Calcium transfer across the outer mantle epithelium in the Pacific oyster, Crassostrea gigas.

Calcium transport is essential for bivalves to be able to build and maintain their shells. Ionized calcium (Ca2+) is taken up from the environment and eventually transported through the outer mantle epithelium (OME) to the shell growth area. However, the mechanisms behind this process are poorly understood. The objective of the present study was to characterize the Ca2+ transfer performed by the OME of the Pacific oyster, Crassostrea gigas, as well as to develop an Ussing chamber technique for the functional assessment of transport activities in epithelia of marine bivalves. Kinetic studies revealed that the Ca2+ transfer across the OME consists of one saturable and one linear component, of which the saturable component fits best to Michaelis-Menten kinetics and is characterized by a Km of 6.2 mM and a Vmax of 3.3 nM min-1 The transcellular transfer of Ca2+ accounts for approximately 60% of the total Ca2+ transfer across the OME of C. gigas at environmental Ca2+ concentrations. The use of the pharmacological inhibitors: verapamil, ouabain and caloxin 1a1 revealed that voltage-gated Ca2+-channels, plasma-membrane Ca2+-ATPase and Na+/Ca2+-exchanger all participate in the transcellular Ca2+ transfer across the OME and a model for this Ca2+ transfer is presented and discussed.

Dilution of Seawater Affects the Ca2 + Transport in the Outer Mantle Epithelium of Crassostrea gigas.

Varying salinities of coastal waters are likely to affect the physiology and ion transport capabilities of calcifying marine organisms such as bivalves. To investigate the physiological effect of decreased environmental salinity in bivalves, adult oysters (Crassostrea gigas) were exposed for 14 days to 50% seawater (14) and the effects on mantle ion transport, electrophysiology and the expression of Ca2+ transporters and channels relative to animals maintained in full strength sea water (28) was evaluated. Exposure of oysters to a salinity of 14 decreased the active mantle transepithelial ion transport and specifically affected Ca2+ transfer. Gene expression of the Na+/K+-ATPase and the sarco(endo)plasmic reticulum Ca2+-ATPase was decreased whereas the expression of the T-type voltage-gated Ca channel and the Na+/Ca2+-exchanger increased compared to animals maintained in full SW. The results indicate that decreased environmental salinities will most likely affect not only osmoregulation but also bivalve biomineralization and shell formation.

Calcium mobilisation following shell damage in the Pacific oyster, Crassostrea gigas.

Shell growth of oysters requires calcium uptake from the environment and transport to the area of shell formation. A shell regeneration assay in combination with radiolabelled calcium was used to investigate uptake and distribution of calcium to different tissues and hemolymph fractions in Pacific oysters, Crassostrea gigas (Bivalvia, Ostreoida). Oysters were notched at the shell margin and subsequently sampled for hemolymph and grading of shell regeneration during a two week experimental period. Half of the oysters were additionally exposed to (45)Ca and sampled for hemolymph and tissues. Total plasma calcium concentrations increased in notched oysters compared to controls on 1, 2 and 7days after notching. A decrease in plasma calcium levels was apparent on day 4, for both total and ionic calcium. The shell regeneration assay in the notched oysters resulted in a visible deposition of CaCO3 onto the regenerate from day 7 onwards. This was coinciding with an increased uptake of total calcium on days 11 and 14 as well as free, i.e. ionic and ligand-bound calcium, on day 14. At day 1, notching also increased calcium uptake into the mantle tissues, in areas above the notch and near the hinge. During the experiment, both the total hemocyte count and the number of granulocytes increased in notched compared to control oysters. The present study suggests that induced shell damage results in a dynamic regulation of the calcium uptake from the environment and the distribution of calcium within the body, starting directly after notching. Increases in both total calcium concentrations and uptake rates coincided with the visible depositions of CaCO3 on the regenerate shell. C. gigas was found to transport calcium mainly in the ionic form in the hemolymph, with only minor parts being bound to proteins or smaller ligands. Hemolymph measurement also revealed that C. gigas is able to regulate the extracellular concentrations of calcium and potassium. The changes in plasma calcium concentrations and speciation, concomitant with increases in granulocytes indicate that multiple calcium transport processes are activated after induced shell damage.

