Factor V Leiden mutation screened by PCR and detected with lanthanide-labeled probes.

The Factor V Leiden mutation is an important human polymorphism, responsible for increased risk of venous thrombosis in heterozygotes as well as homozygotes. Therefore, screening is a useful possibility, and many detection systems have been described for PCR products. We have developed a simplified and robust assay using oligonucleotide probes for normal and mutant sequences, labeled with europium and samarium, respectively, and measured by time-resolved fluorescence. Populations consisting of 233 Welsh and 148 Irish subjects were examined by both restriction fragment length polymorphism (RFLP) analysis and our assay. The allele frequency was 14/466 in the Welsh and 5/296 in the Irish population, in line with other surveys of European populations. Results were not obtained in 2/381 samples by RFLP, compared with 1/381 with our method. We conclude that our method represents an improved system capable of considerable throughput at reasonable cost.

Mitochondrial DNA does not appear to influence the congenital onset type of myotonic dystrophy.

Neither the maternal inheritance pattern nor the early onset of congenital myotonic dystrophy are fully explained. One possible mechanism is that mitochondrial DNA (mtDNA) mutations might interact with the DM gene product, producing an earlier onset than would otherwise occur. We have used Southern hybridisation to show that high levels of major rearrangements of mtDNA are not present in muscle of five and in blood of 35 patients with congenital myotonic dystrophy. We used sequence analysis to show that no one particular mtDNA morph appears to cosegregate with congenital onset. A minor degree of depletion of mtDNA compared with nuclear DNA was present in the muscle of five patients with congenital DM, but we propose that this is not the primary cause of the muscle pathology but secondary to it. We have not found evidence that mtDNA is involved in congenital myotonic dystrophy.

Variation in mitochondrial DNA levels in muscle from normal controls. Is depletion of mtDNA in patients with mitochondrial myopathy a distinct clinical syndrome.

Recent studies have identified a group of patients with cytochrome oxidase (COX) deficiency presenting in infancy associated with a deficiency of mtDNA in muscle or other affected tissue (Moraes et al 1991). We used a novel approach to compare the level of mitochondrial (mtDNA) compared to nuclear DNA in skeletal muscle from a group of patients and controls, based on dot blots that were hybridized with a mtDNA probe labelled with 35S[dCTP] and a reference nuclear DNA probe labelled with [32P]dCTP. The ratio of mtDNA to nuclear DNA varied in samples from different muscles of the same individual. Secondly, fetal muscle had very low levels of mtDNA compared to nuclear DNA, and data from older controls (cross-sectional rather than sequential) suggest that this increases rapidly over the first 3 months after birth and thereafter more slowly. Four patients with COX deficiency had levels of mtDNA that were below the age-specific range defined by 'normal' quadriceps muscle. The clinical features to two of these patients were similar to earlier case reports of mtDNA depletion. In three patients the clinical course was relatively benign compared to cases that have previously been described. Levels of mtDNA in skeletal muscle from some patients with other forms of muscle disease were also found to be low, suggesting that mtDNA depletion, possibly related to depletion of mitochondria, may be a relatively non-specific response of muscle to various pathological processes. However, there does appear to be a distinctive group of young patients with reduced cytochrome oxidase activity in muscle, in whom marked mtDNA depletion reflects the primary defect.

The beaded form of myelinated nerve fibers.

When the nerves are lightly stretched and fixed by freeze-substitution, their fibers show the form-change termed "beading" which consists of a series of undulating constrictions and swellings in the internodes. This form change has not ordinarily been seen in chemically fixed nerves, or when it has, it has been ascribed to a pathological change or an artifact. We now report that beading is also retained in normal nerves when, following a light maintained stretch, they are fixed with aldehydes at a temperature close to 0 degrees C. The degree of beading in single fibers teased from the aldehyde fixed nerves was graded and found to be maximal at 0 degrees C, falling off with increased temperature until, at temperatures above 16 degrees C, most fibers showed no beading or a very mild beading. The fibers of nerves cold-fixed at 0 degrees C displayed the characteristics as freeze-substituted fibers, but with a somewhat smaller number of maximally beaded fibers and an 18% reduction in microtubule numbers in the axons. Desheathing or slitting the sheaths of the nerves before cold-fixation increased the probability of retaining beading. Exposure of stretched nerves to the aldehyde fixative at room temperatures for times as short as 3-5 min before they were cold-fixed showed a diminished degree of beading, indicating that aldehydes can have a deleterious effect on the beading mechanism which we hypothesize to be present in the fiber. This action is distinct from the general cross-linking action of aldehydes.

Quantification of specific mRNA by flatbed scintillation counting of dual-labeled dot blots.

