Dihydroagarofuran sesquiterpene alkaloids from Maytenus putterlickoides.

Two new dihydroagarofuran sesquiterpene alkaloids, putterines A (2) and B (3), and a known alkaloid, mayteine (1), were isolated from the root of Maytenus putterlickoides. The structures of these compounds were elucidated by various spectroscopic techniques, including 1H and 13C NMR, COSY, ROESY, HMBC, and high-resolution FABMS.

Application of automation to the detection of radiation damage using FISH technology.

A whole chromosome painting approach was employed to highlight the damaging effects of low-to-moderate doses of ionizing radiation. A detailed tally of damage involving the painted chromosomes 1 and 2 was compiled from visual analysis and compared with the results of an automatic image processing approach, where the possible outcomes were 'normal', 'abnormal', or 'rejected'. The performance of the automatic approach was tested using a set of 9000 bicolour metaphase images harvested from whole-blood cell culture following irradiation levels of 0.0, 0.5, 1.0 and 2.0 Gy. Every metaphase image in the set was analysed visually. The automatic analysis model was based on two simple image criteria to distinguish normal from abnormal; either an increase in the number of painted objects or a large asymmetry in the area distribution of the expected number of painted objects. A result was obtained without a full karyotype analysis. In practice, automatic analysis produced a set of images for review that were enriched by a factor of 3-4 in true abnormal images. Fast visual review of these images (approximately 200/h) selected the true abnormals. A comparison of the automatic analysis with the visual analysis showed that automated analysis correctly identified 60% of normal cells, 59% of abnormal cells and 73% of rejected cells.

Automated linkage analysis in psychiatric disorders.

A genome-wide search for linkage of microsatellite markers to chromosomal loci containing genes responsible for the major psychoses is a laborious task which can be carried out with greater speed and economy by introducing automation to several steps in the procedure. We describe the use of the Automated Linkage Preprocessor (ALP) program for the computer analysis of the waveform generated by fluorescein-labelled markers after electrophoretic separation. (To obtain a copy send a request to A.F. Brown at the below MRC address or use Anonymous FTP to ftp.hgu.mrc.ac.uk. Software is in directory pub/ALP). The program runs on a PC in the Microsoft Windows environment, and is used in conjunction with an automated laser fluorescence (ALF) sequencer (Pharmacia) and its Fragment Manager software to detect and size the PCR products, filter out peaks of fluorescence due to nonallele fragments, and generate genotypes in a format suitable for direct input to standard linkage analysis programs. The method should offer the advantages of speed, accuracy, and reduced cost. Its use in linkage studies in a large family with manic-depressive illness is discussed.

Automation of genetic linkage analysis using fluorescent microsatellite markers.

Automation of the typing of genetic markers offers advantages of speed, accuracy, and cost in the mapping of genetic traits and the construction of high-resolution linkage maps. We have developed an automated linkage analysis system by (i) using a robotic pipettor to set up polymerase chain reactions (PCR) to amplify microsatellites with incorporation of a single fluorescent label; (ii) using an automated sequencing apparatus for detection of the PCR products; (iii) sizing alleles automatically by the use of internal and external standards; (iv) iteratively filtering out nonallelic fragments and checking for Mendelian consistency; (v) calculating the probabilities of selected genotypes; and (vi) automatically formatting the results for input to linkage analysis programs. The method provides accurate sizing of alleles, minimizes the risk of error during manual reading and transcription of data, and increases the throughput of reliable data. It brings any inconsistencies or ambiguities in the data to the attention of the user and facilitates examination of the raw data. The ALF/ALP system, together with new, optimized microsatellite sets, particularly tetranucleotide repeats, is likely to be well-suited to fully automatic genetic linkage analysis.

Driving multiple pulsed field electrophoresis devices from one personal computer.

A PC has been programmed to control more than one pulsed field electrophoresis device (e.g. clamped homogeneous electric field, CHEF; field inversion gel electrophoresis, FIGE). Each device is assumed to have a field controller requiring signals to (i) switch the field on/off, (ii) re-orient the field and (iii) invert the field. An interface between the computer parallel port and up to four devices is suggested and this was built from standard TTL logic and optical couplers. Software has been written to drive all four devices in parallel. Each pulsed field device may have a totally independent pulse regime, may be stopped or started at random and may be monitored at any time during an experiment. Setting up and running each device is almost entirely by mouse control and frequently used pulse regimes may be saved and rapidly retrieved from hard or floppy disk. The software can be configured for any combination of CHEF and FIGE devices.