A method for determining the gantry angle for megavoltage cone beam imaging.

Accurate knowledge of gantry angle is essential in megavoltage cone beam imaging (MVCBI) with an electronic portal imager. We present a method for determining the gantry angle by detecting multileaf collimator (MLC) leaf positions in projection images. During image acquisition the gantry moves continuously and the MLC operates in dynamic arc mode. Our algorithm detects the leaf positions in the images and compares them with a stationary reference leaf. Comparison of the algorithm against angles determined from the locations of fiducial markers shows the accuracy (0.26 degrees rms error) to be sufficient for MVCBI.

Developments in megavoltage cone beam CT with an amorphous silicon EPID: reduction of exposure and synchronization with respiratory gating.

We have studied the feasibility of a low-dose megavoltage cone beam computed tomography (MV CBCT) system for visualizing the gross tumor volume in respiratory gated radiation treatments of nonsmall-cell lung cancer. The system consists of a commercially available linear accelerator (LINAC), an amorphous silicon electronic portal imaging device, and a respiratory gating system. The gantry movement and beam delivery are controlled using dynamic beam delivery toolbox, a commercial software package for executing scripts to control the LINAC. A specially designed interface box synchronizes the LINAC, image acquisition electronics, and the respiratory gating system. Images are preprocessed to remove artifacts due to detector sag and LINAC output fluctuations. We report on the output, flatness, and symmetry of the images acquired using different imaging parameters. We also examine the quality of three-dimensional (3D) tomographic reconstruction with projection images of anthropomorphic thorax, contrast detail, and motion phantoms. The results show that, with the proper choice of imaging parameters, the flatness and symmetry are reasonably good with as low as three beam pulses per projection image. Resolution of 5% electron density differences is possible in a contrast detail phantom using 100 projections and 30 MU. Synchronization of image acquisition with simulated respiration also eliminated motion artifacts in a moving phantom, demonstrating the system's capability for imaging patients undergoing gated radiation therapy. The acquisition time is limited by the patient's respiration (only one image per breathing cycle) and is under 10 min for a scan of 100 projections. In conclusion, we have developed a MV CBCT system using commercially available components to produce 3D reconstructions, with sufficient contrast resolution for localizing a simulated lung tumor, using a dose comparable to portal imaging.

Multiple QTL mapping in related plant populations via a pedigree-analysis approach.

QTL mapping experiments in plant breeding may involve multiple populations or pedigrees that are related through their ancestors. These known relationships have often been ignored for the sake of statistical analysis, despite their potential increase in power of mapping. We describe here a Bayesian method for QTL mapping in complex plant populations and reported the results from its application to a (previously analysed) potato data set. This Bayesian method was originally developed for human genetics data, and we have proved that it is useful for complex plant populations as well, based on a sensitivity analysis that was performed here. The method accommodates robustness to complex structures in pedigree data, full flexibility in the estimation of the number of QTL across multiple chromosomes, thereby accounting for uncertainties in the transmission of QTL and marker alleles due to incomplete marker information, and the simultaneous inclusion of non-genetic factors affecting the quantitative trait.

Use of EPID for leaf position accuracy QA of dynamic multi-leaf collimator (DMLC) treatment.

