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At least 649 records · Page 36Linked to original sources
PubMed1992

Natural course of Crohn's disease after ileocolic resection: endoscopically visualised ileal ulcers preceding symptoms.

Forty two Crohn's disease patients were followed up after ileocolic resection with regard to symptoms and endoscopic appearance of the ileocolic anastomosis. Twenty eight patients resected because of colonic neoplasm served as controls. In all the Crohn's disease patients the ileal resection margin was disease free macroscopically at operation. In addition, intraoperative ileoscopy was performed in 13 and no sign of residual inflammation in the neoterminal ileum was seen. Endoscopy soon after surgery often showed preanastomotic ileal ulceration before symptoms appeared, whereas no anastomotic lesions were observed in the controls. Thus, 22 of 30 Crohn's disease patients examined had ulceration of the anastomotic area after three months, but only 10 had developed symptoms indicating relapse (73 v 33%). Corresponding figures in the 30 patients examined after one year were 93 v 37%, and in 14 patients after three years they were 100 and 86% respectively. The inflammatory lesions in all cases were preanastomotic, in the neoterminal ileum, and showed time related progression from aphthae to larger ulcers and stricture. The study suggests that endoscopically observed inflammatory lesions that appear soon after ileocolic resection for Crohn's disease signify new inflammation and not residual, persistent disease or incomplete anastomotic healing. The data further suggest that despite clinical remission after apparently radical intestinal resection, the bowel is permanently inflamed in Crohn's disease.

Adolescent↗
PubMed2004

Effect of laryngoplasty on respiratory noise reduction in horses with laryngeal hemiplegia.

REASONS FOR PERFORMING STUDY: Laryngoplasty is the technique of choice for treatment of laryngeal hemiplegia, with the aim of improving airway function and/or eliminating respiratory noise. However, there are no quantitative data in the literature describing the effect of laryngoplasty on upper airway noise or its relationship to upper airway mechanics in horses with laryngeal hemiplegia. OBJECTIVES: To determine whether laryngoplasty reduces respiratory noise in exercising horses with laryngeal hemiplegia; and to establish whether the degree of upper airway obstruction can be predicted by upper airway noise, or the degree of arytenoid abduction correlated with airway obstruction and noise production. METHODS: Six Standardbred horses with normal upper airways during maximal exercise were used. Respiratory sounds and inspiratory transupper airway pressure (Pui) were measured in all horses before and after induction of laryngeal hemiplegia and 30, 60 and 90 days after laryngoplasty. Inspiratory sound level (SL) and the sound intensity of the 3 inspiratory formants (F1, F2 and F3, respectively) were measured using a computer-based sound analysis programme. The degree of abduction was graded by endoscopic visualisation 1, 30, 60 and 90 days post operatively. Linear regression analysis was used to determine correlations between Pui, sound indices and grades of arytenoid abduction. RESULTS: In laryngeal hemiplegia-affected horses, Pui, inspiratory SL and the sound intensity of F1, F2 and F3 were significantly increased. At 30 days following laryngoplasty, the sound intensity of F1 and Pui returned to baseline values. The sound intensities of F2, F3 and SL were significantly improved from laryngeal hemiplegia values at 30 days post operatively, but did not return to baseline at any measurement period. Sound level, F2 and F3 were significantly correlated with Pui (P<0.05), but the correlations were weak (r2 = 0.26, 035 and 0.40, respectively). Grade of abduction and F2 were positively and significantly correlated (P<0.006, r2 = 0.76). Grade of arytenoid abduction and Pui were not correlated (P = 0.12). CONCLUSIONS: Laryngoplasty reduced inspiratory noise in laryngeal hemiplegia-affected horses by 30 days following surgery, but did not return it to baseline values. While upper airway noise and Pui were correlated, this relationship was insufficiently strong to predict Pui from noise in individual animals. The degree of arytenoid abduction was not correlated with Pui, but was positively correlated with noise production. POTENTIAL RELEVANCE: Laryngoplasty reduces upper airway noise in horses with laryngeal hemiplegia, but is not as effective as bilateral ventriculocordectomy in this regard, although respiratory noise reduction occurs more rapidly than with bilateral ventriculocordectomy. Residual noise during exercise cannot be used as a predictor of improvement in upper airway function in individual horses following laryngoplasty. The degree of arytenoid abduction obtained following surgery does not affect upper airway flow mechanics. Interestingly, we found that the greater the arytenoid abduction, the louder the respiratory noise.

