Multiple cis-regulatory elements and the yeast sulphur regulatory network are required for the regulation of the yeast glutathione transporter, Hgt1p

HGT1 encodes a high-affinity glutathione transporter in the yeast Saccharomyces cerevisiae that is induced under sulphur limitation. The present work demonstrates that repression by organic sulphur sources is under the control of the classic sulphur regulatory network, as seen by the absence of expression in a met4 background. Cysteine appeared to be the principal regulatory molecule, since elevated levels were seen in str4 strains (deficient in cysteine biosynthesis) that could be repressed by elevated levels of cysteine, but not by methionine or glutathione. Investigations into cis-regulatory elements revealed that the previously described motif, a 9-bp cis element, CCGCCACAC, located at the –356 to –364 region of the promoter could in fact be refined to a 7-bp CGCCACA motif that is also repeated at –333 to –340. The second copy of this motif was essential for activity, since mutations in the core region of the second copy completely abolished activity and regulation by sulphur sources. Activity, but not regulation, could be restored by reintroducing an additional copy upstream of the first copy. A third region, GCCGTCTGCAAGGCA, conserved in the HGT1 promoters of the different Saccharomyces spp, was observed at –300 to –285 but, while mutations in this region did not lead to any loss in repression, the basal and induced levels were significantly increased. In contrast to a previous report, no evidence was found for regulation by the VDE endonuclease. The strong repression at the transport level by glutathione seen in strains overexpressing HGT1 was due to a glutathione-dependent toxicity in these cells.     

Chondromyxoid fibroma resembles in vitro chondrogenesis, but differs in expression of signalling molecules

Chondromyxoid fibroma is a rare benign cartilaginous bone tumour characterized by morphological features that resemble different steps of chondrogenesis in terms of both cellular morphology, ranging from spindled to rounded cells, and the extracellular matrix formed, which ranges from fibrous to cartilaginous. The presence in chondromyxoid fibroma of signalling molecules that regulate the spatial expression of proteins involved in normal cartilage proliferation and differentiation was investigated in samples from 20 patients and compared with articular chondrocytes from 11 normal donors cultivated in 3D pellet culture. Sections were stained with safranin-O and H&E, and immunohistochemistry was performed for p16, cyclin D1, FGFR3, BCL2, p21, PTHLH, PTHR1 and N-cadherin. Expression patterns were analysed using hierarchical clustering. In chondromyxoid fibroma, specific morphological features correlated with a distinct pattern of expression. Comparison with normal chondrocytes in pellet culture showed a striking morphological resemblance, but with an unmistakably different pattern of expression. N-cadherin, PTHLH, and PTHR1 were expressed to a significantly higher level (p < 0.01) in articular chondrocyte pellets but, conversely, there was significantly lower expression of cyclin D1, p16 and BCL2 (p < 0.05) in these cells. Morphological similarities reflect common steps in cartilage differentiation, albeit driven by different molecular mechanisms. The proteins we have found to be differentially expressed seem crucial for neoplastic chondrogenesis.     

Using probabilistic and decision-theoretic methods in treatment and prognosis modeling

Causal probabilistic networks, also called Bayesian networks, allow both qualitative knowledge about the structure of a problem and quantitative knowledge, derived from case databases, expert opinion and literature to be exploited in the construction of decision support systems for diagnosis, treatment and prognosis. This mixing of qualitative and quantitative knowledge will be illustrated, using the selection of antibiotics for a subset of patients with severe infections. The subset consists of patients where bacteria or fungi have been found in the blood. A simple pathophysiological model of infection is used to calculate a prognosis, dependent on the choice of antibiotics. A decision-theoretic approach is used to balance the therapeutic benefit of antibiotic treatment against the cost of antibiotics in the form of direct monetary cost, side effects and ecological cost. A retrospective trial on patients with bacteria or fungi in the blood stemming from the urinary tract indicates that with this approach, it may be possible to suggest balanced choices of antibiotics that not only achieve greater therapeutic benefit, but also reduce the cost of therapy.     

Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis

Pseudomonas aeruginosa causes a chronic infection in the lungs of cystic fibrosis (CF) patients by establishing an alginate-containing biofilm. The infection has been studied in several animal models; however, most of the models required artificial embedding of the bacteria. We present here a new pulmonary mouse model without artificial embedding. The model is based on a stable mucoid CF sputum isolate (NH57388A) with hyperproduction of alginate due to a deletion in mucA and functional N-acylhomoserine lactone (AHL)-based quorum-sensing systems. Chronic lung infection could be established in both CF mice (Cftr(tmlUnc-/-)) and BALB/c mice, as reflected by the detection of a high number of P. aeruginosa organisms in the lung homogenates at 7 days postinfection and alginate biofilms, surrounded by polymorphonuclear leukocytes in the alveoli. In comparison, both an AHL-producing nonmucoid revertant (NH57388C) from the mucoid isolate (NH57388A) and a nonmucoid isolate (NH57388B) deficient in AHL were almost cleared from the lungs of the mice. This model, in which P. aeruginosa is protected against the defense system of the lung by alginate, is similar to the clinical situation. Therefore, the mouse model provides an improved method for evaluating the interaction between mucoid P. aeruginosa, the host, and antibacterial therapy.     

