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PubMed1997

Characterisation of basic proteins from Spiroplasma melliferum using novel immobilised pH gradients.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has become the method of choice for efficient separation of complex protein mixtures. Previously, analysis of the Spiroplasma melliferum proteome (protein complement of a genome) has been performed with pH 3-10 and narrow range pH 4-7 IPG gel strips. We report here on the use of novel 18 cm basic (pH 6-11) immobilised pH gradients (IPG) to increase the resolution of protein spots visible within 2-D gels. These gradients were synthesised to emulate the gradient of commercially available IPG gel strips in a 5 cm region of overlap so as to attempt construction of a more complete map of cellular protein expression. Approximately 50 additional gene products were detected from S. melliferum that were not previously well-resolved or visible using wide-range pH 3-10 IPG gel strips. Twenty-seven of these were electrotransferred to polyvinylidene difluoride (PVDF) membrane and analysed by N-terminal protein microsequencing. Protein spots with an initial peak yield of as little as 100 femtomoles (fm) were sequenced to 5-10 amino acid residues, demonstrating the importance of improved sample handling procedures and analytical technologies. Many essential metabolic enzymes were shown to have basic pI, including: glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, carbamate kinase and lactate dehydrogenase. A very basic protein (pI approximately 11.0) was identified as uridylate kinase, an enzyme indirectly associated with pyrimidine biosynthesis and thought be absent in some members of the bacterial class Mollicutes. The advent of novel basic (pH 6-11) IPGs has allowed the visualisation of a significantly greater percentage of the 'functional proteome', that portion of the total protein complement of a genome actively translated within a specific time frame, on 2-D electrophoresis gels. This will aid in the characterisation of translated gene products in conjunction with genome sequencing initiatives.

Amino Acid Sequence↗

PubMed1990

Immunohistochemical localisation of nerve growth factor in a subpopulation of chick spinal ganglion neurons.

We have previously isolated and sequenced the chicken nerve growth factor (NGF) gene and now, from the deduced amino acid sequence, selected and produced peptides suitable for use as antigens. Anti-sera raised against these peptides inhibit the biological activity of a partially purified preparation of native chicken NGF and, when used in immunocytochemical studies, allow the visualisation of sensory neurons accumulating endogenous NGF. Immunoreactive cells form a distinct population of small neurons which may correspond to the well-described neurons generated in the dorsomedial area of spinal ganglia. We conclude that two subpopulations of neurons exist within dorsal root ganglia, whose requirements for, and use of, NGF may be quite distinct.

Amino Acid Sequence↗

PubMed1997

Development of phrenic motoneuron morphology in the fetal rat.

This study examined the morphological changes that a homogeneous mammalian spinal motoneuron population undergoes during foetal development. Retrograde labelling of the phrenic nerve with the carbocyanine dye, DiI, was used to visualise developmental changes in phrenic motoneuron morphology within the cervical spinal cord of perinatal rats from embryonic day (E) 13.5 to birth (ca. E21). Groups of intimately associated phrenic somata had migrated into the ventromedial region of cervical segments C3-C6 by E14. This migration was followed by their progressive compaction into a tightly aligned column by E18. During this period, close contact was maintained between phrenic somata throughout the motor pool, suggestive of the presence of gap junctions. From E15 to E18, extensive dendritic arborisations fanned out dorsolaterally and ventromedially into the white matter and the floor plate. By E19, however, dendritic fasciculation and retraction and the extension of newly formed rostrocaudally projecting dendrites had resulted in the approximation of the dendritic morphology observed at birth. These data demonstrate that morphological maturation of phrenic motoneurons occurs subsequently to the onset of functional recruitment and the arrival of central processes of dorsal root ganglion neurons within the ventral horn (ca. E17). By birth, a number of immature features remain, including a larger proportion of neurites that project into the white matter and into the floor plate, the presence of growth cones on a number of dendrites, and close contact between populations of contralaterally derived dendrites.

Animals↗

PubMed1992

3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus).

3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗

PubMed1997

A multidimensional partition analysis of SSFP image pulse sequences.