Animals↗
PubMed1999

CT data sets surface extraction for biomechanical modeling of long bones.

In modelling applications such as custom-made implants design is useful to have a surface representation of the anatomy of bones rather than the voxel-based representation generated by tomography systems. A voxel-to-surface conversion process is usually done by a 2D segmentation of the images stack. However, other methods allow a direct 3D segmentation of the CT or MRI data set. In the present work, two of these methods, namely the Standard Marching Cube (SMC) and the Discretized Marching Cube (DMC) algorithms, were compared in terms of local accuracy when used to reconstruct the geometry of a human femur. The SMC method was found to be more accurate than the DMC method. The SMC method was capable of reconstructing the inner and outer geometry of a human femur with a peak error lower than 0.9 mm and an average error comparable to the pixel size (0.3 mm). However, the large number of triangles generated by the algorithm may limit its adoption in many modelling applications. The peak error of the DMC algorithm was 1.6 mm but it produced approximately 70% less triangles than the SMC method. From the results of this study, it may be concluded that three dimensional segmentation algorithms are useful not only in visualisation applications but also in the creation of geometry models.

Algorithms↗
PubMed2003

ER quality control can lead to retrograde transport from the ER lumen to the cytosol and the nucleoplasm in plants.

Quality control in the secretory pathway is a fundamental step in preventing deleterious effects that may arise by the release of malfolded proteins into the cell or apoplast. Our aims were to visualise and analyse the disposal route followed by aberrant proteins within a plant cell in vivo using fluorescent protein technology. A green fluorescent protein (GFP) fusion was detected in the cytosol and the nucleoplasm in spite of the presence of an N-terminal secretory signal peptide. In contrast to secreted GFP, the fusion protein was retained in the cells where it was degraded slowly, albeit at a rate much higher than that of the endoplasmic reticulum (ER)-retained derivative GFP-HDEL. The fusion protein could not be stabilised by inhibitors of transport or the cytosolic proteasome. However, the protein is a strong lumenal binding protein (BiP) ligand. Complete signal peptide processing even after long-term expression in virus-infected leaves rules out the possibility that the documented accumulation in the cytosol and nucleoplasm is because of the bypassing of the translocation pores. The data are consistent with the hypothesis that the fusion protein is disposed off from the ER via a retrograde translocation back to the cytosol. Moreover, accumulation in the nucleoplasm was shown to be microtubule dependent unlike the well-documented diffusion of cytosolically expressed GFP into the nucleoplasm. The apparent active transport of the GFP fusion into the nucleoplasm may indicate an as yet undiscovered feature of the ER-associated degradation (ERAD) pathway and explain the insensitivity to degradation by proteasome inhibitors.

Biological Transport↗
PubMed1987

Cross sectional echocardiographic assessment of great artery diameters in infants and children.

The pulmonary trunk and aortic root were measured on cross sectional echocardiograms in 173 normal subjects aged from one day to 15 years. Fifteen neonates were reexamined 3-6 days later. The great vessels were visualised in the parasternal long axis and short axis views. All measurements were made in end diastole and end systole by the leading edge method. The internal diameter (inner surface to inner surface) of the pulmonary trunk was also measured. The diameters of the great vessels correlated best with the square root of body surface area. Individual variability in cardiac growth gave a wide scatter of normal values. This was controlled for by calculating the ratio of the pulmonary trunk to aortic root for each subject. This ratio showed little individual variability and, except for the neonatal period, was remarkably constant throughout infancy and childhood (1.06 (0.06)). In the first 24 hours of life the ratio of the pulmonary trunk to the aortic root was significantly larger (1.29 (0.12)) but within one week it decreased to the "normal" ratio found in the older age groups. These normal data should be useful in assessing patients with congenital heart disease, particularly those in whom pulmonary blood flow is abnormal.

