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A simple and rapid method for detecting human immunodeficiency virus by PCR.

A simple, sensitive and specific method using the polymerase chain reaction (PCR) for amplification of human immunodeficiency virus type 1 (HIV-1) is described. The method involves minimal manipulations. Peripheral blood mononuclear cells (PBMC) were prepared by a rapid Ficoll-Paque gradient method. Lymphocytes were lysed in PCR buffer containing Proteinase K and detergents, and subjected to amplification under stringent conditions, using two primer pairs. Amplified DNA sequences were hybridized with a 3'-end labelled probe, electrophoresed on agarose gels and visualised by ethidium bromide staining. Identification of amplified HIV-1 proviral DNA sequences was confirmed by autoradiography. HIV-1 sequences were amplified in all samples from 103 HIV-1 seropositive individuals, but not in 40 HIV-1 seronegative controls. The absence of contamination may be attributable in part to minimisation of manipulations before amplification.

Base Sequence

RT97: a marker for capsaicin-insensitive sensory endings in the rat skin.

The mouse monoclonal antibody RT97, which recognises the 200-kDa neurofilament subunit in its phosphorylated form, selectively labels the somata of sensory A-fibres (large light cells) in the dorsal root ganglion of the rat. We have tested the hypothesis that this antibody also visualises large diameter sensory fibres and their end structures in peripheral tissue, in particular in the skin. RT97 immunoreactivity is found in endings that are known to be served by myelinated afferent fibres, including Meissner-like endings, Merkel discs, hair follicle receptors, Pacinian corpuscles and free nerve endings. RT97 immunoreactivity has not, however, been observed in endings of presumably unmyelinated sensory fibres (intraepidermal fibres immunoreactive for substance P and calcitonin gene-related peptide) or in sympathetic fibres innervating sweat glands and blood vessels. In addition, neither systemic (100-150 mg/kg as adults) nor perineural capsaicin pre-treatment affects RT97 immunoreactivity in the skin. The data indicate that RT97 is a useful marker in the study of the capsaicin-insensitive sensory innervation of the skin and possibly other peripheral organs.

Animals

DIPLOMO: the tool for a new type of evolutionary analysis.

A package of computer programs called DIPLOMO (DIstance PLOt MOnitor) has been developed for making pairwise comparisons of different estimates of the distances between a set of taxa by plotting them against each other in a simple scatter plot. Taxa with similar relative distance characteristics are thereby grouped graphically. Groupings of different taxa may be directly identified, and the distance characteristics of chosen groups visualised and compared using devices to give them different colours or symbols. The program is particularly useful for detecting and analysing subtle trends in gene sequence evolution. This is done by comparing different components of change, for example synonymous versus non-synonymous nucleotide changes, transversions versus transitions and changes in different genes of the same set of taxa, etc. The program has a wide range of other uses, for example comparing different methods of sequence analysis, assessing which components of genetic change correlate best with phenotypic change or with geographical separation. This paper describes the DIPLOMO package, and illustrates typical DIPLOMO analyses using lentivirus gene sequence data.

Biological Evolution

Discriminant functions.

Discriminant Functions (DFs), first described by Fisher in 1936, have been applied to the classification of microcytic disorders such as iron deficiency and heterozygous thalassemia. Mathematically DFs are weighted linear combinations of variables. If the underlying assumption of multivariate normality is valid DFs provide the best possible classification. Variables may need to be transformed before the DF is derived. When two groups have to be classified it is easy to visualise the DF. With one variable the DF is represented by the point which provides the best separation. In the bivariate situation the two groups form ellipses and the DF is the best line of separation whilst in the trivariate case the two groups are ellipsoids and a plane forms the best separation. Ratios and power functions are equivalent to DFs but they are less efficient and less rigorously derived. To apply DFs in hematological practice it is necessary to carefully select the measurements to be included and to define the case selection criteria. Once the DF has been derived it should be tested on a new data set and its transferability assessed. Like any single test the DF will have sensitivity and specificity which may need to be adjusted by changing the "cut-off" if the DF is used for screening rather than for differential diagnosis.

Diagnosis

In situ hybridisation and in situ polymerase chain reaction detection of parvovirus B19 DNA within cells.

