Endothelium-derived relaxing factor activates calcium-activated potassium channels of resistance vessel smooth muscle cells

Direct observation was made by using the patch-clamp technique with a specially designed microperfusion system to investigate the effect of acetylcholine (Ach 10(-6) mol/L) elicited endothelium-derived relaxing factor (EDRF) on the calcium-activated potassium channel (IK(Ca)) in the smooth muscle cells of mesenteric resistance vessels in Wistar rats. Activation of IK(Ca) was firstly observed by inducing the elicited EDRF or sodium nitroprusside (SNP 10(-8) mol/L) under various clamping voltages in cell-attached configuration. While the pipette solution contained KCl 126 mmol/L and the bath solution contained KCl 5.9 mmol/L, two types of conductances of calcium-activated potassium current being 76.4 +/- 2.3 pS (mean +/- S.E. n = 7) and 160.3 +/- 7.5 pS (mean +/- S.E. n = 7) were recorded during the EDRF activation, one type of conductance being 100.5 +/- 2.8 pS (mean +/- S.E. n = 6) was activated by nitric oxide (NO) which is an effective component from SNP. Differences in kinetic characteristics of these channels between EDRF and NO activation were found, particularly the probability of the channel being open in EDRF activation was obviously greater than that in NO stimulation. It has been shown that the potassium channel mechanisms involved in the EDRF and NO actions might be different.     

Primary ciliary dyskinesia: ultrastructural defects and clinical features

Golden hamsters were exposed to conditions of 2.5 times normal gravity (hypergravity, HG) for 4 months. During this period, tests were carried out to study equilibrium maintenance, swimming behaviour and open-field behaviour of these HG hamsters and of control hamsters living in a normal-gravity environment. The tests proved to be useful devices for detecting differences in perceptive-motor behaviour between HG hamsters and control hamsters. The HG hamsters had more difficulties in balancing on tubes and orientation during swimming. In the open-field study, the HG hamsters showed less locomotor activity than control hamsters. However, no differences were observed between the groups in washing, rearing and number of times having defaecation. These findings indicate that the daily transition from 2.5 to 1 g was not experienced as stressful by the hamsters, although performance on several perceptive-motor tasks was decreased, especially during the first weeks.     

Cre/loxP-mediated excision and amplification of large segments of the Escherichia coli genome

The isolation and amplification of large, predetermined segments of a genome from its host have been explored. The prototype of our approach was the excisional replication of some viruses such as the lambda-lysogen. Similar machinery was used to excise and amplify large genomic segments of Escherichia coli in its host. Two loxP sequences for a site-specific recombinase Cre, together with a conditional replication origin (pi-dependent gamma-ori), were inserted into the genome by homologous recombination at predetermined sites, 50-100 kb apart. Cre and pir200 which encodes the site-specific recombinase Cre and an ori-specific replication protein pi, respectively, were also introduced into the genome. The predetermined genomic segments flanked by the loxP sequences were excised and amplified upon induction of the cre and pir200 genes which were under the control of the tet promoter. This excised and amplified DNA could be easily purified as a large plasmid. This procedure can provide an alternative to conventional cloning methods by obtaining predetermined large genomic segments directly from the original organisms. In this study, using the Cre/loxP site-specific recombination and pi/gamma-ori replication system of plasmid R6K, a procedure was devised that could isolate a large segment of the E. coli genome and demonstrated the feasibility of the procedure by excising and amplifying the 50-kb trg-narZ and 100-kb trg-hipA regions of the E. coli W3110 genome.     

Extended life of human corneal endothelial cells transfected with the SV40 large T antigen

PURPOSE: To transfect human corneal endothelial cells with a plasmid vector coding for the SV40 large T antigen to extend the life of the cells in culture. METHODS: Human corneal endothelial cells were transfected with the SV40 large T antigen-coding plasmid pSV3neo using the electroporation method. Transfected and control cells were propagated in culture until senescence. Polymerase chain reaction and immunofluorescence were used to demonstrate messenger RNA and protein, respectively, for the Simian virus 40 large T antigen in the transfected cells. Polymerase chain reaction and hot blotting were used to demonstrate messenger RNA coding for several growth factors and receptors in transfected and control cells. RESULTS: The transfected cells continued to proliferate to 38 passages (more than 120 population doublings) in culture (control cells, 8 population doublings). Transfected cells, but not control cells, expressed messenger RNA coding for the Simian virus 40 large T antigen. Similarly, immunofluorescent staining with monoclonal antibodies demonstrated that the Simian virus 40 large T antigen protein was present in the nucleus of the transfected cells. Transfected cells were shown to produce messenger RNA coding for epidermal growth factor, epidermal growth factor receptor, basic fibroblast growth factor, fibroblast growth factor receptor-1, interleukin-1 alpha, the interleukin-1 receptor, transforming growth factor beta-1, and the glucocorticoid receptor. Qualitative expression of the messenger RNA coding for each of these modulators was similar in proliferating primary corneal endothelial cells and proliferating or confluent transfected corneal endothelial cells. CONCLUSIONS: In culture, the life of human corneal endothelial cells transfected with a plasmid vector coding for the Simian virus 40 large T antigen is extended. This study suggests that human corneal endothelial cells have the capacity for extensive proliferation, but the proliferation of untransfected cells is regulated through mechanisms that have not yet been characterized.     