Adolescent↗
PubMed2004

The genes for the helix-loop-helix proteins Id6a, Id6b, Id1 and Id2 are specifically expressed in the ventral and dorsal domains of the fish developing somites.

Muscle differentiation is inhibited by members of the Id family that block the transcriptional effect of myogenic bHLH regulators by forming inactive heterodimers with them. Also, Id proteins promote cell proliferation by interacting with key regulators of the cell cycle. In order to determine the role of Id-encoding genes during fish development and especially in early myogenesis, we examined the expression patterns of Id1, Id2 and two nonallelic Id6 (Id6a and Id6b)-encoding genes in developing trout embryos. These four Id paralogs were found to exhibit discrete expression in the developing nervous system and in the eye rudiment. During the segmentation process, Id6a, Id6b and Id1 were expressed in the tail bud, the paraxial mesoderm and the ventral and dorsal domains of neoformed somites. As the somite matured in a rostrocaudal progression, the labelling for Id1 transcripts rapidly faded whereas labelling for Id6 transcripts was found to persist until at least the completion of segmentation. By contrast, Id2 transcripts were visualised transiently only in dorsal domains of neoformed somites and strongly accumulated in the pronephros. The preferential localisation of Id6a, Id6b, Id1 and Id2 transcripts within ventral and/or dorsal extremes of the developing somites, suggests that these areas, which were the last ones to express muscle-specific genes, contain dividing cells involved in somite expansion.

Amino Acid Sequence↗
PubMed1988

Dual-label autoradiographic analysis of human skin fibroblast and myoblast proteins by two-dimensional polyacrylamide gel electrophoresis using immobilised pH gradients in the first dimension.

Horizontal two-dimensional polyacrylamide gel electrophoresis with immobilised pH gradients in the first dimension has been applied to the analysis of human skin fibroblast and muscle myoblast total cell proteins. Excellent two-dimensional separations of skin fibroblast proteins were obtained using pH 4-10 immobilised pH gradient gels with a long interelectrode distance (16 cm), but resolution was degraded, particularly of the more acidic proteins, by the use of shorter (10 cm) gels. Improved resolution of acidic and basic proteins was obtained using separate pH 4-7 and pH 7-10 immobilised pH gradient gels respectively in the first dimension. Two-dimensional protein maps of skin fibroblast proteins were visualised both by silver staining and by autoradiography of samples labelled synthetically with [35S]methionine. Horizontal two-dimensional electrophoresis, using pH 4-7 and pH 7-10 immobilised pH gradient gels in the first dimension, was applied to the analysis of protein samples from skin fibroblasts and muscle myoblasts dual-labelled synthetically with [35S]methionine and [75Se]selenomethionine in an attempt to identify sets of proteins specific to each cell type. In addition, two-dimensional maps or protein samples derived from normal individuals and patients with Duchenne muscular dystrophy were compared to search for protein changes associated with the disease state. Although sets of qualitative protein spot differences were observed by visual inspection of the two-dimensional gels, more rigorous qualitative and quantitative analysis of the patterns using a computerised analysis system will be required to obtain the maximum amount of information from these data.

Autoradiography↗
PubMed1993

Specific atrial overexpression of G protein coupled human beta 1 adrenoceptors in transgenic mice.

