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Biomedical subjects

A T Sumner

Publications and source records attributed to A T Sumner.

At least 55 records · Page 3Linked to original sources
PubMed1988

Cytochemical applications of X-ray microanalysis.

X-ray microanalysis (XRMA) has been applied to a wide variety of cytochemical problems, but the most valuable applications have been to the validation of cytochemical methods (by the qualitative or quantitative analysis of reaction products), and to the simultaneous localization of more than one substance, which cannot easily be achieved by using alternative methods. The latter applications involve stoichiometric studies (the quantitative relationships between reaction products and substrates), and distribution studies. Ultrastructural cytochemistry with XRMA is limited by the need to use high-brightness electron sources. Apart from the limited availability of such sources, they may cause unacceptable damage to the specimen. Preparation methods for cytochemistry using XRMA are reviewed; in principle these do not differ from those used for other cytochemical applications, but it is important not to introduce extraneous elements (from fixative, buffer, or embedding medium) into the specimen, where the additional X-ray peaks may interfere with the analysis. Quantification in XRMA of cytochemical preparations poses special problems, because the addition of the reaction product to the specimen alters the yield of continuum X rays, used for assessing the mass of the specimen, and also dilutes endogenous elements. However, measurement of ratios between characteristic elemental peaks is a useful method in X-ray microanalytical cytochemistry, and it is concluded that one of the most important attributes of XRMA for cytochemical purposes is the ease with which the substances of interest can be measured.

Animals↗
PubMed1988

Effect of HpaII and MspI restriction endonucleases on chronic myelogenous leukemia chromosomes. Detection of CpG dinucleotide demethylation in situ.

The restriction endonucleases HpaII and MspI both cleave the nucleotide sequence CCGG, but the action of HpaII is inhibited if the internal cytosine is methylated. HpaII and MspI were used on fixed chromosomes from bone marrow cells of individuals suffering from chronic myelogenous leukemia and healthy individuals. We found that MspI acts with the same efficiency on all chromosome samples, whereas HpaII extracts more DNA from the chromosomes of leukemic individuals than from the chromosomes of nonleukemic individuals. We postulate that demethylation of cytosine in the CpG dinucleotide of leukemic cell DNA accounts for our findings.

Cytidine Monophosphate↗
PubMed1988

Ageing of fixed cytological preparations produces degradation of chromosomal DNA.

When fixed chromosome preparations were allowed to age for 1-72 h, they became progressively more susceptible to digestion by exonuclease III and by S1 nuclease. Analysis of DNA from these aged preparations on agarose gels showed that the molecular weight of the DNA decreased as ageing progressed. We conclude that DNA in fixed chromosome preparations becomes progressively degraded as the preparations age.

Cell Survival↗
PubMed1987

Immunocytochemical labelling of the kinetochore of human synaptonemal complexes, and the extent of pairing of the X and Y chromosomes.

An immunocytochemical method was used to label the kinetochores on human synaptonemal complexes. Synaptonemal complex spreads were labelled with autoimmune CREST serum, followed by a second antibody labelled with colloidal gold, and examined by electron microscopy. Clusters of gold particles were found at discrete sites which were identified as kinetochores on the autosomal synaptonemal complexes, as well as on the XY pair. This method was used to investigate the extent of pairing of the human X and Y chromosomes at pachytene. Our observations confirm earlier work, based purely on measurements, that the pairing of the sex chromosomes sometimes extends beyond the centromere of the Y chromosome into the long arm. At the same time we showed that the centromeric indices of the X and Y at pachytene are highly variable, so that measurements alone are not sufficient to estimate the degree of pairing of the sex chromosomes.

Centromere↗
PubMed1987

Immunocytochemical demonstration of kinetochores in human sperm heads.

CREST sera have been used to identify kinetochores in mature mammalian sperm heads. It is necessary to decondense the sperm heads artificially to permit access of the reagents before the kinetochores can be demonstrated immunocytochemically. The distribution of kinetochores in the sperm heads appears to be random. These results show that the kinetochore antigen recognized by the CREST sera used here is retained during spermiogenesis and is passed on to the zygote at fertilization.

Autoantibodies↗
PubMed1987

Post-fertilization polyploidy in human preimplantation embryos fertilized in-vitro.