Modification of an in situ polymerase chain reaction (ISPCR) technique is described for the detection of B19 parvovirus infection. Specific amplification of B19 DNA inside fixed cells was followed by hybridisation with a digoxigenin-labelled probe and then visualised by immunochemical reaction. The assay had higher sensitivity compared to direct in situ hybridisation and still allowed cellular localisation and characterisation of infected cells. This assay can be used as a confirmatory method for PCR in tissues and will allow further identification of tissues permissive for B19 parvovirus infection.

Base Sequence

Linkage studies of X-linked mental retardation: high frequency of recombination in the telomeric region of the human X chromosome (fragile site/linkage/recombination/X chromosome).

One of the commonest forms of X-linked mental retardation is associated with a fragile site at Xq27 on the human X chromosome which can be visualised structurally after culturing cells in folate-deficient media. Unusually, the mutation can be transmitted through a phenotypically normal male. There is already some evidence that the gene loci for G6PD and factor IX are linked to this mental retardation locus. We have followed the inheritance of a DNA sequence 52A, in fragile site families that are also informative for factor IX. We demonstrate that these probes are localised at Xq27/Xq28-Xqter, close physically to the fragile site. We did not find close linkage between 52A, factor IX, and the fragile site in the families studied despite 52A and factor IX showing linkage in normal families. We discuss the importance of these data for the genetic mapping of this region of the human X chromosome and the implication for the use of these DNA probes for clinical diagnosis.

Animals

Visualisation by low-angle shadowing of the leucocyte-common antigen. A major cell surface glycoprotein of lymphocytes.

The leucocyte-common antigen (L-CA) from rat thymocytes is a cell surface glycoprotein of 180 000 apparent mol. wt. with an 80-kd cytoplasmic domain. This paper reports the molecular dimensions of the molecule visualised by electron microscopy after low-angle shadowing. The L-CA monomer consists of a globular head region of approximately 12 nm diameter and a short tail approximately 18 nm long. In deoxycholate both monomers and multimers are seen with aggregation occurring at the head groups. When the detergent is removed, larger clusters are formed with tails extending from a central aggregate. A 100-kd tryptic fragment of L-CA that is known to include the extracellular parts of the molecule also exists in monomer and multimer forms and is seen to have a rod-like structure of length 28 nm without evidence of the head group. Altogether the data indicate that the rod-like structure is found outside the cell and that the extra sequence that forms the head is inside. The tryptic fragment is likely to be derived by cleavage after the transmembrane sequence.

Animals

Spatial scale interactions in stereo sensitivity and the neural representation of binocular disparity.

How are binocular disparities encoded and represented in the human visual system? An 'encoding cube' diagram is introduced to visualise differences between competing models. To distinguish the models experimentally, the depth-increment-detection function (discriminating disparity d from d +/- delta d) was measured as a function of standing disparity (d) with spatially filtered random-dot stereograms of different centre spatial frequencies. Stereothresholds degraded more quickly as standing disparity was increased with stimuli defined by high rather than low centre spatial frequency. This is consistent with a close correlation between the spatial scale of detection mechanisms and the disparities they process. It is shown that a simple model, where discrimination is limited by the noisy ratio of outputs of three disparity-selective mechanisms at each spatial scale, can account for the data. It is not necessary to invoke a population code for disparity to model the depth-increment-detection function. This type of encoding scheme implies insensitivity to large interocular phase differences. Might the system have developed a strategy to disambiguate or shift the matches made at fine scales with those made at the coarse scales at large standing disparities? In agreement with Rohaly and Wilson, no evidence was found that this is so. Such a scheme would predict that stereothresholds determined with targets composed of compounds of high and low frequency should be superior to those of either component alone. Although a small stereoacuity benefit was found at small disparities, the more striking result was that stereothresholds for compound-frequency targets were actually degraded at large standing disparities. The results argue against neural shifting of the matching range of fine scales by coarse-scale matches posited by certain stereo models.

Computer Graphics

Alterations of CAP audiogram by increased endolymphatic pressure and its relation to hydrops.