Quality improvement versus quality assurance?

These suggestions for applications of QI philosophies and considerations for structural integration of QA and QI are not intended to convey that organizationwide adoption of QI merely involves use of QI tools and techniques, or that instilling QI philosophy in an organization is easily accomplished. Achieving continuous quality improvement on an organizationwide basis requires long-term, senior-level commitment, extensive training, adoption of the philosophies at all management levels, and behavioral and cultural change within the organization. The adoption of QI methods and philosophies in health care organizations does not preclude the use of or eliminate the need for QA approaches. Quality improvement and quality assurance are complementary endeavors for attaining continual improvement in health care quality. Improvement of the quality of care provided is and always has been the fundamental goal of health care quality assurance. Attainment of that goal can be advanced through building on the strengths of traditional quality assurance efforts and adopting philosophies and methods of quality improvement as the core forces of total quality management programs.

In vivo loss of telomeric repeats with age in humans.

Telomeric DNA in the skin cells of 21 human subjects aged between 0 and 92 years was quantified by determining the length of the telomeric smear and the relative amount of TTAGGG repeat sequences. Both telomere length and quantity of telomeric repeat sequences were found to decrease significantly with age. Telomere loss has previously been postulated to be a caused of cell senescence.

Semiconductor-controlled contour-clamped homogeneous electric field apparatus.

The design and construction of a transistor-driven hexagonal contour-clamped homogeneous electric field (CHEF) apparatus is discussed in detail. The addition of computer control of pulsed-field timings and experiment duration gives rise to an efficient electrophoresis tool designed to achieve separation of DNA molecules in different size groupings. In particular, pulse time regimes which lead to the monotonic separation of DNA molecules ranging from 90 kbp to over a megabase pair are demonstrated. Theoretical treatment of electric field clamping with transistor-driven multiple electrodes is supported by measurements and by the actual performance of electrophoretic separation of yeast chromosomes. The large sample capacity of gels run in this apparatus coupled with the modest power requirements necessary to provide a homogeneous electric field offer significant advantages over earlier CHEF designs.

Analysing and sorting human chromosomes.

Analysing and sorting human chromosomes by flow cytometry is a powerful tool in the hands of the molecular biologist. Because of the large number of chromosomes analysed by flow, typically 10(5) for each sample, estimates of the distribution of DNA throughout the chromosome complement of an individual can be made to within three megabase pairs. Rearrangements of DNA in this size range can often be clearly seen and measured by flow cytometry in situations where it was not obvious by traditional cytogenetics. The production of enriched samples of a particular chromosome by flow sorting and the subsequent construction of DNA libraries has played an important part in mapping genes to particular hereditary diseases. Techniques now exist which allow the hybridization of a few thousand sorted chromosomes with characterized or uncharacterized DNA probes to give relatively quick answers about specific chromosome genes. The process of obtaining a sorted chromosome sample from a growing population of peripheral blood lymphocytes or lymphoblastoid cells is compared with other methods of achieving similar results in molecular biological terms. Advances in flow cytometric techniques, which include slit-scanning and hybridization of DNA probes in suspension, are likely to improve the enrichment quality of specific sorted human chromosomes.

Telomere reduction in human colorectal carcinoma and with ageing.

We have hypothesized that end-to-end chromosome fusions observed in some tumours could play a part in genetic instability associated with tumorigenesis and that fusion may result from the loss of the long stretches of G-rich repeats found at the ends of all linear chromosomes. We therefore asked whether there is telomere loss or reduction in common tumours. Here we show that in most of the colorectal carcinomas that we analysed, there is a reduction in the length of telomere repeat arrays relative to the normal colonic mucosa from the same patient. We speculate on the consequences of this loss for tumorigenesis. We also show that the telomere arrays are much smaller in colonic mucosa and blood than in fetal tissue and sperm, and that there is a reduction in average telomere length with age in blood and colon mucosa. We propose that the telomerase is inactive in somatic tissues, and that telomere length is an indicator of the number of cell divisions that it has taken to form a particular tissue and possibly to generate tumours.

Human chromosome analysis and sorting.