The humeroscapular motion interface

Motion between the humerus and scapula commonly is described as glenohumeral motion. However, humeroscapular motion occurs at two distinct sites. In addition to the motion at the diarthrodial glenohumeral joint, movement occurs between the proximal humerus and related structures and the surrounding sleeve of structures, including the acromion, deltoid, coracoid, coracoacromial ligament, and the muscles attached to the coracoid. This site of nonarticular shoulder motion is defined as the humeroscapular motion interface. Nonarticular humeroscapular motion can be documented and measured using standard magnetic resonance imaging techniques. The maximum average interfacial motion using axial images was 29.1 mm, which occurred at the level of the maximum diameter of the humeral head. Interfacial motion varied depending on the site measured. If pathologic conditions such as adhesions secondary to trauma or surgery interfere with or obliterate this space at sites of significant sliding motion, overall shoulder motion will be limited. Successful treatment of shoulder stiffness related to humeroscapular restraints is likely to require restoration of the normal sliding motion at the humeroscapular motion interface, in addition to resolving restraints affecting the glenohumeral joint motion.     

A modified repair technique for tricuspid incompetence in Ebstein's anomaly

OBJECTIVE: A modified technique for tricuspid valve repair in Ebstein's anomaly restructures the valve mechanism at the level of the true tricuspid anulus by using the most mobile leaflet for valve closure without plication of the atrialized chamber. Midterm results of this therapeutic approach for patients with Ebstein's anomaly and tricuspid valve incompetence are reported. METHODS: Between October 1988 and April 1997, the incompetent tricuspid valve was repaired with our technique in 19 patients (12 female, 7 male; 2 to 54 years, mean 21 years). The indication for operation was congestive heart failure of various degrees in all patients. Tricuspid incompetence was grade II in two patients, grade III in 14, and grade IV in three. Associated congenital malformations were simultaneously repaired (interatrial communication in 18, ventricular septal defect in two, pulmonary stenosis in two, mitral valve prolapse in one). Follow-up ranged between 10 and 103 months (median 28 months) and was complete for all patients. RESULTS: There were no operative deaths. One patient with active endocarditis and pulmonary abscess died 2 months after the operation of recurrent sepsis; there were no late deaths. During follow-up, New York Heart Association functional class improved from 2.8 before the operation to 1.9 without recurrent cyanosis, and tricuspid incompetence decreased from a mean grade of 3.1 to one of 0.9, without any echocardiographic deterioration of the tricuspid valve function or right ventricular dilation. CONCLUSIONS: Our technique allows tricuspid valve repair in patients with Ebstein's anomaly, even in cases usually reserved for primary valve replacement, without late functional deterioration.     

Hepatitis C virus E2 protein purified from mammalian cells is frequently recognized by E2-specific antibodies in patient sera

The envelope protein of hepatitis C virus (HCV) is composed of two membrane-associated glycoproteins, E1 and E2. To obtain HCV E2 protein as a secretory form at a high level, we constructed a recombinant chinese hamster ovary (CHO) cell line expressing a C-terminal truncated E2 (E2t) fused to human growth hormone (hGH), CHO/hGHE2t. The hGHE2t fusion protein was purified from the culture supernatant using anti-hGH mAb affinity chromatography at approximately 80% purity. The purified hGHE2t protein appeared to be assembled into oligomers linked by intermolecular disulfide bond(s) when density gradient centrifugation and SDS-polyacrylamide gel electrophoresis were employed. When the purified fusion protein was used for testing its ability to bind to antibodies specific for HCV by enzyme-linked immunosorbent assay, the protein was recognized by antibodies in sera from 90% of HCV-positive patients. Treatment of hGHE2t protein by beta-mercaptoethanol, but not by heat and SDS, significantly reduced its reactivity to the antibodies of patient sera, suggesting that intermolecular and/or intramolecular disulfide bonds are important for its ability to recognize its specific antibody and that the E2 protein contains discontinuous antigenic epitope(s).     

Characterization of two rice MADS box genes that control flowering time

This study utilised positron emission tomography (PET) to identify the cortical areas involved in verbal initiation and suppression in normal subjects whilst performing a sentence completion test (the Hayling Test). In the first condition (response initiation) subjects were required to complete a sentence from which the last word was omitted, whereas in the second condition (response suppression) subjects were asked to complete a sentence with a word which made no sense in the context of the sentence. Subjects were also required to perform a control task in which they had to read out the last word of given sentences. Compared to the control task, response initiation was associated with left-sided activation of the frontal operculum, inferior frontal gyrus, middle temporal gyrus and the right anterior cingulate gyrus, whereas response suppression was associated with left frontal operculum, inferior frontal gyrus and right anterior cingulate gyrus activation. The difference in activation between the two conditions of the Hayling Test lay in the increased activation of the left middle temporal gyrus and the left inferior frontal gyrus during response initiation.     

Identifying 5-methylcytosine and related modifications in DNA genomes

Intense interest in the biological roles of DNA methylation, particularly in eukaryotes, has produced at least eight different methods for identifying 5-methylcytosine and related modifications in DNA genomes. However, the utility of each method depends not only on its simplicity but on its specificity, resolution, sensitivity and potential artifacts. Since these parameters affect the interpretation of data, they should be considered in any application. Therefore, we have outlined the principles and applications of each method, quantitatively evaluated their specificity,resolution and sensitivity, identified potential artifacts and suggested solutions, and discussed a paradox in the distribution of m5C in mammalian genomes that illustrates how methodological limitations can affect interpretation of data. Hopefully, the information and analysis provided here will guide new investigators entering this exciting field.