'Vanishing embryo syndrome' in IVF/ICSI

BACKGROUND: In a Danish population-based cohort study assessing the risk of cerebral palsy in children born after IVF, we made some interesting observations regarding 'vanishing co-embryos'. METHODS and RESULTS: All live-born children born in Denmark from 1 January 1995 to 31 December 2000 were included in this analysis. The children conceived by IVF/ICSI (9444) were identified through the IVF Register, the children conceived without IVF/ICSI (395 025) were identified through The Danish Medical Birth Register. Main outcome measure was the incidence of cerebral palsy. Within the IVF/ICSI children we found indications of an increased risk of cerebral palsy in those children resulting from pregnancies, where the number of embryos transferred was higher than the number of children born. CONCLUSIONS: The association between vanishing embryo syndrome and incidence of cerebral palsy following IVF requires further investigation in larger, adequately powered, studies.     

Immunohistochemical differentiation between lymphangiographically verified lymphatic vessels and blood vessels

Summary In order to investigate the antigen profile in human lymphatic vessels when compared with blood vessels, postmortem retrograde lymphangiography was done via the thoracic duct on six patients. Formalin fixed, paraffin embedded tissue was stained immunohistochemically for Factor VIII-Related antigen (F VIII R:Ag), with Ulex Europaeus 1 lectin (UEA-1) and for laminin. The results show that the endothelium of blood vessels and lymphatics at all levels of the lymphatic system react positively following staining for Factor VIII-R:Ag and with UEA-1 lectin. The staining for F VIII R:Ag was generally weaker in the endothelial cells lining lymphatic vessels. Staining for the basement membrane component laminin can be used to distinguish lymphatic capillaries and smaller lymphatic collecting vessels from blood vessels.     

Design and application of sensor for recording sounds over human eye and nose

The recording of sounds over the oribt of the eye has been found to be useful in the detection of intracranial aneurysms. A hydrophone for auscultation over the eye has been developed and is tested under controlled conditions. The tests consist of measurement over the eyes in three healthy volunteers at rest, during voluntary breathing, during eyeball movements and during sustained orbicular muscular contractions. Furthermore, measurements are performed at the side of the nose. Major features of the hydrophonic transducer are high sensitivity to physiological sounds and a high degree of insensitivity to environmental sounds propagated through the air. It can be concluded that the hydrophone may be useful for the early detection of intracranial aneurysms and also for apnoea detection.     

Ion channel regulation of the dynamical instability of the resting membrane potential in saccular hair cells of the green frog (Rana esculenta)

AIMS: We investigated the ion channel regulation of the resting membrane potential of hair cells with the aim to determine if the resting membrane potential is poised close to instability and thereby a potential cause of the spontaneous afferent spike activity. METHODS: The ionic mechanism and the dynamic properties of the resting membrane potential were examined with the whole-cell patch clamp technique in dissociated saccular hair cells and in a mathematical model including all identified ion channels. RESULTS: In hair cells showing I/V curves with a low membrane conductance flanked by large inward and outward rectifying potassium conductances, the inward rectifier (K(IR)), the delayed outward rectifier (K(V)) and the large conductance, calcium-sensitive, voltage-gated potassium channel (BK(Ca)) were all activated at rest. Under current clamp conditions, the outward current through these channels balanced the inward current through mechano-electrical transduction (MET) and Ca2+ channels. In 45% (22/49) of the cells, the membrane potential fluctuated spontaneously between two voltage levels determined by the voltage extent of the low membrane conductance range. These fluctuations were not influenced by blocking the MET channels but could be reversibly stopped by increasing [K+]o or by blocking of K(IR) channels. Blocking the BK(Ca) channels induced regular voltage oscillations. CONCLUSIONS: Two intrinsic dynamical instabilities of V(m) are present in hair cells. One of these is observed as spontaneous voltage fluctuations by currents through K(IR), K(V) and h-channels in combination with a steady current through MET channels. The other instability shows as regenerative voltage changes involving Ca2+ and K(V) channels. The BK(Ca) channels prevent the spontaneous voltage fluctuations from activating the regenerative system.     

Significance of skin temperature changes in surface electromyography

Summary Differing results have been reported concerning the direction and quantity of the electromyogram (EMG) amplitude response to changes in tissue temperature. The EMG signals from the soleus muscle of six healthy human subjects were therefore recorded during dynamic exercise (concentric contractions) at ambient temperatures of 30°C and 14°C. The mean skin temperature above the muscle investigated was 32.9° C and 21.7° C, respectively. The core temperature, estimated by rectal temperature, was unchanged. The cooling of the superficial tissues caused approximately a doubling of the EMG amplitude. For the probability level 0.9 in the amplitude probability distribution function, the average signal level increased from 73 V to 135 V (P=0.02). The average mean power frequency of the EMG signal was reduced from 142 Hz to 83 Hz (P=0.004). The amplitude increase was not due to shivering but other possible explanations are presented. As the changes in T _sk investigated were within the range which may occur normally during the working hours, it was concluded that T _sk should be carefully controlled in vocational EMG studies.