The k-space description, of MRI pulse sequences, has been combined with a partition model in order to model the image reconstruction and the contrast behaviour found in SSFP pulse sequences. A partition represents the magnetisation created, due to excitation by a given rf pulse. In the present model, it is visualised as a set of parameters rather than a vector sum taken over a collection of spins. A multidimensional parameter space, where each dimension is associated with one of the partition parameters, is introduced in order to describe the interaction between partitions and pulse sequence events (e.g., rf pulses and gradients). The three k-space dimensions form the first three dimensions and higher orders are used to handle phase dispersions due to diffusion and main field inhomogeneities. The model makes it possible to perform fast simulation of images resulting from general SSFP pulse sequences. A computer implementation generates images (256 matrix), containing more than 10 different T1/T2 combinations, in less than 45 s on a 120 MHz Pentium computer. The contrast behaviour and signal intensities found in simulated images show excellent agreement with data generated using a clinical MRI scanner system.

Computer Simulation↗

PubMed1992

Localization and function of the D3 dopamine receptor.

A novel dopamine receptor has been recently cloned, which differs from D1 and D2 receptors by its sequence, localization, pharmacology and possibly signalling system, hence its designation as D3 receptor. The D3 receptor cDNA was obtained by a combination of screenings of DNA libraries and PCR polymerase chain reaction experiments. It encodes a protein with a predicted structure consisting in 7 transmembrane domains indicating that it belongs to the G-protein coupled receptor family. Its global homology is 52% with the D2 receptor but 78% if only transmembrane domains are considered. Two shorter transcript variants, in addition to the full-length cDNA were detected by PCR in various rat brain regions. The shorter transcripts are generated by alternative splicing and encode two putative proteins respectively interrupted after the second transmembrane domain and lacking the second extracytoplasmic loop. After transfection of this latter isoform into cultured cells, no dopaminergic activity could be detected. These shorter splice variants may regulate the number of active D3 receptors. The human D3 receptor was also cloned using similar approaches with rat D3 receptor cDNA probes and was found highly homologous to the rat receptor, except in the third intracytoplasmic loop. The human D3 receptor gene was assigned to the chromosome 3 at q13.3 band. Visualisation of D3 receptor mRNA in rat brain by in situ hybridization indicated a predominant expression of the message in the ventral striatum and other "limbic" areas. There is no overlap in the distributions of D2 and D3 receptor mRNAs in discrete structures, suggesting that the two transcripts are expressed by different cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1998

[PET in neuroendocrine tumors].

With the radionuclide tracers available today, 50-90 per cent of neuroendocrine tumours of the gastro-intestinal tract can be visualised with PET (positron-emission tomography). PET also enables the effect of tumour treatment to be monitored in terms of biochemical and functional variables, which is not possible with other radiological techniques. Owing to the very good tumour resolution possible with PET, it serves as a complement to other routine methods such as computed tomography and ultrasonography, and can be used to screen the chest and abdomen for small primary tumours that can not be detected with other methods. In several pre-operative trials PET has been shown to demonstrate more changes in the pancreas and liver than was possible with other methods. In the near future it will be possible to demonstrate the presence of and quantify growth factor receptors, hormones, enzymes, DNA synthesis, mRNA synthesis and protein synthesis. Access to these tumour biological data will be of crucial importance to the individualisation of treatment.

Forecasting↗

PubMed1984

[2-dimensional echography in the preoperative evaluation of mitral stenosis].

2D echocardiography has become one of the most important investigations in the preoperative assessment of mitral stenosis. This study was undertaken to determine the reliability of the information so obtained, by comparison with the surgical appearances. The study population consisted of 104 patients (average age 45 years, 76% women) undergoing open heart surgery for pure mitral stenosis (72%) or mixed mitral valve disease (28%) between 1980 and 1981. All underwent 2D echo using a phased array Aloka SSD 800 80 degrees sector scanner. Cardiac catheterisation was performed in 102 cases and left ventricular angiography in 89 cases. The echocardiogramme was interpreted by an observer who had no knowledge of the surgical results. The mitral surface area, the condition of the valves and subvalvular apparatus and the predictive value of the possible surgical technique were analysed. The 2D echo mitral surface area was estimated by planimetry and quantitatively by using the Gorlin formula during catheterisation and by the surgical description preoperatively. 2D echo was more sensitive than M mode in the detection of severe mitral stenosis (90% vs 73%, p less than 0,01). The 2D echo-Gorlin correlation was quite good (R = 0,70, p less than 0,01) but was worse when the valves were very thickened. When compared with the surgical observations, 87% of the 2D echo data was correct. The thickness of the valves, their amplitude, the diastolic bowing of the anterior leaflet and the presence of calcification were assessed by 2D echo. The echo-surgical results matched perfectly in 76% of cases. The usual cause of error was underestimation of the degree of valvular damage. The valvular bowing and thickness were the most useful signs. Dense, brilliant echos of valvular calcification were found in only 58% of surgically proven cases of valvular calcification, but the error was often related to fine calcification, not visible on fluoroscopy, or to its localisation on the posterior leaflet. The subvalvular apparatus was evaluated in over 93% of patients, a complete study being possible in 73% of them. The 2D echo-surgical correlations were excellent in 90% of the cases in which it had been completely visualised. The chordal thickening was correctly predicted in 79% of cases. The surgical assessment was more pessimistic in 1/3 of cases in which the chordae appeared to be of normal thickness. The length of the chordae was correctly predicted in 68% of cases. The surgical assessment was more pessimistic in 1/2 of cases in which the chordae appeared to be of normal length.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