Most current theories regarding the inner ear pathology of Menières disease assume that there is an augmentation of the endolymphatic pressure due to the presence of hydrops. In this study normal hearing pigmented guinea pigs were employed to investigate the effect of increased endolymphatic pressure on the compound action potential (CAP) audiogram. All animals were implanted with an electrode on the round window and the CAP audiogram was determined prior to further surgery. The endolymphatic canal was then visualised by a posterior fossa intra-dural surgical approach. A hole was pierced in the canal and a cannula inserted. The CAP audiogram was again determined before, and at frequent intervals after, the application of hydrostatic pressure (0.5-1 cm Hg). A similar sequence of CAP sensitivity losses was observed within 2 h for 0.5 cm Hg or 15 min for 1 cm Hg. There was at first a very high frequency loss, followed by a very low frequency loss and finally a mid frequency sensitivity loss rendered the audiogram flat and lying around 50 dB sound pressure level. Given that the first characteristic index for experimental hydrops is a low frequency loss the present data suggest that an increase in endolymphatic pressure, as in these experiments, is likely to be a rather late pathological feature of hydrops. Indeed we have shown that a high frequency loss develops at a second phase during the evolution of hydrops.

Action Potentials

Fatal head injury in children: a new approach to scoring axonal and vascular damage.

As part of a multidisciplinary study of brain damage in children fatally injured in motor vehicle accidents, a simple method to quantify and visualise the distribution and extent of injury has been developed. Vascular and axonal injury were assessed using coronal brain sections stained for haematoxylin and eosin, or reacted immunohistochemically for beta-amyloid precursor protein. Subsequent analysis was carried out using NIH Image software, and the resulting information is displayed in schematic diagrams. These summary diagrams simply and clearly show the distribution of injury in both the coronal and horizontal planes. This technique offers an advantage over previous scoring methods in that it provides both a quantitative and a visual summary of the distribution and extent of brain injury. This information can then be used to compare the injury distribution and severity with estimated impact points and acceleration data.

Accidents, Traffic

[New data in cardiology: the electric charge of the heart].

This study emerging from profound consideration of the basis of vectorcardiography reveals a new electric model of the heart, the starting point for a computer programme of which only the principle is described. Noting inadequacies of vectorcardiography linked to necessary but possibly excessive simplifications, the author suggests a solution based only on Einthoven's postulate of a single dipole: at each instant during the cardiac revolution, the positions of point N, the centre of negative charges and origin of the dipole, of point P, the centre of positive charges and extremity of the dipole, and the value of the load borne by this dipole are calculated. The classical orientations of septal, parietal and basal vectors are thus found. It is shown that electric charge follows a bell-shaped curve and that the velocity of the dipole is compatible with that of the depolarisation wave, in contrast to velocities given by vectorcardiography. The trajectory of the dipole, a veritable "dipologram" visualises breaks in continuity which are interpreted. This new method has the advantage of being entirely confirmable: the dipole provided by the programme enables the calculation of potentials. Comparison between measured potentials and calculated potentials ensures the reliability of results provided by this programme. This method is totally in contrast with conventional vectorcardiography: the dipole is entirely mobile regarding both its origin and extremity, its site in the thorax is precisely identified, and its length is calculated, together with its velocity and the charge which it carries.(ABSTRACT TRUNCATED AT 250 WORDS)

Electricity

High-resolution magnetic resonance imaging of arthritic pathology in the rat knee.

High-resolution magnetic resonance imaging (MRI) has been used to visualise the changes that occur in both soft tissue and bone during antigen-induced, monoarticular arthritis (AIMA) of the rat knee. Extensive optimisation studies were performed in order to minimise the time of the experiments and to maximise both the signal-to-noise ratio and the contrast in the MR images. The study was cross-sectional rather than longitudinal and at each of the 13 time points studied during the progression of the disease, corresponding X-radiographs and histological sections were obtained. Interpretation of the spin echo MR images was aided by the use of chemical shift-selective imaging, magnetisation transfer contrast and relaxation time experiments, as well as by correlation with the histology and X-radiography data. The MR images clearly show invasion of the synovium by an inflammatory pannus which spreads over the articular cartilage and invades the bone, leading to erosion and later remodelling. Two distinct types of bony erosion were observed: focal erosions, especially at the margins of the joint, and subchondral erosions. It is concluded that MRI provides a sensitive, non-invasive method for investigating both early-stage inflammatory changes and late-stage bony changes in the knee joints of the arthritic rat.

Animals

A novel protein antigen of the malaria parasite Plasmodium falciparum, located on the surface of gametes and sporozoites.