A dual-labeling technique was developed for direct quantification of specific mRNA using a flatbed liquid scintillation counter. This method simultaneously measures cpm of 32P- and 35S-labeled probes bound to RNA dot blots and subtracts counts due to nonspecific background radioactivity bound to the filter. Probes for T-cell receptor and beta-actin (as the internal standard) were hybridized both separately and simultaneously to RNA isolated from five different sources. There was concordance between the radioactivity measured from single- and dual-hybridizations for each combination of 35S- and 32P-labeled probes. This methodology directly quantifies specific mRNA sequences bound to membranes and has potential for measuring gene dosage, without the need for re-probing or densitometric analysis.

Papillomavirus screening in cervical cell samples using dual-label dot-blot analysis.

The presence of human papillomavirus (HPV) in cervical cells is closely related to the development of cervical carcinoma. Detection of virus may be by Southern blot, dot blot or the highly sensitive polymerase chain reaction. Whatever method is employed, there are problems of false negatives due to poor clinical samples in which the DNA may be degraded or is absent altogether. Here we describe a new method of dual labelling for dot blots using a 32P-labelled probe for HPV and a 35S-labelled probe for human actin genes. The samples were counted on a Beta-plate flat-bed scintillation counter and the data analysed to separate the activities of the two isotopes. The counts from the actin probe show whether human DNA is present or not and false negatives from this cause may thereby be eliminated. The counts due to HPV when compared with those for actin give a quantitative measure of HPV abundance for the particular sample and this may have clinical relevance.

Quantification of oncogene dosage in tumours by simultaneous dual-label hybridization.

Gene amplification and allele loss occur in a variety of human tumours and some have prognostic value. Therefore, techniques which facilitate detection and quantification of gene dosage could have wide applicability in cancer research. Using the INT-2 gene as a model system, a quantitative procedure has been developed for measuring gene copy number using dual-label hybridization to DNA dot blots. A probe specific for the INT-2 gene was labelled with [alpha-32P]dCTP and a probe to beta-actin, the control locus, was labelled with [alpha-35S]dATP. Flat-bed scintillation counting was used to detect and separate the emissions resulting from each bound probe, and gene dosage was calculated from the ratio of INT-2 to the beta-actin probe compared with the ratio derived from constitutional DNA. Calculated ratios of greater than 1.22 and less than 0.78 indicated gene amplification and allelic loss respectively, at the 99% confidence limit derived from the population of 35 constitutional DNAs. The results were validated by RFLP analysis. It is expected that this technique will permit precise gene dosage quantification in many areas.

Quantification of 32P-labeled samples in gel fragments using the flat-bed liquid scintillation counter.

Quantification of 32P in bands after gel electrophoresis was performed using the flat-bed scintillation counter (Betaplate). The most convenient system involved placing fragments of dried gel between two glass fiber sheets, each previously sealed in a thin plastic bag with liquid scintillant. Good pulse-height spectra and counting efficiencies were obtained with low cross talk and background. The method has been used to quantify mRNA in RNA antisense-protection assays that were linear over a wide range (1-20000 cpm). Cross talk and background could be reduced further by an alternative technique utilizing plastic trays with shallow wells in which a solid scintillant had been melted. Fragments were immersed in the molten scintillant (90 degrees C), which was allowed to solidify, by cooling, before counting.

Hypoglycemic neuropathy in experimental diabetes.

Morphological and electrophysiological observations were made over 4 weeks on 5 groups of 8-week-old male Sprague-Dawley rats. These were comprised of controls, untreated diabetics, and diabetic animals in which sustained hypoglycemia, moderate hypoglycemia, or normoglycemia was induced by continuous subcutaneous insulin infusion (CSII) therapy. Teased fiber studies showed a marked increase in the number of myelinated fibers undergoing axonal degeneration and regeneration in the tibial nerve of severe hypoglycemic and also in moderate hypoglycemic animals but not in controls, untreated diabetic and normoglycemic groups. There was also a significant correlation between episodes of hypoglycemia (less than or equal to 2.0 mmol/l) and the prevalence of axonal degeneration and regeneration in CSII-treated diabetics. Motor nerve conduction velocity was significantly reduced in the moderate and severe hypoglycemic groups and also in untreated diabetic animals when compared with controls. However, it was significantly improved in the normoglycemic group over the untreated diabetic and severe hypoglycemic groups. In conclusion, this study has demonstrated that severe or even mild hypoglycemia produced a detrimental effect on peripheral nerve structure and function in experimental diabetes. Therefore, it may be desirable to avoid even asymptomatic hypoglycemia in the management of diabetes.

Lymphocyte proliferation and cytotoxic assays using flat-bed scintillation counting.

Lymphocyte 51Cr release and [3H]thymidine uptake assays were evaluated with respect to measurement of sample radioactivity using the flat-bed scintillation counter. 51Cr lysates were spotted onto a glass fibre filter sheet while [3H]thymidine-labelled cells were filtered onto a similar sheet using a cell harvester. The 96 samples were rapidly processed for counting, without removal of individual sample areas. Either form of preparation showed good linearity of count rate with the quantity of material on the filter. Reproducibility was good; the coefficient of variation for 96 samples being within 5%. The low background and high efficiency of this counter results in increased assay sensitivity and allows considerable economies in materials to be made. A commercial version of the counter has six counting heads permitting a high rate of sample throughput.

