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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↗

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↗

[Imaging of the anterior eye chamber with optical coherence tomography].

BACKGROUND: Optical coherence tomography (OCT) represents a high-resolution diagnostic method which can be used to precisely image the anterior eye segment. Further developments in scanning technology and data evaluation allowed to visualize the entire anterior eye segment in one image, and the purpose of this study was to clinically evaluate this new system. PATIENTS AND METHODS: The studied OCT system was adapted to a slit-lamp and allowed us to image the anterior segment with an axial resolution of 10 microm at a scanning rate of 200 Hz with a depth of 7 mm and a maximal width of 15 mm. In selected cases the clinical value of anterior segment OCT was assessed. The reproducibility and level of agreement of anterior chamber depth measurements were assessed during the preoperative examination in 49 cataract surgery patients and compared to ultrasound (US) biometry (10 MHz). RESULTS: The studied anterior segment OCT allowed the cross-sectional imaging of the entire anterior eye segment. Changes of the anterior chamber before and after cataract surgery or surgical iridectomy were visualised. Furthermore, the findings of the anterior chamber after implantation of an iris-fixated intraocular lens (IOL) and an iris prosthetic system could be assessed. The reproducibility of the anterior chamber depth measurement was +/- 22 microm (OCT) and +/- 76 microm (US). The mean difference between optical and acoustic values was 1 microm (0.03 % p = 0.921) and the limits of agreement (95 % confidence interval) were 260 microm (8.58 %). CONCLUSIONS: The anterior segment OCT proved to be a helpful diagnostic method. The cross-sectional visualisation of the entire anterior chamber allowed us to assess important values for the implantation of iris-fixated IOL and other changes after surgical procedures. The resolution and reproducibility were higher than for conventional ultrasound biometry.

Aged↗

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↗

Magnetic Resonance Enteroclysis imaging of Crohn's.

AIM: The aim of our prospective study was to evaluate the diagnostic accuracy of MR Enteroclysis (MRE) to assess the extension and complications of Crohn's Disease (CD) in comparison with conventional enteroclysis (CE). MATERIALS AND METHODS: The study comprised 30 consecutive patients affected by Crohn's disease (18 women and 12 men; age range 16-76, mean age 40.6 years), who underwent conventional and MR enteroclysis. The MR enteroclysis protocol includes three sequences: coronal and axial FIESTA and ssFSE sequences and T1-3D-FSPGR sequences before and after intravenous injection of gadolinium, acquired after administration of 1.6-2 l of iso-osmolar polyethylene glycol solution via a nasojejunal catheter. Two radiologists blindly scored each sequence for opacification of the lumen, small bowel distension and image quality and evaluated the following parameters: presence of wall ulcers, pseudopolyps, stenoses and fistulae. RESULTS: The accuracy of the FIESTA sequence was significantly higher (p<0.01) than the ssFSE and 3D FSPGR sequences in the evaluation of lumen opacification and bowel distension, and for the overall quality of the images (p<0.01). The sensitivity and specificity of MRE were 82% and 100% for the visualisation of parietal ulcers, 87% and 100% for pseudopolyps, 100% and 88% for stenoses, 75% and 100% for parietal fistulae. High-grade stenoses with prestenotic dilation (n=6) and low-grade stenoses (n=9) were visualised both by MRE and CE. MRE also showed abscesses in two patients, not seen at CE. The FIESTA and ssFSE sequences had higher accuracy in the detection of wall ulcers and fistulae, whereas the 3D FSPGR sequences showed higher accuracy in the evaluation of wall thickening. CONCLUSIONS: MRE is able to demonstrate the spectrum of superficial, intramural and exoenteric alterations in Crohn's disease and closely correlates with CE in demonstrating alterations of the bowel lumen and wall.

Adolescent↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

Suppression of the ribosomal L2 gene reveals a novel mechanism for stress adaptation in soybean.

Pseudomonas syringae pv. glycinea bacteria or zoospores of the fungus Phytophthora sojae were used to trigger a hypersensitive reaction (HR) in cell cultures of soybean (Glycine max [L.] Merr. cv. Williams 82). During a screen for genes that show an altered expression as a response to dying neighbour cells we have identified a gene fragment that is specifically but transiently down-regulated in an HR. The corresponding cDNA codes for the ribosomal protein L2 (rpL2) of 80S ribosomes, which is essential for the peptidyl-transferase activity. Two gene copies of rpL2 exist in soybean and both genes are transcribed. The temporary down-regulation of the rpL2 genes is followed by a transient block in the synthesis of new proteins as visualised by pulse-labelling experiments using 35S-amino acids. The same basic phenomenon was also found after treatment of soybean cells with other stress-causing compounds such as elicitors or heavy metals. It is suggested that the transient block in protein synthesis allows a more rapid depletion of, for example, signal molecules with a short half-life time and thus leads to a faster adaptation of the cellular protein inventory to the new environmental conditions.

Adaptation, Physiological↗

A universal procedure for primer labelling of amplicons.