OBJECTIVE: The aim was to develop a transgenic mouse model of atrial beta 1 adrenoceptor overexpression in order to create atrial alteration of the receptor transduction system. METHODS: Transgenic founders were generated after microinjection of the transgene construct into the pronucleus of fertilised mouse eggs. Heterozygous progeny were screened for RNA expression of the human beta 1 adrenoceptor gene under the control of a 0.56 kb proximal promoter of the human atrial natriuretic factor. One line, out of the three obtained, was selected and further characterised for overexpression of the human beta 1 adrenoceptor. Polymerase chain reaction was employed to detect beta 1 adrenoceptor mRNA, and 125I-cyanopindolol (ICYP) binding assays were used to quantify receptors in heart membranes. A quantitative autoradiographic ICYP binding technique was also used to visualise atrial and ventricular beta adrenoceptors in heart sections. RESULTS: The human beta 1 adrenoceptor was overexpressed specifically in the atria of transgenic mice. The level of the beta 1 adrenoceptor was 5-10-fold higher in transgenic mice compared to basal murine beta 1 adrenoceptors in non-transgenic control mice. Left and right atrial receptor overexpression was confirmed by in vitro autoradiography. The human receptors were able to couple to the murine stimulatory G proteins (Gs), as shown by high affinity binding site dosage using the beta adrenoceptor agonist isoprenaline. Isoprenaline displacement studies allowed the determination of two different affinity sites, one of high affinity (KH = 5.8 nM), and one of low affinity (KL = 520 nM). When expressed in terms of protein density (fmol.mg-1), atrial transgenic beta 1 adrenoceptors displayed a threefold increase in high affinity sites (KH) as compared to control mice. Preliminary electrocardiographic data showed supraventricular premature beats in 6/14 transgenic mice v 2/16 control mice. CONCLUSIONS: These transgenic mice may provide a useful pharmacological tool to investigate the pathophysiological consequences of the overactivation of atrial beta 1 adrenoceptor-adenylyl cyclase signalling system.

Animals↗
PubMed1988

The major protein of bull seminal plasma: biosynthesis and biological function.

We isolated the major protein of apparent Mr of 15,000-16,000 from seminal plasma as well as from seminal vesicle secretion of bull and proved by amino acid analysis and tryptic peptide mapping that the two proteins were identical. An antiserum against this major protein was employed to quantitate and identify the major protein in seminal plasma as well as seminal vesicle secretion. The antiserum did not cross-react with proteins from bovine or human plasma or follicular fluid respectively. Cell-free translation of poly(A)RNA from seminal vesicle tissue and immunoprecipitation yielded one major species with apparent Mr of 18,000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. Cloning and sequencing of a major protein-specific cDNA led to the identification of clone pMP17, encoding a precursor of the major protein of 128 amino acid residues. We proved that the major protein is identical to protein PDC 109 (Esch et al., Biochem. Biophys. Res. Comm. 113:861-867, 1983). The seminal vesicles synthesize major protein in an androgen-dependent fashion. In addition to intraluminal secretion of the vas deferens, ampullary spermatozoa revealed an intense immunoreaction which was restricted to the neck region of the sperm head and the middle piece, while the principal piece of the tail as well as the sperm head were devoid of immunoreactive material. Epididymal epithelium (as well as calf seminal vesicle epithelium) showed no immunoreactivity with major protein antiserum. Immunoelectron microscopy demonstrated that only spermatozoa devoid of a plasma membrane around the middle piece were able to bind the antiserum against major protein. After removal of the plasma membrane from epididymal spermatozoa, binding of major protein to subplasmalemmal binding sites was visualised using gold-labeled MP. Transblotting with gold-labeled MP demonstrated a protein of about 66 kDa which appears to represent the major protein-receptor. Binding of major protein to the receptor (after loss of the plasma membrane in the mid-piece region of the spermatozoa after contact with secretions from seminal vesicles) is interpreted as a physiological process presumably related to the onset of sperm motility.

Amino Acid Sequence↗
PubMed2000

Isolation and cloning of Streptomyces terminal fragments.

