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.     

An integrative approach to CTL epitope prediction: a combined algorithm integrating MHC class I binding, TAP transport efficiency, and proteasomal cleavage predictions

Reverse immunogenetic approaches attempt to optimize the selection of candidate epitopes, and thus minimize the experimental effort needed to identify new epitopes. When predicting cytotoxic T cell epitopes, the main focus has been on the highly specific MHC class I binding event. Methods have also been developed for predicting the antigen-processing steps preceding MHC class I binding, including proteasomal cleavage and transporter associated with antigen processing (TAP) transport efficiency. Here, we use a dataset obtained from the SYFPEITHI database to show that a method integrating predictions of MHC class I binding affinity, TAP transport efficiency, and C-terminal proteasomal cleavage outperforms any of the individual methods. Using an independent evaluation dataset of HIV epitopes from the Los Alamos database, the validity of the integrated method is confirmed. The performance of the integrated method is found to be significantly higher than that of the two publicly available prediction methods BIMAS and SYFPEITHI. To identify 85% of the epitopes in the HIV dataset, 9% and 10% of all possible nonamers in the HIV proteins must be tested when using the BIMAS and SYFPEITHI methods, respectively, for the selection of candidate epitopes. This number is reduced to 7% when using the integrated method. In practical terms, this means that the experimental effort needed to identify an epitope in a hypothetical protein with 85% probability is reduced by 20-30% when using the integrated method.The method is available at http://www.cbs.dtu.dk/services/NetCTL. Supplementary material is available at http://www.cbs.dtu.dk/suppl/immunology/CTL.php.     

Genetic structure of population of Bacillus cereus and B. thuringiensis isolates associated with periodontitis and other human infections

The genetic diversity and relationships among 35 Bacillus cereus and Bacillus thuringiensis isolates recovered from marginal and apical periodontitis in humans and from various other human infections were investigated using multilocus enzyme electrophoresis. The strains were isolated in Norway, except for three strains isolated from periodontitis patients in Brazil. The genetic diversity of these strains was compared to that of 30 isolates from dairies in Norway and Finland. Allelic variation in 13 structural gene loci encoding metabolic enzymes was analyzed. Twelve of the 13 loci were polymorphic, and 48 unique electrophoretic types (ETs) were identified, representing multilocus genotypes. The mean genetic diversity among the 48 genotypes was 0.508. The genetic diversity of each source group of isolates varied from 0.241 (periodontal infection) to 0.534 (dairy). Cluster analysis revealed two major groups separated at a genetic distance of greater than 0.6. One cluster, ETs 1 to 13, included solely isolates from dairies, while the other cluster, ETs 14 to 49, included all of the human isolates as well as isolates from dairies in Norway and Finland. The isolates were serotyped using antiflagellar antiserum. A total of 14 distinct serotypes were observed. However, little association between serotyping and genotyping was seen. Most of the strains were also analyzed with pulsed-field gel electrophoresis, showing the presence of extrachromosomal DNA in the size range of 15 to 600 kb. Our results indicate a high degree of heterogeneity among dairy strains. In contrast, strains isolated from humans had their genotypes in one cluster. Most strains from patients with periodontitis belonged to a single lineage, suggesting that specific clones of B. cereus and B. thuringiensis are associated with oral infections.     

Lysine 322 in the human IgG3 C(H)2 domain is crucial for antibody dependent complement activation

The classical complement activation cascade of the immune system is initiated by multivalent binding of its first component, C1q, to the Fc region of immunoglobulins in immune complexes. The C1q binding site on mouse IgG2b has been shown to contain the amino acids Glu 318, Lys 320 and Lys 322 in the C(H)2 domain (Duncan, A.R., Winter, G.,1988. The binding site for C1q on IgG. Nature 322 738-740). Identical or closely related motifs are found on all IgGs in all species, and the binding site has therefore been thought to be universal. However, the results from another study indicate that the site is different in human IgG1 molecules (Morgan, A., Jones, N.D., Nesbitt, A.M., et al., 1995. The N-terminal end of the C(H)2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, Fc gamma RI and Fc gamma RIII binding. Immunology 86 319-324). To determine the site(s) responsible for complement activation in anti-NIP-mouse/human IgG3 antibodies, we have mutated amino acids Lys 276, Tyr 278, Asp 280, Glu 318, Lys 320 and Lys 322 in two beta-strands in the C(H)2 domains of human IgG3. In addition, we mutated the Glu 333, which resides in close proximity to the postulated C1q-binding site of mouse IgG2b, as well as Leu 235 in the lower hinge region. All mutants were tested in Antibody Dependent Complement Mediated Lysis (ADCML)(4) assays, where the antigen concentration on target cells was varied and human serum was complement source. Only the mutants that lacked the positively charged side chain of lysine in position 322 showed strong reduction in ADCML, particularly at low antigen density on target cells. Alanine scanning of positions 318 and 320 did not affect ADCML, contrary to what was observed for mouse IgG2b. Neither did a leucine to glutamic acid mutation in position 235 have the effect that has been reported for human IgG1. These results suggest that the complement binding site on human IgG3 molecules is different from that found on mouse IgG2b, and possibly on human IgG1 as well. Thus the contact site may not be conserved.     