PubMed1997

Numerical chromosomal aberrations in Hodgkin's disease detected by in situ hybridisation on routine paraffin sections.

AIMS: To visualise directly numerical chromosomal aberrations and polyploidy in both Hodgkin and Reed Sternberg (HRS) cells and background cells from cases of Hodgkin's disease using in situ hybridisation. METHODS: Non-isotopic DNA in situ hybridisation was applied to interphase cell nuclei of Hodgkin's disease within routine paraffin embedded tissue sections. Two a satellite DNA probes, specific for chromosomes 3 and 12, were used to evaluate the feasibility of this approach. Double labelling with immunocytochemical detection of the CD30 antigen was used to identify HRS cells. Cytogenetic normal diploid and triploid placental tissue served as controls. RESULTS: The eight cases of Hodgkin's disease investigated displayed frequent polysomy, while the majority of background cells showed disomy signals. CONCLUSIONS: Numerical chromosomal aberrations were detected in HRS cells from eight cases of Hodgkin's disease by in situ hybridisation. These data show that in Hodgkin's disease HRS cells frequently display polyploidy compared with background cells and are, therefore, probably the only neoplastic component in this disease. Correlations between polysomy and tumour type or grade could not be made from these data owing to the limited number of cases examined and to problems with interpreting data from truncated nuclei.

Adolescent↗

PubMed1994

Structural determinants of substrate selection by the human insulin-receptor protein-tyrosine kinase.

Using NMR spectroscopy to visualise tyrosine phosphorylation kinetics in real time, we have investigated the sequence-dependent determinants of the selectivity of the human insulin receptor protein-tyrosine kinase for different tyrosine residues. The peptides used encompass the multiple-tyrosine-containing autophosphorylation site sequences from the insulin receptor kinase core domain (Tyr1158, Tyr1162 and Tyr1163) and from its specific C-terminal tail domain (Tyr1328 and Tyr1334). Comparison of the phosphorylation kinetics with those found for the tyrosine residues on a peptide comprising the regulatory tyrosine phosphorylation site of cdc2 points to the role of the primary sequence context of the phosphate acceptor. The particularly deleterious influence of a basic residue immediately C-terminal to the tyrosine is discussed in relation to the autophosphorylation properties of the regulatory loop regions of the insulin and epidermal growth factor receptor kinases. The data further suggest that receptor tyrosine kinase active sites and their substrate targets act in concert to ensure that specific downstream effects are activated.

Amino Acid Sequence↗

PubMedDate not supplied

The microanatomy of the alveolar duct of the human lung imaged by confocal microscopy and visualised with computer-based 3D reconstruction.

The most widely accepted model of human lung alveolar duct systems is that they are constructed from central helical fibres between the turns of which lie alveolar opening. Experimental difficulties of handling and sectioning lung tissue have made it difficult to confirm this. Confocal laser scanning microscopy (CLSM) was therefore used to generate optical serial sections of the lung that were reconstructed in three dimensions and displayed using volume rendering techniques. From images of the reconstructions, a new structure is proposed in which alveolar ducts consist of collections of connected oval, twisted loop structures with eccentric openings.

Adult↗

PubMed1993

Assessment of the influence of subacute phenobarbitone administration on multi-tissue cell proliferation in the rat using bromodeoxyuridine immunocytochemistry.