Detection and visualisation of nucleic acids is integral to genome analyses. Exponential amplification procedures have provided the means for the manipulation of nucleic acid sequences, which were otherwise inaccessible. We describe the development and application of a universal method for the labelling of any PCR product using a single end-labelled primer. Amplification was performed in a single reaction with the resulting amplicon labelled to a high specific activity. The method was adapted to a wide range of PCRs and significantly reduced the expense of such analyses.

DNA Primers↗

How the horse moves: 1. Significance of graphical representations of equine forelimb kinematics.

The kinematics of 24 two-year-old Dutch Warmblood horses were recorded at the trot (4 m/s) on a high-speed treadmill to study the coordination of joints within the equine forelimb. Joint angle-time, angle-angle, stick, and marker diagrams were used to show forelimb motion graphically. Because the kinematic data referred to the joint angles of the horse standing squarely and were time-standardised to the duration of the stride cycle, mean joint curves could be calculated for the total group. The motion of each segment in the equine forelimb during a complete stride is described and its function in intralimb coordination evaluated. It appeared that the rotation of the scapula and the cranio-caudal movement of the distal forelimb are synchronous and pendular. The carpal joint rapidly snaps into overextension at the beginning of the stance phase to enable the forelimb to work as a propulsive strut. The fetlock joint acts as an elastic spring, thereby conserving energy and, at the same time, absorbs oscillations generated by initial ground contact. Furthermore, the coordination between carpal and fetlock joints in the swing phase appears to be strongly influenced by inertia. Using the graphic tools evaluated in this paper, we were able to visualise the kinematics of the equine forelimb and relate these to specific functions of the forelimb in locomotion. This information can be used to select kinematic variables for clinical studies in which equine forelimb function has to be described and quantified.

Animals↗

CoXpress: differential co-expression in gene expression data.

BACKGROUND: Traditional methods of analysing gene expression data often include a statistical test to find differentially expressed genes, or use of a clustering algorithm to find groups of genes that behave similarly across a dataset. However, these methods may miss groups of genes which form differential co-expression patterns under different subsets of experimental conditions. Here we describe coXpress, an R package that allows researchers to identify groups of genes that are differentially co-expressed. RESULTS: We have developed coXpress as a means of identifying groups of genes that are differentially co-expressed. The utility of coXpress is demonstrated using two publicly available microarray datasets. Our software identifies several groups of genes that are highly correlated under one set of biologically related experiments, but which show little or no correlation in a second set of experiments. The software uses a re-sampling method to calculate a p-value for each group, and provides several methods for the visualisation of differentially co-expressed genes. CONCLUSION: coXpress can be used to find groups of genes that display differential co-expression patterns in microarray datasets.

Algorithms↗

Strategies for examination of Alzheimer's disease amyloid precursor protein isoforms.

We describe a proteomics procedure using bioinformatics, immunoprecipitation, two-dimensional gel electrophoresis, Western blotting, in-gel digestion, LC-MS, MALDI-MS, and MS-MS for isolation and identification of amyloid precursor protein (APP) isoforms APP695, APP751, and APP770. Retinoic acid-induced Ntera 2 cell line, derived from a human teratocarcinoma cells, was the in-vitro source of APP. Initial isolation of whole APP was performed by immunoprecipitation, using AB10, a monoclonal antibody raised to amino acids 1-17 of the beta-amyloid peptide sequence, which is present in all three alpha secretase-cleaved isoforms of interest. The next stage was separation of whole APP into its isoform components by two-dimensional gel electrophoresis. Because of low APP concentrations, detection by the usual staining methods, for example Sypro Ruby, able to detect low picomole concentrations, did not enable visualisation of the isoforms. Western analysis, however, enabled primary detection of APP, because of the inherent sensitivity of antibodies raised to specific isoform regions. This initial visualization acted as a template for excision of isoforms from 2D gels, which were then subjected to peptide mass mapping. Initial theoretical digestion of each isoform revealed the presence of specific peptides, which were then used as "tags" for isoform detection.

Alzheimer Disease↗

Multiplex PCR for identifying mycobacterial isolates.

AIMS: To develop a multiplex polymerase chain reaction (PCR) method to facilitate identification of mycobacterial isolates. METHODS: Type strains of 14 species of mycobacteria and 56 clinical isolates were lysed by boiling in TE Triton. The lysate (5 microliters) was used directly in a PCR reaction incorporating three pairs of PCR primers expected to amplify fragments from the genome of (a) all mycobacteria, (b) Mycobacterium tuberculosis complex only and (c) M avium only. PCR products were visualised by electrophoresis on agarose gels. RESULTS: Multiplex PCR applied to 14 type strains yielded patterns on electrophoresis which permitted identification of the mycobacterial isolates as M tuberculosis complex, M avium or as mycobacteria other than the former. The identification of 56 clinical isolates by multiplex PCR was consistent with other methods and was accomplished in less than one working day. CONCLUSIONS: This method may facilitate rapid and convenient identification of most clinical isolates of mycobacteria by PCR and gel electrophoresis. Further evaluation is warranted.

Base Sequence↗