We describe in this paper an alternative method for routine dynamic multi-leaf collimator (DMLC) quality assurance (QA) using an electronic portal imaging device (EPID). Currently, this QA is done at our institution by filming an intensity-modulated radiotherapy (IMRT) test field producing a pattern of five 1-mm bands 2 cm apart and performing a visual spot-check for leaf alignment, motion lags, sticking and any other mechanical problems. In this study, we used an amorphous silicon aS500 EPID and films contemporaneously for the DMLC QA to test the practicality and efficacy of EPID vis-à-vis film. The EPID image was transformed to an integrated dose map by first converting the reading to dose using a calibration curve, and then multiplying by the number of averaged frames. The EPID dose map was then back-projected to the central axis plane and was compared to the film measurements which were scanned and converted to dose using a film dosimetry system. We determined the full-width half-maximum (FWHM) of each band for both images, and evaluated the dose to the valley between two peaks. We also simulated mechanical problems by increasing the band gap to 1.5 mm for some leaf pairs. Our results show that EPID is as good as the film in resolving the band pattern of the IMRT test field. Although the resolution of the EPID is lower than that of the film (0.78 mm/pixel vs 0.36 mm/pixel for the film), it is high enough to faithfully reproduce the band pattern without significant distortion. The FWHM of the EPID is 2.84 mm, slightly higher than the 2.01 mm for the film. The lowest dose to the valley is significantly lower for the EPID (15.5% of the peak value) than for the film (28.6%), indicating that EPID is less energy independent. The simulated leaf problem can be spotted by visual inspection of both images; however, it is more difficult for the film without being scanned and contrast-enhanced. EPID images have the advantage of being already digital and their analysis can easily be automated to flag leaf pairs outside tolerance limits of set parameters such as FWHM, peak dose values, peak location, and distance between peaks. This automation is a new feature that will help preempt MLC motion interlocks and decrease machine downtime during actual IMRT treatment. We conclude that since EPID images can be acquired, analyzed and stored much more conveniently than film, EPID is a good alternative to film for routine DMLC QA.

Carvedilol and atenolol once daily in the treatment of hypertension.

Fifty milligrams of carvedilol and 100 mg atenolol were administered in a random order once a day for 2 months to 43 patients with mild to moderate hypertension, in a double-blind crossover study. Blood pressure, heart rate and peripheral blood flow parameters (n = 11) were recorded 2 and 24 h after the drug administration. Supine blood pressure was the same 2 h after both carvedilol and atenolol administration, but carvedilol caused a greater decrease in standing systolic blood pressure 2 h after the administration (P less than 0.05). The heart rate decreased less with carvedilol (P less than 0.01). There was no difference in the effects exerted by the two therapies on systolic blood pressure and the heart rate 24 h after drug administration, but the diastolic blood pressure was higher in patients given carvedilol (92 versus 88 mmHg; P less than 0.05). Forearm blood flow, forearm vascular resistance and calf blood flow did not change significantly with either of the therapies. In conclusion, 50 mg carvedilol once a day is an effective antihypertensive therapy, though its duration of action did not reach that of 100 mg atenolol once a day. Peripheral vasodilation was similar with both therapies.

An implementation of the HACCP system in the production of food-packaging material.

The Hazard Analysis Critical Control Point (HACCP) system according to the Codex Alimentarius model was applied to the processes of five paper and paperboard mills and four plants further converting paper or board intended for contact with foodstuffs. The generalised flow diagrams of the processes are presented. Each of the overall processes contained 40-150 process steps. Normally three to five sessions with HACCP teams and additional private negotiations were needed for each mill or plant. Hazards leading to critical control points (CCPs) were microbiological (handling/storage, circulation water, starch, process environment) and physical (process environment) in mills, and microbiological (storage, lacquers or glues, packaging and process environment), chemical (printing) and physical (storage of products, packaging and process environment) in plants. Specifications, critical limits (e.g., based on different kinds of reports and instructions), monitoring methods (microbiological and visual) and frequency, responsibilities and corrective actions of the processes are presented. Most of the improvements focused on improving the process environment. In five cases, hygiene training was included in the implementation of the HACCP system.

Potential microbiological hazards in the production of refined paper products for food applications.