The development of in-vitro fertilization (IVF) and embryo transfer (ET) as a treatment for certain forms of infertility has emphasized the need for more information on why so many preimplantation embryos fail to develop beyond the very early cleavage stages. Analysis of nuclei from embryos that had ceased to grow or that were morphologically abnormal was carried out using one of two techniques: measurement of the DNA content of their nuclei, or observation of the number of Y chromosomes per nucleus. It was found that there was a correlation between the length of time in culture and abnormal development of polyploidy in a number of the cells. On the other hand, a small proportion of polyploid cells observed in a morphologically normal expanded blastocyst probably represents the normal precursors of polyploid trophoblast cells as observed in other mammalian species.

Blastocyst↗
PubMed1987

Correlation of pachytene chromomeres and metaphase bands of human chromosomes, and distinctive properties of telomeric regions.

By means of double staining with DAPI and chromomycin A3, we show that the chromomeres of human pachytene chromosomes are generally DAPI positive and chromomycin negative, like the G- or Q-bands of mitotic chromosomes. Thus we have demonstrated, using an objective technique not based on morphological comparisons, that chromomeres and G-bands are equivalent. However, terminal chromomeres and the ends of mitotic chromosomes, as well as a few other sites, are chromomycin positive and DAPI negative. The ends of human chromosomes appear, therefore, to contain a distinctive class of GC-rich DNA.

Chromomycins↗
PubMed1986

Electron microscopy of the parameres formed by the centromeric heterochromatin of human chromosome 9 at pachytene.

The structure and arrangement of the parameres, which are small bodies representing part of the heterochromatin of human chromosome 9 at pachytene, were studied using transmission and scanning electron microscopy. Parameres appear to be denser than other parts of the chromosomes but have a similar fibrous substructure. The most common arrangement is clusters on the axis of the bivalent, consisting of varying numbers of parameres of variable size. The parameres are joined to each other and to the rest of the chromosome by interconnecting fibres. No evidence was obtained for the organisation of parameres into paired lateral loops, as proposed by previous workers using light microscopy. The combination of osmium impregnation of pachytene chromosomes with a backscattered electron detector in the scanning electron microscope produced very clear images of the pattern of chromomeres. This procedure may prove valuable for pachytene mapping of chromosomes because of the greatly improved resolution compared with light microscopy.

Centromere↗
PubMed1986

Mechanisms of quinacrine binding and fluorescence in nuclei and chromosomes.

The mechanisms has been investigated whereby quinacrine binds to the DNA of nuclei and chromosomes in cytological preparations fixed in methanol-acetic acid. A variety of evidence is consistent with the idea that the quinacrine binds by intercalation. This is supported by a high value for the affinity of quinacrine for DNA, together with a saturation value of 0.2 quinacrine molecules/nucleotide; binding in the presence of strong salt solutions; and inhibition of fluorescence and banding by denaturation or depurination of DNA. At high quinacrine concentrations, weak binding of quinacrine to nuclei and chromosomes also occurs, but this is not relevant to the production of strong fluorescence or Q-banding patterns. A number of factors were tested which might have affected quinacrine fluorescence and banding. These included: pH; blocking protein amino groups by acetylation or benzoylation; introduction of hydrophobic groups by benzoylation; and dephosphorylation. All these treatments were without effect. However, comparison of the quinacrine fluorescence of human and onion nuclei, which differ substantially in the base composition of their DNA, shows that quinacrine fluorescence can be enhanced in cytological preparations by AT-rich DNA.

Allium↗
PubMed1985

The distribution of quinacrine on chromosomes as determined by X-ray microanalysis. II. Comparison of heterochromatic and euchromatic regions of mouse chromosomes.

The distribution of quinacrine and protein sulphur has been compared with that of DNA in euchromatic and heterochromatic regions of mouse chromosomes stained with the fluorescent dye quinacrine, using X-ray microanalysis. Heterochromatin tends to bind relatively more quinacrine than euchromatin, and contains a greater concentration of sulphur. Measurements of quinacrine fluorescence, when compared with quinacrine binding, show that the excitation of fluorescence is more efficient when the dye is bound to euchromatin than when it is bound to heterochromatin. Although this observation is consistent with the hypothesis that the dull quinacrine fluorescence of mouse centromeres is due to quenching by guanine residues, two other factors should also be considered: the lower absolute amount of dye bound to the centromeres, and a concentration-dependent quenching of fluorescence.

Animals↗
PubMed1985

Some factors affecting the action of restriction endonucleases on human metaphase chromosomes.