Streptomyces species have a linear chromosome of approximately 8 Mb in size. Many strains also carry linear plasmids. Most of these linear elements contain terminal proteins covalently bound to the 5' ends of the DNA. Using a method for the visualisation of terminal DNA fragments in agarose gels, it was possible to see three fragments in S. rimosus and five fragments in S. avermitilis. The method was also used to clone the 298 bp BamHI fragment carrying the left end of plasmid SLP2. Analysis of the sequence showed that the end resembled other Streptomyces chromosome and plasmid ends, but there were eight palindromes (instead of seven) and a tandem duplication of a 14 bp sequence.

Base Sequence↗
PubMed2004

The management of short saphenous varicose veins: a survey of the members of the vascular surgical society of Great Britain and Ireland.

OBJECTIVE: The outcome of short saphenous vein surgery is often unsatisfactory and the high litigation rate reflects this. The aim of this study was to explore the current management of short saphenous varicose veins in Great Britain and Ireland. METHODS: This was a postal questionnaire survey of the surgical members of the Vascular Surgical Society of Great Britain and Ireland. Of 532 questionnaires 379 were returned (71.2%). RESULTS: There was diversity of opinion about the management of short saphenous veins. Eighty nine per cent of surgeons requested duplex imaging for all patients and over 50% arranged additional duplex marking of the saphenopopliteal junction preoperatively. Only 10.4% formally exposed and identified the popliteal vein during saphenopopliteal ligation, the majority (75.7%) dissected down the short saphenous vein to visualise the junction. The short saphenous vein was stripped routinely by 14.5% of surgeons, the majority preferring to excise a proximal segment of up to 10 cm (55.1%). Compared with long saphenous vein surgery, surgeons were generally more likely to warn patients of nerve damage but equally likely to warn of deep vein thrombosis. A small number of surgeons failed to warn patients of these complications. CONCLUSION: The variation in management of short saphenous veins may be explained by the lack of definitive clinical trials in this area.

Data Collection↗
PubMed1995

Correlation between functional, radiological and anatomical abnormalities in upper airway obstruction (UAO) due to tracheal stenosis.

Few data are available on the relative sensitivity of different lung function tests in upper airway obstruction (UAO) and on the correlation of these tests with radiological estimations of tracheal stenosis. This paper is based on patients with a goiter or tracheal tumor and in healthy subjects breathing through rigid resistors, as well as on literature data. Lung function abnormalities present a specific pattern in UAO: this is characterised by a typical shape of the maximal flow-volume curve, a marked reduction in peakflow (PEF) and only minor changes in routine spirometry (e.g. FEV1, which thus tends to underestimate the stenosis). The clinical relevance of the lung function abnormalities in UAO can be estimated from their relationship with the exercise limitation: a moderate exercise limitation to about 60% pred is found if PEF is reduced to about 40% pred, and Raw is increased to about 600% pred (in which instance FEV1 is still 75% pred). This limitation grossly corresponds to a UAO of about 6 mm diameter. No good correlation is found between lung function abnormalities and the radiological stenosis (CT-scan or X-ray): CT-scan or X-ray are only moderately correlated with each other. Moreover, stenosis can be visualised through X-ray in only about 60% of the cases.

Adult↗
PubMed1994

Molecular dynamics simulations of oligonucleotides in solution: visualization of intrinsic curvature.

We have undertaken molecular dynamics simulations on the d(CGCAAAAAAGCG).d(CGCTTTTTTGCG) dodecamer in solution. In this study, we focus on aspects of conformation and dynamics, including the possibility of cross-strand hydrogen bonds. We compare our results with those from crystallography as well as infrared, Raman and NMR spectroscopy and cyclization kinetics. Our method of analysis allows us to visualise the curvature of the helix as a function of time during the simulation. We find that the major distortions of the helix axis path occur at the junctions between the (essentially straight) A-tract and the CG- and GC-tracts, although at one junction this is due to hyperflexibility (i.e., regions of high flexibility with no preferred direction of curvature), while at the other junction a static curvature is found (i.e., a preferred, sustained direction of curvature).