This study sought to investigate the significance of raw materials (starch-based glues, raw material papers) at different microbiologically critical stages in the manufacturing process of refined paper products. The study examined the occurrence of microorganisms in the process and in end-product samples. Microbiological surveys verified that the production and use of pasteurized starch-based glue was the most important factor threatening the process hygiene and product safety. Subsequently, the production and use of starch-based glue was changed, and a follow-up programme targeting the microbiological quality of glue was developed as part of a hygiene and safety management system. A total of 33 spore-forming bacterial and 15 enterobacterial isolates were ribotyped, and 22 and 10 different ribogroups (ribotypes), respectively, were generated. These isolates from starch-based glue, raw material paper and end products were atypical and, thus, in many cases physiological, chemotaxonomic (FAME) and molecular (partial 16S rDNA) results did not correspond. The most common spore-forming bacteria (55% of the isolates) were Paenibacillus sp. and within this genus several new species were also proposed. The most common enterobacteria (87%) were Enterobacter cloacae and Citrobacter freundii belonging to bacteria in hazard group 2, or species closely related to them. It was demonstrated that the same spore-forming bacteria (ribotypes) were present in both the glue samples and the end products (45% of isolates). All RiboPrint patterns were saved at the VTT identification library for future use.

Effect of work with visual display units on musculo-skeletal disorders in the office environment.

BACKGROUND: The increase in computer and mouse use has been associated with an increased prevalence of disorders in the neck and upper extremities. Furthermore, poor workstation design has been associated with an increased risk of developing these symptoms. Aim The aims of this study were (i) to estimate the prevalence of musculo-skeletal disorders among full-time visual display unit (VDU) users; (ii) to examine how the prevalence varies by work environment; and (iii) to explore the association with work factors. METHOD: A survey was carried out on the effect of work with VDUs on musculo-skeletal disorders in workers in the office environment of 56 workplaces. Office workers (n = 298), customer service workers (n = 238) and designers (n = 247) were studied. RESULTS: For all the occupations combined, the 12 month prevalences of musculo-skeletal symptoms in the neck, shoulders, elbows, lower arms and wrists, and fingers were 63, 24, 18, 35 and 16%, respectively. The study indicated that musculo-skeletal pain is common among computer workers in offices. There was no strong association between the duration of computer work and pain or between the duration of mouse use and pain, but workers' perception of their workstation as being poor ergonomically was strongly associated with an increased prevalence of pain. CONCLUSIONS: Musculo-skeletal symptoms are common, but the duration of daily keyboard and mouse use had no connection with musculo-skeletal symptoms. Instead, more consideration should be paid to the ergonomics of workstations, the placing of the mouse, the postures of the upper extremities and the handling of the mouse.

Inhibition of the adherence of Escherichia coli strains to basement membrane by Lactobacillus crispatus expressing an S-layer.

AIMS: This study aimed to evaluate the efficiency with which Lactobacillus crispatus JCM 5810 inhibited the adhesion of enteric pathogens to a synthetic basement membrane and to elucidate the mechanism underlying the inhibition. METHODS AND RESULTS: Lactobacillus crispatus JCM 5810 inhibited the adhesion of three diarrhoeagenic Escherichia coli strains to a reconstituted basement membrane preparation called Matrigel, used as a model of a damaged intestinal tissue site. Inhibition was also observed with the use of immobilized laminin, a major component of Matrigel, but diminished after the removal of S-layer protein (CbsA) from JCM 5810 cells. The isolated CbsA inhibited the adhesion of E. coli to both Matrigel and immobilized laminin. Lactobacillus crispatus JCM 5810 and CbsA seem to inhibit pathogenic E. coli from adhering to basement membrane via competition with laminin molecules for binding sites. CONCLUSIONS: These results suggested that not only Lact. crispatus JCM 5810 cells but CbsA alone might prevent pathogens from colonizing damaged intestinal tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to show the applied aspect of Lactobacillus S-layer protein.