A Plasmodium falciparum cDNA clone was isolated of which the insert is transcribed at high rates as a 1.4-kb mRNA in the sexual stages of the malaria parasite. The cDNA clone contains a copy of a non-interrupted gene which codes for a protein of 157 amino acids (Mr = 16607). This 16-kDa protein does not contain repetitive sequences and is characterised by a putative N-terminal signal sequence, a hydrophobic membrane anchor sequence and a highly hydrophilic C-terminal region suggesting that it is an integral membrane protein. Rabbit antisera raised against a synthetic peptide covering amino acids 31-47 of the 16-kDa protein and against recombinant fusion proteins recognised the 16-kDa antigen in protein extracts of gametocytes, macrogamete/zygotes and sporozoites by Western blot analysis. The rabbit antisera also reacted with gametes, gametocytes and sporozoites in a standard immunofluorescence assay. By immunoelectron microscopy using the protein A-gold method the 16-kDa protein could be clearly visualised on the surface of macrogametes and sporozoites, whereas the antigen was not detectable in the asexual erythrocytic stages of the parasite. The 16-kDa antigen of P. falciparum therefore might have the potential to elicit a dual protective immune response against the sporozoite and sexual stage parasites.

Amino Acid Sequence

Laparoscopic laser cholecystectomy: our first 200 patients.

A series of 200 consecutive patients were considered for laparoscopic laser cholecystectomy. Laparoscopic laser cholecystectomy was attempted in 195 cases and was performed in 192 cases. Laparoscopy was performed in five patients, but laparoscopic cholecystectomy was not attempted owing to dense adhesions (3), cholangiocarcinoma (1) and an absent gallbladder (1). The indications for operation were symptomatic gallstones which included biliary colic (142), acute cholecystitis (49) and gallstone pancreatitis (9). The median duration of operation was 75 min. Operative cholangiography was attempted in 151 (77%) of cases, and was successful in 85% of attempts. Laparoscopic common bile duct visualisation was performed three times with successful stone extraction twice. The other common bile duct was normal. The median duration of postoperative hospital stay was 2 days, for return to normal activity 6 days, and for return to work 10 days. Mean analgesic and antiemetic requirements were approximately one-third of those for open cholecystectomy. Of the patients, 94% reported good or excellent overall satisfaction and 96% reported excellent cosmetic results. Seven complications occurred (4%). Three patients had immediate conversion to laparotomy owing to haemorrhage (2) and gallbladder rupture (1). Four patients required laparotomy for postoperative complications (common bile duct damage, slipped clips from cystic duct, perforated duodenum and leaking accessory hepatic duct). No complications occurred in the last 140 cases. These data suggest that laparoscopic laser cholecystectomy reduces the discomfort of laparotomy and allows a shorter postoperative recovery. The operation has a learning curve, but will ultimately be applicable to the majority of patients with symptomatic gallstones.

Adult

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

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

RuvAB-mediated branch migration does not involve extensive DNA opening within the RuvB hexamer.

The Escherichia coli RuvA and RuvB proteins promote the branch migration of Holliday junctions during the late stages of homologous recombination and DNA repair (reviewed in [1]). Biochemical and structural studies of the RuvAB-Holliday junction complex have shown that RuvA binds directly to the Holliday junction [2] [3] [4] [5] [6] and acts as a specificity factor that promotes the targeting of RuvB [7] [8], a hexameric ring protein that drives branch migration [9] [10] [11]. Electron microscopic visualisation of the RuvAB complex revealed that RuvA is flanked by two RuvB hexamers, which bind DNA arms that lie diametrically opposed across the junction [8]. ATP-dependent branch migration occurs as duplex DNA is pumped out through the centre of each ring. Because RuvB possesses well-conserved helicase motifs and RuvAB exhibits a 5'-3' DNA helicase activity in vitro [12], the mechanism of branch migration is thought to involve DNA opening within the RuvB ring, which provides a single strand for the unidirectional translocation of the protein along DNA. We have investigated whether the RuvB ring can translocate along duplex DNA containing a site-directed interstrand psoralen crosslink. Surprisingly, we found that the crosslink failed to inhibit branch migration. We interpret these data as evidence against a base-by-base tracking model and suggest that extensive DNA opening within the RuvB ring is not required for DNA translocation by RuvB.

Adenosine Triphosphate

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