Base Sequence↗
PubMed2000

Real-time RT-PCR fluorescent detection of tomato spotted wilt virus.

A real-time reverse transcription-polymerase chain reaction assay based on TaqMan chemistry was developed for the detection and quantification of tomato spotted wilt virus (TSWV). This method enabled sensitive, reproducible and specific detection of TSWV in 'leaf soak' and total RNA extracts from infected plants. TaqMan reliably detected TSWV in as little as 500 fg total RNA. The assay was 10-fold more sensitive than visualisation of ethidium bromide-stained bands following agarose gel electrophoresis. TSWV isolates from various crops and locations were detected with a cycle threshold of 20-26 in 1 ng total RNA extracted from fresh or freeze-dried leaves. A dilution series of in vitro transcripts from a cloned 628 base pair fragment of TSWV S RNA served as standard for quantification of viral template in infected leaf samples. The TaqMan assay detected reproducibly 1000 molecules of the target transcript.

Base Sequence↗
PubMedDate not supplied

Investigating extended regulatory regions of genomic DNA sequences.

MOTIVATION: Despite the growing volume of data on primary nucleotide sequences, the regulatory regions remain a major puzzle with regard to their function. Numerous recognising programs considering a diversity of properties of regulatory regions have been developed. The system proposed here allows the specific contextual, conformational and physico-chemical properties to be revealed based on analysis of extended DNA regions. RESULTS: The Internet-accessible computer system RegScan, designed to analyse the extended regulatory regions of eukaryotic genes, has been developed. The computer system comprises the following software: (i) programs for classification dividing a set of promoters into TATA-containing and TATA-less promoters and promoters with and without CpG islands; (ii) programs for constructing (a) nucleotide frequency profiles, (b) sequence complexity profiles and (c) profiles of conformational and physico-chemical properties; (iii) the program for constructing the sets of degenerate oligonucleotide motifs of a specified length; and (iv) the program searching for and visualising repeats in nucleotide sequences. The system has allowed us to demonstrate the following characteristic patterns of vertebrate promoter regions: the TATA box region is flanked by regions with an increased G+C content and increased bending stiffness, the TATA box content is asymmetric and promoter regions are saturated with both direct and inverted repeats. AVAILABILITY: The computer system RegScan is available via the Internet at http://www.mgs.bionet.nsc. ru/Systems/RegScan, http://www.cbil.upenn.edu/mgs/systems/r egscan/.

Algorithms↗
PubMed2003

Depletion of the highly abundant protein albumin from human plasma using the Gradiflow.

Analysis of complex protein samples by two-dimensional electrophoresis (2-DE) is often more difficult in the presence of a few predominant proteins. In plasma, proteins such as albumin mask proteins of lower abundance, as well as significantly limiting the amount of protein that can be loaded onto the immobilized pH gradient strip. In this paper the Gradiflow, a preparative electrophoresis system, has been used to deplete human plasma of the highly abundant protein albumin under native and denatured conditions. A three step protocol incorporating a charge separation to collect proteins with an isoelectric point greater than albumin and two size separations to isolate proteins larger and smaller than albumin, was used. When the albumin depleted fractions were analysed on pH 3-10 2-DE gels, proteins that were masked by albumin were revealed and proteins not seen in the unfractionated plasma sample were visualised. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry analysis confirmed the identification of the protein that lies beneath albumin to be C4B-binding protein alpha chain. The liquid fractions from the Gradiflow separations were also analysed by liquid chromatography-tandem mass spectrometry to confirm the proteins were separated according to their size and charge mobility in an electric field.

Albumins↗
PubMed1986

Regulatory peptides of the gastrointestinal and respiratory tracts.