Expression of cbsA encoding the collagen-binding S-protein of Lactobacillus crispatus JCM5810 in Lactobacillus casei ATCC 393(T).

The cbsA gene encoding the collagen-binding S-layer protein of Lactobacillus crispatus JCM5810 was expressed in L. casei ATCC 393(T). The S-protein was not retained on the surface of the recombinant bacteria but was secreted into the medium. By translational fusion of CbsA to the cell wall sorting signal of the proteinase, PrtP, of L. casei, CbsA was presented at the surface, rendering the transformants able to bind to immobilized collagens.

A Collagen-Binding S-Layer Protein in Lactobacillus crispatus.

Two S-layer-expressing strains, Lactobacillus crispatus JCM 5810 and Lactobacillus acidophilus JCM 1132, were assessed for adherence to proteins of the mammalian extracellular matrix. L. crispatus JCM 5810 adhered efficiently to immobilized type IV and I collagens, laminin, and, with a lower affinity, to type V collagen and fibronectin. Strain JCM 1132 did not exhibit detectable adhesiveness. Within the fibronectin molecule, JCM 5810 recognized the 120-kDa cell-binding fragment of the protein, while no bacterial adhesion to the amino-terminal 30-kDa or the gelatin-binding 40-kDa fragment was detected. JCM 5810 but not JCM 1132 also bound (sup125)I-labelled soluble type IV collagen, and this binding was efficiently inhibited by unlabelled type IV and I collagens and less efficiently by type V collagen, but not by laminin or fibronectin. L. crispatus JCM 5810 but not L. acidophilus JCM 1132 also adhered to Matrigel, a reconstituted basement membrane preparation from mouse sarcoma cells, as well as to the extracellular matrix prepared from human Intestine 407 cells. S-layers from both strains were extracted with 2 M guanidine hydrochloride, separated by electrophoresis, and transferred to nitrocellulose sheets. The S-layer protein from JCM 5810 bound (sup125)I-labelled type IV collagen, whereas no binding was seen with the S-layer protein from JCM 1132. Binding of (sup125)I-collagen IV to the JCM 5810 S-layer protein was effectively inhibited by unlabelled type I and IV collagens but not by type V collagen, laminin, or fibronectin. It was concluded that L. crispatus JCM 5810 has the capacity to adhere to human subintestinal extracellular matrix via a collagen-binding S-layer.

Efficacy and tolerability of felodipine ER and diltiazem SR as monotherapy in primary hypertension: a double-blind randomized study.

PURPOSE: Efficacy, tolerability, and optimal doses of felodipine ER (FER) and diltiazem SR (DSR), given as monotherapy, were evaluated in patients with mild or moderate primary hypertension. METHODS: This was a multicenter, double-blind, parallel-group study of 98 hypertensive patients. Following a 4 weeks placebo run-in period, patients were randomized to either FER 5 mg once daily (qd) or DSR 90 mg twice daily (bid). If supine DBP was > 90 mmHg after 2 and 4 weeks treatment, the dose was increased to 10 mg FER qd or 120 mg DSR bid plus 20 mg FER qd or 180 mg DSR bid, respectively. The double-blind treatment lasted 8 weeks. RESULTS: After 8 weeks FER treatment 70% of the patients responded (DBP < or = 90 mmHg or DBP decrease > or = 10 mmHg) and 50% became normotensive (DBP < or = 90 mmHg); the corresponding figures for DSR were 63% and 37%, respectively. No statistical significant differences in BP reduction and HR were found between the two compounds. HR did not change during the study. Seven patients discontinued due to adverse events (AEs). Five patients received FER and two patients received DSR. The AEs were similar in the two groups and generally mild. CONCLUSIONS: At the highest dose levels of FER and DSR, no further BP reduction was observed, but there was a tendency to report more AEs. Both FER and DSR can be used as first-line therapy in hypertension.

Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus.

The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.