Flow cytometry has provided the cytogeneticist with a fast and accurate method of measuring the quantity of DNA in each human chromosome (1). Almost all the chromosomes in the human complement can now be resolved and abnormal chromosomes and aneuploidies (13,21, and X) recognized. A flow karyotype shows a pattern of peaks and troughs that is unique for each individual or cell line because of the variation in heterochromatic regions of the chromosomes (2). When combined with family studies, flow cytometry has been able to resolve homologues differing in DNA content by as little as 1/2000 of the human genome (3,4), less than a metaphase band. In addition, the sorting capabilities of most flow machines have provided a method for the purification of small but useful quantities of individual chromosomes, for example, 2×10(6) average sized human chromosomes are equivalent to 500 ng of DNA. Using recombinant DNA techniques, this material can be used to generate a large number of DNA probes to produce a chromosome-specific library, which can be used for the molecular analysis of genetic disease (5,6). More recently, molecular biologists have experimented with gene mapping by sorting small quantities of individual chromosomes onto filters for spot-blot hybridization with DNA probes (7).

Flow cytometry measurements of human chromosome kinetochore labeling.

A method for the preparation and measurement of immunofluorescent human chromosome centromeres in suspension is described using CREST antibodies, which bind to the centromeric region of chromosomes. Fluorescein isothiocyanate (FITC)-conjugated antihuman antibodies provide the fluorescent label. Labeled chromosomes are examined on microscope slides and by flow cytometry. In both cases a dye which binds to DNA is added to provide identification of the chromosome groups. Sera from different CREST patients vary in their ability to bind to chromosome arms in addition to the centromeric region. Flow cytometry and microfluorimetry measurements have shown that with a given CREST serum the differences in kinetochore fluorescence between chromosomes are only minor. Flow cytometry experiments to relate the number of dicentric chromosomes, induced by in vitro radiation of peripheral blood cells to the slightly increased number of chromosomes with above-average kinetochore fluorescence did not produce decisive radiation dosimetry results.

Bladder cancer flow cytometry profiles in relation to histological grade and stage.

Flow cytometry was used as an additional measurement to monitor 59 untreated patients with bladder cancer. The findings indicate that tumours belonging to the same stage and grade can demonstrate different flow cytometric profiles. Using this technique, together with routine histological assessment, we were able to subclassify bladder tumours in respect of their progress. Flow cytometric profiles are recommended for additional information about the aggressiveness of the tumour and the potential response to treatment.

Automatic reading of DNA sequencing gel autoradiographs using a large format digital scanner.

We have developed a large format digital scanner for several applications in nucleic acid analysis. Here we describe the scanning of autoradiographs of DNA sequencing gels and a set of programs for reading the base sequence. The programs correct distortions in the gel, recognize bands by their characteristic shape and assign bases to bands by weighting band position and intensity. The sequence read in this way is as accurate as that read by an expert.

DNA/RNA ratio in bladder cancer: a factor indicating the recurrence rate?

Flow cytometry (FCM) was used to monitor 24 patients receiving intravesical mitomycin C (MMC) therapy. The patients were selected for MMC therapy because of multiple low stage bladder tumours which had not been cleared by previous endoscopic management. The response to treatment was monitored with cystoscopy, cytology, mucosal biopsies and flow cytometry. Follow-up was for a minimum of 12 months and 19 patients (79%) remained free of tumour. Four patients developed recurrences 6 months after therapy (16.5%) and one showed no response. There were no partial responders. The flow patterns of the four patients who developed recurrence showed an increased RNA/DNA ratio; this was detected after the third MMC instillation and remained throughout the follow-up period. The patients who did not develop recurrence demonstrated either a 1:1 or decreased RNA/DNA ratio. The increased RNA/DNA ratio appears to precede cystoscopic, urine cytology or biopsy evidence of recurrence. If this is confirmed in a larger series it could be used to select patients at high risk for recurrence who would be suitable for alternative therapy.

Karyotyping and identification of human chromosome polymorphisms by single fluorochrome flow cytometry.

Frequency distributions of fluorescence intensity of ethidium bromide stained human chromosomes from nine phenotypically normal males are cross correlated and autocorrelated following repeated flow cytometric measurements. It is shown that each individual donor produces a fluorescence profile which is both visually and numerically different from those of other individuals in the set. The wide variety of chromosome heteromorphisms which occur to varying degrees for chromosomes 1, 9, 13, 14, 15, 16, 21, 22 and Y give rise to the uniqueness of a given fluorescence profile. Estimates of chromosome heteromorphisms for each individual in the set were made and then compared with parallel results obtained from inspection of Q-banded and C-banded conventional metaphase preparations. Fluorescence profiles identifiable with each individual were also obtained for Hoechst 33258 stained chromosomes.