Evaluation of a communication and stress management training programme for infertile couples

OBJECTIVE: This study evaluates a patient education programme focussed on improving communication and stress management skills among couples in fertility treatment. METHODS: In total, 37 couples completed the intervention. Two teachers conducted all the five courses offered. The effectiveness regarding communication and infertility-related stress was assessed by questionnaires immediately before (time T1) and after the intervention (time T2). Seeking of information and professional support was assessed at a 12-month follow-up (time T3); response rates were: T1, 93.2%; T2, 85.1%; T3, 74.3%. Data were compared at baseline (T1) and at the 12-month follow-up (T3) with a prospective cohort of Danish people in fertility treatment. RESULTS: There were no differences in infertility-related stress at base line between the two groups studied. We estimated the bi-directional changes in communication, e.g., changes from talking often to talking less frequently and vice versa. More intervention participants started to talk often with their partner about infertility and its treatment after the intervention compared to those who stopped to talk often. Women and men changed occurrence, frequency and content of communication with close other people. Among women marital benefit increased significantly. Infertility-related stress was not reduced significantly. Significantly more intervention participants than in the comparison group had contacted support groups, a psychologist and/or agencies for adoption at the 12-month follow-up. CONCLUSION: The intervention resulted in important perceived improvement in the participants' competence to actively manage changes in marital communication and in communication in different social arenas. PRACTICE IMPLICATIONS: We recommend fertility clinics to develop and evaluate different interventions for those fertility couples who ask for more psychosocial support.     

Chemokines and soluble adhesion molecules in renal transplant recipients with cytomegalovirus infection

Cytomegalovirus (CMV) infection is associated with leucocyte infiltration in various organs, which supports a role for chemokines and adhesion molecules in the pathogenesis of CMV infection. In a prospectively conducted study of renal transplant recipients, 10 patients with CMV disease, five patients with asymptomatic CMV infection and 10 patients who did not have any CMV infection were included. During CMV infection, and in particular during CMV disease, plasma levels of the chemokines IL-8, macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1) and the soluble adhesion molecules vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and L-selectin increased and were positively correlated with the degree of CMV pp65 antigenaemia. Furthermore, a decrease in plasma levels of these chemokines and adhesion molecules was observed following ganciclovir therapy in the patients with CMV disease. This could suggest a role for these molecules in the pathogenesis of CMV infection.     

Apical and basolateral expression of Aquaporin-1 in transfected MDCK and LLC-PK cells and functional evaluation of their transcellular osmotic water permeabilities

 Aquaporin-1 is present in the apical and basolateral membranes in proximal tubules and descending limbs of Henlé’s loop. In order to be able to study the routing of Aquaporin-1 and the regulation of Aquaporin-1-mediated transcellular water flow, we stably transfected LLC-PK₁ and MDCK-HRS cell lines with an Aquaporin-1 expression construct. LLC-PK₁ clone 7 and MDCK clone K integrated two and one copies, respectively, which was reflected in the amount of Aquaporin-1 mRNA expressed in both clones. The Aquaporin-1 protein levels, however, were similar. In both clones, immuno-electronmicroscopy showed extensive labelling of Aquaporin-1 on the basolateral plasma membrane, endosomal vesicles and the apical plasma membrane, including the microvilli. To measure transcellular water permeation, a simple method was applied using phenol-red as a cell-impermeant marker of concentration. In contrast to the native cell lines, both clones revealed a high transcellular osmotic water permeability, which could not be influenced by forskolin add/3-isobutyl-1-methylxanthine (IBMX) or the phorbol ester 12-O-tetradecanoyl 13-acetate (TPA). After glutaraldehyde fixation, it was inhibitable by HgCl₂. These results indicate that targeting of Aquaporin-1 to the apical and basolateral plasma membrane is independent of cell type and show for the first time that water flow through a cultured epithelium can be blocked by mercurial compounds. Received: 9 October 1996 / Received after revision: 3 January 1997 / Accepted: 8 January 1997     

Faster activation of polymorphonuclear neutrophils in resistant mice during early innate response to Pseudomonas aeruginosa lung infection

Polymorphonuclear neutrophils (PMNs) are crucial for the outcome of Pseudomonas aeruginosa lung infection in patients with cystic fibrosis. We compared PMNs and inflammatory cytokines in the lungs and blood from susceptible BALB/c and resistant C3H/HeN mice 1 and 2 days after intratracheal challenge with alginate embedded P. aeruginosa. These parameters were correlated with the quantitative bacteriology and histopathology of the lungs. After challenge, the content of granulocyte colony-stimulating factor (G-CSF) and macrophage inflammatory protein-2 (MIP-2) was increased in the lungs and the sera and the percentage of PMNs was increased in the blood. However, 2 days after challenge the concentration of G-CSF and MIP-2 was higher in the lungs and sera of BALB/c mice. CD11b expression was higher on the PMNs of the C3H/HeN mice. The expression of CD62L on PMNs of both strains of mice was decreased 1 day after bacterial challenge, whereas the expression was increased after 2 days of challenge on PMNs of C3H/HeN mice only. These changes were accompanied by a more severe lung inflammation in BALB/c mice and faster clearance of the bacteria in C3H/HeN mice. In conclusion, the rapid early bacterial clearance in the lungs of C3H/HeN mice could be explained by faster activation of the PMNs, as indicated by the higher up-regulation of CD11b. The severe lung inflammation in BALB/c mice may be caused by the early higher content of G-CSF in the sera mobilizing PMNs from the bone marrow and the persistent chemotactic gradient provided by MIP-2 in the lungs.