We have investigated whether restriction endonucleases produce bands on human chromosomes by extracting DNA, using staining methods which are stoichiometric for DNA. Restriction enzymes that produce C-band patterns appear to remove DNA extensively from chromosome arms. In general, however, those restriction enzymes that produce G-bands do not extract DNA from chromosomes, and their effects are believed to be due to conformational change in the chromosomal DNA; in these cases, the chromosomal regions affected appear to be determined by the chromosome structure and not by the specificity of the enzyme. DNA loss from chromosomes due to digestion by restriction enzymes may in some cases be uniform, although a G-banding pattern is visible after Giemsa staining.

Azure Stains↗
PubMed1984

Further evidence for the DNA base specificity of light-induced banding.

Experiments have been carried out to investigate the DNA base specificity of light-induced banding (LIB) produced by photo-oxidation of chromosomes followed by Acridine Orange staining to detect denatured DNA. Nuclei of different base composition, human and onion, and fluorochromes of different base specificities and modes of binding to DNA were used. Our results indicate that specific destruction of guanine residues is the main effect of photo-oxidation under the conditions used, and that LIB is a base-specific phenomenon. In addition, photo-oxidation may also cause DNA-protein cross-linking which affects the binding of some dyes, while prolonged photo-oxidation appears to cause more general damage to DNA.

Base Composition↗
PubMed1984

Distribution of protein sulphydryls and disulphides in fixed mammalian chromosomes, and their relationship to banding.

Fixed mammalian mitotic chromosomes, when stained with a variety of sensitive, sulphydryl-specific fluorochromes directly or following reduction of disulphide groups, show uniform fluorescence. Thus sulphydryl and disulphide groups are uniformly distributed along the length of the chromosomes, and do not show patterns related to chromosome bands. Performance of G- or C-banding procedures oxidizes sulphydryls to disulphides, but does not produce an inhomogeneous distribution of these groups. Cross-linking sulphydryl groups has no effect on G-, C- or Q-banding of chromosomes. Thus there appears to be no connection between chromosome bands and the distribution or state of oxidation of sulphur in chromosomal proteins.

Animals↗
PubMed1984

X-ray microanalysis of protein sulphydryl groups in chromatin.

A variety of evidence suggests that the protein sulphur in condensed chromatin tends to form disulphide bonds, whereas that in dispersed chromatin tends to exist as sulphydryl groups. X-ray microanalysis of mouse chromosomes showed a greater concentration of sulphur in heterochromatin than in euchromatin. Without the use of specific cytochemical reagents, it cannot be determined whether this sulphur in chromosomes is in the form of sulphydryls or disulphides. Two maleimides containing distinctive elements have been studied as X-ray cytochemical reagents for sulphydryl groups. Although adequate X-ray signals can be obtained from both, they give some non-specific staining, and do not appear to bind to all the available sulphydryl groups.

Animals↗
PubMed1983

Standards for measuring spatial resolution in biological X-ray microanalysis.

Lateral resolution in biological X-ray microanalysis is usually severely limited by the necessity to obtain an adequate number of X-ray counts, which requires the use of a relatively large electron beam spot size. Resolution is further degraded by spreading of the beam within the specimen. Although it is possible to make theoretical calculations of resolution, no straightforward and general method of measuring lateral resolution in biological X-ray microanalysis appears to have been proposed hitherto. In this paper, standards are proposed consisting of distinctive protein-bound elements embedded in araldite, and their use is described.

Egg Proteins↗
PubMed1982

Some observations on the mechanisms of blocking of nuclear staining by cisplatin.

The effect of cisplatin (cis-dichloro-diamminoplatinum II) treatment on staining of nuclei with various basic dyes and with the Feulgen reaction has been studied. Although cisplatin is reported to show negligible reaction with DNA phosphates, it hs a substantial blocking effect on staining with most dyes. Short treatment with cisplatin results in binding mainly to guanine bases of DNA, causing partial blocking of the Feulgen reaction and almost complete blocking of ethidium intercalation; binding of neutral red and crystal violet is enhanced, apparently as a result of cisplatin-induced denaturation of DNA. Very prolonged cisplatin treatment does not completely block the Feulgen reaction, indicating that reaction of cisplatin with purine bases is not complete. Since attachment of cisplatin to DNA bases is unlikely to prevent binding of most basic dyes, it is suggested that the blocking of their staining may result from steric hindrance caused by formation of DNA-protein cross-links by cisplatin. Whatever the mechanism, it is incapable of producing complete blocking of staining with certain dyes. As a practical tool, it appears that rapid and almost complete blocking of staining by cisplatin may be used as an indicator of intercalative binding of dyes to DNA.

Cell Nucleus↗