA) and 3D reconstruction in patients with aortic coarctation (CoA). Eighteen patients with suspected or surgically proven coarctation were examined by spiral CT. In addition to the axial slices, 3D reconstructions, such as shaded surface display (SSD) and maximum intensity projection (MIP), were used to determine the diameters of the CoA and the pre- and poststenotic aorta and to visualise the collateral vessels. Diameters derived from cardiac catheterization were compared with those from CTA in 8 patients. The degree of aortic stenosis was correlated with blood pressure gradients (BPG) in 12 patients. The difference between the diameters of the CoA and the pre- and poststenotic aorta derived from MIP and angiography was not statistically significant (p = 0.69). With SSD the internal thoracic artery was detected in 16 and the posterior intercostal artery in 13 cases. The degree of aortic stenosis correlated poorly with the BPG (r = 0.51, r ² = 0.26). CTA with 3D reconstruction represents a reliable noninvasive technique for the assessment of the degree of CoA and the visualisation of collateral vessels. It may serve as a follow-up investigation after intervention or surgical treatment. Received 22 November 1996; Revision received 5 February 1997; Accepted 10 March 1997     

Role of aspartate-96 in proton translocation by bacteriorhodopsin.

Proton transfer reactions in bacteriorhodopsin were investigated by Fourier transform infrared spectroscopy, using a mutant protein in which Asp-96 was replaced by Asn-96. By comparison of the BR - K, BR - L, and BR - M difference spectra (BR indicating bacteriorhodopsin ground state and K, L, and M indicating photo-intermediates) of the wild-type protein with the corresponding difference spectra of the mutant protein, detailed insight into the functional role of this residue in the proton pump mechanism is obtained. Asp-96 is protonated in BR, as well as another aspartic residue, which is tentatively assigned to be Asp-115. Asp-96 is not affected in the primary photoreaction. During formation of the L intermediate it is subjected to a change in the H-bonding character of its carboxylic group, but no deprotonation occurs at this reaction step. Also, in the mutant protein a light-induced structural change of the protein interior near the Asn-96 residue is probed. The BR - M difference spectrum of the mutant protein lacks the negative carbonyl band at 1742 cm-1 of Asp-96 and in addition a positive band at about 1378 cm-1, which is most likely to be caused by the carboxylate vibration of Asp-96. This argues for a deprotonation of Asp-96 in the time range of the M intermediate during its photostationary accumulation. On the basis of these results, it is suggested that the point mutation does not induce a gross change of the protein structure, but a proton-binding site in the proton pathway from the cytoplasmic side to the Schiff base is lost.     

Role and possible mechanisms of clenbuterol in enhancing reverse remodelling during mechanical unloading in murine heart failure

AIMS: Combined left ventricular assist device (LVAD) and pharmacological therapy has been proposed to favour myocardial recovery in patients with end-stage heart failure (HF). Clenbuterol (Clen), a beta(2)-adrenoceptor (beta(2)-AR) agonist, has been used as a part of this strategy. In this study, we investigated the direct effects of clenbuterol on unloaded myocardium in HF. METHODS AND RESULTS: Left coronary artery ligation or sham operation was performed in male Lewis rats. After 4-6 weeks, heterotopic abdominal transplantation of the failing hearts into normal recipients was performed to induce LV unloading (UN). Recipient rats were treated with saline (Sal) or clenbuterol (2 mg/kg/day) via osmotic minipumps (HF + UN + Sal or HF + UN + Clen) for 7 days. Non-transplanted HF animals were treated with Sal (Sham + Sal, HF + Sal) or clenbuterol (HF + Clen). LV myocytes were isolated and studied using optical, fluorescence, and electrophysiological techniques. Clenbuterol treatment improved in vivo LV function measured with echocardiography (LVEF (%): HF 35.9 +/- 2 [16], HF + Clen 52.1 +/- 1.4 [16]; P < 0.001; mean +/- SEM [n]). In combination with unloading, clenbuterol increased sarcomere shortening (amplitude (microm): HF + UN + Clen 0.1 +/- 0.01 [50], HF + UN + Sal 0.07 +/- 0.01 [38]; P < 0.001) by normalizing the depressed myofilament sensitivity to Ca(2+) (slope of the linear relationship between Ca(2+) transient and sarcomere shortening hysteresis loop during relaxation (microm/ratio unit): HF + UN + Clen 2.13 +/- 0.2 [52], HF + UN + Sal 1.42 +/- 0.13 [38]; P < 0.05). CONCLUSION: Clenbuterol treatment of failing rat hearts, alone or in combination with mechanical unloading, improves LV function at the whole-heart and cellular levels by affecting cell morphology, excitation-contraction coupling, and myofilament sensitivity to calcium. This study supports the use of this drug in the strategy to enhance recovery in HF patients treated with LVADs and also begins to elucidate some of the possible cellular mechanisms responsible for the improvement in LV function.