The effects of daily administration of phenobarbitone on the mitotic rates of several tissues were investigated by bromodeoxyuridine (BrdU) immunocytochemistry. Phenobarbitone (80 mg/kg per day) was dosed to AP Wistar male rats for up to 7 days and BrdU (10 mg/ml) was given by infusion at a rate of 10 microliters/h via subcutaneously implanted osmotic minipumps for 2 days prior to necropsy on days 1, 2, 3, 5 and 7. BrdU-labelled nuclei were visualised by peroxidase-antiperoxidase immunocytochemistry and counts of the numbers of labelled cells (labelling index, LI%) made from at least 1000 cells per tissue section(s). The LIs of several tissues (testis, adrenal cortex and medulla, kidney distal convoluted tubule and exocrine pancreas) showed no statistical difference by comparison with controls. Several tissues exhibited characteristic responses to phenobarbitone administration. Pituitary and endocrine pancreas LIs were decreased while those of thyroid, liver and kidney proximal convoluted tubule were increased. The pattern of LI increase was unique to each tissue with liver (median and lateral lobes) increased two-fold on day 3 and returning to control levels thereafter while kidney proximal tubule LI rose gradually with time and remained elevated on day 7. Thyroid LI on day 1 was almost double that of day 0 control and increased steadily thereafter. These data illustrate the varied responses of different tissues to phenobarbitone exposure, namely, depression and stimulation of mitosis. The causation of these functional changes is discussed in relation to direct and indirect effects on functional parameters, especially enzyme induction, alterations in hormonal and growth factor status and receptor regulation.

Adrenal Glands↗

PubMedDate not supplied

Direct diagnosis of carriers of Duchenne and Becker muscular dystrophy by amplification of lymphocyte RNA.

Rapid detection of deletion and duplication mutations that cause Duchenne and Becker muscular dystrophy was achieved in patients and carriers after amplification of small amounts of mRNA from peripheral blood lymphocytes. The entire coding region of the dystrophin mRNA was amplified in 10 sections by reverse transcription and nested polymerase chain reaction, and the products were directly visualised on acrylamide minigels with ethidium staining. Major structural gene mutations were identified by the appearance of a band of different size to that of the wild type. The altered band was readily detected in all patients and heterozygous relatives. This non-radioactive test of venous blood samples can be used for unambiguous and rapid identification of virtually all carriers of deletions or insertions within the dystrophin gene.

Base Sequence↗

PubMed1996

Combined use of proacrosin immunocytochemistry and autosomal DNA in situ hybridisation for evaluation of human ejaculated germ cells.

The recently reported human pregnancies and births after fertilising oocytes with round spermatids recovered from the ejaculate of men with non-obstructive azoospermia have underscored the need for a more accurate evaluation of the nuclear and cytoplasmic maturation status of ejaculated germ cells. In this study we describe our first experience with a method combining the immunocytochemical visualisation of proacrosin with autosomal DNA fluorescence in situ hybridisation (FISH) to assess ejaculated germ cells from patients with a spermiogenesis defect. The proacrosin immunoreactivity, analysed with the use of the monoclonal antibody 4D4, has been detected in cells of round spermatid size presenting a haploid FISH figure as well as in larger cells whose ploidy corresponds to primary and secondary spermatocytes. These observations are in agreement with previously published results obtained, with the use of the same antibody, by immunocytochemical analysis of histological sections of testicular tissue. All the cells of round spermatid size possessing proacrosin immunoreactivity were found to be haploid by FISH. On the other hand, some of the haploid cells of round spermatid size did not possess proacrosin immunoreactivity. The structural pattern of proacrosin immunoreactivity was highly variable both in spermatids and in younger spermatogenic cells. These data show that cell size is the main criterion to be used for the identification of ejaculated round spermatids, whereas the presence of the developing acrosome represents only an auxiliary criterion. The scoring of acrosomal development in ejaculated spermatids may be useful as part of pre-treatment diagnosis before the inclusion of infertile couples in a spermatid conception programme.

Acrosin↗

PubMed1992

Multiple mitochondrial DNA deletions in an elderly human individual.

We have used the polymerase chain reaction (PCR) to study deletions in the mitochondrial DNA (mtDNA) of an elderly human individual. An extended set of PCR primers has been utilised to identify 10 mitochondrial DNA deletions in a 69-year-old female subject with no known mitochondrial disease. The particular deletions visualised as PCR products depended on the primer pairs used, such that the more distantly separated PCR primers enabled visualisation of larger deletions. Some deletions were common to the heart, brain and skeletal muscle, whereas others were apparently specific to individual tissues. DNA sequencing analysis of PCR products showed that short direct repeat sequences (5 to 13 bp) flanked all deletion breakpoints; in most cases one copy of the repeat was deleted. It is proposed that the accumulation of such multiple deletions is a general phenomenon during the ageing process.

Aged↗

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↗

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↗

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↗