Variation of erythroid and myeloid precursors in the marrow and peripheral blood of volunteer subjects infected with human parvovirus (B19).

Infection of normal individuals with human parvovirus (B19) results in a mild disease (erythema infectiosum) but gives rise to aplastic crises in patients with chronic hemolytic anemias. The effects of this disease on hemopoiesis were investigated following intranasal inoculation of the virus into three volunteers. A typical disease ensued with a viremia peaking at 9 d. Marrow morphology 6 d after inoculation appeared normal but at 10 d there was a severe loss of erythroid precursors followed by a 1-2-g drop in hemoglobin, and an increase in serum immunoreactive erythropoietin. Erythroid burst-forming units (BFU-E) from the peripheral blood were considerably reduced, starting at the time of viremia and persisting for 4-8 d depending on the individual. Granulocyte-macrophage colony-forming units (CFU-GM) were also affected but the loss started 2 d later. Both CFU-GM and BFU-E showed a sharp overshoot at recovery. In the marrow, BFU-E and CFU-E were reduced at 6 and 10 d in the individual having the longest period of peripheral progenitor loss. In contrast, there was an increase in BFU-E and CFU-E in the subject with least change in peripheral progenitors. In the third subject, with an intermediate picture, there was a loss at 6 d but an increase at 10 d of erythroid progenitors. It is suggested that the architecture of the marrow might partially isolate progenitors from high titers of virus in the serum and individual variation in this respect might give the results observed.

A new design for a liquid scintillation counter for micro samples using a flat-bed geometry.

A new design for a liquid scintillation counter based on a flat-bed geometry is described. Micro-samples are dried or filtered onto transfer membranes or glass fibre filters in a 6 X 16 matrix, compatible with 96-well micro-titration plate filtration assays of labelled cells. A prototype counter without lead shielding had low background countrates (2-3 cpm for 3H) giving a figure of merit of 1325 (and 1292 for 14C). Only 5-15 ml of scintillant/96 samples are required and thus the volume of radioactive waste is low.

Long-term haemopoiesis in human fetal liver cell cultures.

Haemopoiesis in human fetal liver is almost entirely restricted to the erythroid series but when fetal liver cells were cultured under conditions established for the long-term maintenance of adult marrow haemopoiesis, a rapid switch to granulopoiesis was observed. Erythroid progenitor cells (BFU-E) rapidly disappeared, even though no humoral or cellular inhibitors of erythropoiesis could be detected, while myeloid progenitors (CFU-GM) increased in number. When the fetal liver cells were seeded onto stromal layers derived from adult marrow, in which endogenous haemopoiesis had ceased, granulopoiesis was established and maintained for more than a year, considerably longer than has previously been achieved with human haemopoietic cells.

Erythroid differentiation in CGL cells from a patient with blast crisis.

A 60-year-old patient with Ph1 + ve CGL presented with blast crisis. The leukaemic blast cells resembled erythroblasts and had 51 chromosomes with two Ph1. Cells obtained from peripheral blood, marrow and a pleural effusion were cultured under a variety of conditions. After 2-3 weeks in culture, the model 51-chromosome line persisted but many of the cells displayed erythroid morphology and differentiated to resemble mature normoblasts, strongly positive on o-tolidine +ve staining. Haemoglobin analysis by starch gel and globin synthesis studies demonstrated only fetal haemoglobin (HbF) synthesis in the cultured cells whilst the patient's reticulocytes synthesized very little HbF. Restriction enzyme mapping of DNA from the cultured cells showed that beta-globin genes were still present in these cells even though they were not expressed.

Cytosine arabinoside deamination in human leukaemic myeloblasts and resistance to cytosine arabinoside therapy.

1. The conversion of cytosine arabinoside into its active metabolite cytosine arabinoside triphosphate, and catabolism by deamination to uracil arabinoside, was measured in intact marrow myeloblasts from patients with acute myeloid leukaemia. The ratio of uracil arabinoside/cytosine arabinoside triphosphate ranged from 0.32 to 19.11. 2. The effect of tetrahydrouridine, and inhibitor of cytosine arabinoside deamination, on cytosine arabinoside triphosphate production was studied. The greatest increase of cytosine arabinoside triphosphate production caused by addition of tetrahydrouridine was 27%. 3. The increase in cytosine arabinoside triphosphate production wad not related to the ratio of uracil arabinoside/cytosine arabinoside triphosphate or to the deaminase activity per 10(6) cells. It wa proportional to the percentage change of cytosine arabinoside in the incubation medium. 4. The sensitivity of DNA synthesis to inhibition by cytosine arabinoside was measured in myeloblasts from 11 patients. Addition of tetrahydrouridine did not increase the sensitivity of the marrow to cytosine arabinoside. 5. Cytosine arabinoside deamination is unlikely to be an important mechanism of resistance in myeloblasts in vivo, although it may produce apparent resistance in vitro.