The gastrointestinal and respiratory tracts contain numerous regulatory peptides produced by and released from specialised epithelial cells and the organ innervation. This complex system of endocrine cells and nerves is generally called "the diffuse neuroendocrine system". Markers are now available which permit the visualisation of the diffuse neuroendocrine system or its individual components. These include antibodies to neuron-specific enolase, chromogranin, neurofilament triplet proteins, the brain protein S100 and antibodies to a variety of regulatory peptides. Peptides present in the gut and lung innervation include: vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), galanin, substance P, calcitonin gene-related peptide (CGRP), neuropeptide tyrosine (NPY), somatostatin and cholecystokinin (the latter two are also localised to endocrine cells of the gut). Bombesin-immunoreactivity is found in nerves in the gut and in endocrine cells of the foetal/neonatal lung. Neuropeptides of the gut and lung originate either from local neurons (e.g. VIP, PHI, galanin) or extrinsic neurons localised in sensory ganglia (e.g. substance P and CGRP) or the sympathetic chain (e.g. NPY). Recent studies point to the involvement of regulatory peptides in diseases of the gut and lung. These, together with detailed distribution studies, provide supportive data on the putative role of the peptides in the control of normal bowel and respiratory functions. The gastrointestinal and respiratory tracts were within the systems investigated by Feyrter during his original observations on the existence of specialised epithelial cells with a putative regulatory function (Feyrter, 1938). These "endocrine/paracrine" cells were found to be scattered in epithelial organs throughout the body. In fact, endocrine cells of the respiratory tract are frequently referred to as "Feyrter's cells". The term "regulatory peptides" was introduced as a generic term (Polak and Bloom, 1983) after the finding that active peptides are produced both by cells of the diffuse endocrine or APUD (amine precursor uptake and decarboxylation) system (Pearse, 1983) and autonomic/sensory nerves. These peptides are released into the circulation from endocrine cells or locally from nerve terminals or paracrine cells. The concept of "gut/brain" peptides was dispelled after the findings that the respiratory tract was provided abundantly with numerous active peptides produced by and released from mucosal endocrine cells and/or the innervation.

Animals↗
PubMed2003

Store operated Ca2+ influx by selective depletion of ryanodine sensitive Ca2+ pools in primary human skeletal muscle cells.

The contraction and relaxation of skeletal muscle is driven by release of Ca2+ from sarcoplasmic reticulum through the ryanodine receptor type 1 and extruding the ion from the cytosol by Ca2+ ATPases. Efficient refilling of the empty Ca2+ stores is essential for repetitive cycles of muscle contraction and relaxation, but not investigated in human skeletal muscle cells. Here we show that under conditions of selective depletion of the ryanodine-sensitive Ca2+ pool Ca2+ influx occurs in differentiated human skeletal muscle cells using the Ca2+ imaging technique. This Ca2+ influx is not due to permeation through the L-type Ca2+ channel and not observed under conditions of inhibited Ca2+ ATPase. The Ca2+ influx was visualised by quenching the intracellular fura2 signal with Mn2+ on single cell level and also using fluorescence photometry of cell suspensions. The Mn2+ influx was inhibited by the Ca2+ channel blockers La(3+) and SKF96356. The delineation of the signalling cascade leading to Ca2+ influx evoked by selective depletion of ryanodine sensitive Ca2+ stores showed that phospholipase C or protein kinase C were not involved. Interestingly, a Mn2+ influx was triggered by the cell-permeant analogue of diacylglycerol and further augmented by the application of RHC80267, a diacylglycerol lipase inhibitor. This signalling pathway could be attributed to the participation of a protein kinase C activity. However, Mn2+ influx evoked by selective depletion of ryanodine sensitive Ca2+ stores was not altered by RHC80267 or protein kinase C inhibitors. Using RT-PCR, correctly spliced mRNA fragments were detected corresponding to human transient receptor potential (TRPC) Ca2+ channels type 1, 3, 4 and 6. These data show that in skeletal muscle at least two independent mechanisms of Ca2+ influx exist. For Ca2+ influx triggered by the selective depletion of ryanodine sensitive Ca2+ stores we propose a phospholipase C independent coupling of ryanodine receptors to voltage insensitive Ca2+ channels.

Caffeine↗