A mechanical model for radial keratotomy: toward a predictive capability.

A detailed three-dimensional finite element model for radial keratotomy has been developed. The model includes the full load-bearing tunic of the eye and employs realistic geometry. Incisions are modeled explicitly, and material parameters are obtained from in vivo data. Calculated results obtained with the model are compared with data from two independent medical studies on radial keratotomy.

Isopycnic centrifugation of mammalian metaphase chromosomes in Nycodenz.

Separation of chromosomes on the basis of buoyant density has proved difficult due to their high density and sensitivity to high ionic strength. Nycodenz is a new non-ionic gradient medium which offers significant advantages over previously described media for separation of chromosomes. The purpose of this study was to examine the banding characteristics of isolated mammalian metaphase chromosomes in Nycodenz gradients. The results indicate that chromosomes can be efficiently concentrated and separated from nuclei using Nycodenz gradients. Furthermore, chromosomes density-labeled with bromodeoxyuridine (BrdU) can be separated from unlabeled chromosomes in Nycodenz gradients. Nycodenz does not appear to alter chromosome morphology or protein complement. The introduction of Nycodenz represents a significant new tool for use in chromosome separation and purification.

The ultrastructural organization of prematurely condensed chromosomes.

In an effort to understand the arrangement of the basic 30 nm chromatin fibre within metaphase chromosomes, changes in the organization of prematurely condensed chromosomes (PCC) were examined as a function of progression through the cell cycle. The structural features of PCC observed under the light microscope were compared with those obtained by scanning electron microscopy. PCC with varying levels of condensation were obtained by fusing mitotic HeLa cells with interphase cells synchronized at different times in the cell cycle. PCC from G1 cells are composed of rather tightly packed bundles of tortuous chromatin fibres. The density of fibre packing along the longitudinal axis of G1-phase PCC is lower and less uniform than that of metaphase chromosomes. Early G1 PCC exhibit gyres suggesting a despiralized chromonema. The condensed domains in G1 PCC appear to be organized as supercoiled loops; whereas fibre-sparse domains consist of longitudinal fibres running along the chromosome axis. As cells progressed towards S phase, a greater proportion of highly extended regions containing prominent longitudinal fibres became evident in the PCC. The pulverized appearance of S-phase PCC under the light microscope corresponded to the highly condensed, looping fibre domains separated by more extended segments containing longitudinal fibres that are visualized using the scanning electron microscope. Active sites of DNA synthesis are implicated to be localized within extended longitudinal fibres. Post-replicative chromosome maturation extends through the G2 period and appears to involve rearrangement of the extended longitudinal fibres into packed looping-fibre clusters, which then coalesce. These observations support the model for packing DNA into chromosomes proposed in 1980 by Mullinger & Johnson. Briefly, this model suggests that the chromonema of each metaphase chromatid contains regions composed of folded longitudinal chromatin fibres as well as looping fibres that emerge from the axis at distinct foci. The final level of chromatin packing in metaphase chromosomes is attained by spiralization of the chromonema.

Cell cycle-specific changes in the ultrastructural organization of prematurely condensed chromosomes.

Prematurely condensed chromosomes (PCC) of HeLa cells synchronized in different phases of the cell cycle were analyzed by high-resolution scanning electron microscopy. The purpose of this study was to examine changes in the arrangement of the basic 30-nm chromatin fiber within interphase chromosomes associated with progression through the cell cycle. These studies revealed that highly condensed metaphase chromosomes and early G1-PCC consisted of tightly packed looping fibers. Early to mid G1-PCC were more extended and exhibited gyres suggestive of a despiralized chromonema. Further attenuation of PCC during progression through G1 was associated with a gradual transition from packed looping fibers to single extended longitudinal fibers. This process occurs prior to the initiation of DNA synthesis which appears to be localized within single longitudinal fibers. Following replication of a chromosome segment, extended longitudinal fibers were rapidly reorganized into packed looping fiber clusters concomitant with the formation of a multifibered chromosome axis. This results in the characteristic "pulverized" appearance of S-PCC when viewed by light microscopy. Subsequently, adjacent looping fiber domains coalesce, resulting in the uniformly packed, looping fiber arrangement observed in G2-PCC. Spiralization of the chromonema during the G2-mitotic transition results in the formation of highly compact metaphase chromosomes.

Proteins of metaphase chromosomes and interphase chromatin.

Metaphase chromosomal and interphase chromatin proteins from cells of two species have been compared by polyacrylamide gel electrophoresis. Consistent, common changes in the quantitative distribution of the nonhistone chromosomal proteins are observed in both species. Proteins of ca. 65,000 and 68,000 MW are enriched in interphase chromatin while proteins of ca. 50,000 and 200,000 are more prominent components of metaphase chromosomes. A group of proteins of 90,000-100,000 are also increased in metaphase chromosomes compared to interphase chromatin. By two dimensional gel analysis, the most abundant proteins from chromosomes of both cell types are similar, suggesting a structural role for these nonhistone proteins (1).

Proteins from metaphase chromosomes treated with fluorochromes.

The biochemical composition of any isolated organelle is affected by the agent(s) to which it has been exposed during the isolation and purification procedures. Many agents are known to extract molecular components either selectively or nonselectively. The protein composition of isolated chromosomes exposed to fluorochromes must be examined before proceeding to the biochemical analysis of the proteins from individual chromosomes purified by flow cytometry techniques. We have examined the polyacrylamide gel electrophoresis protein profiles from from metaphase chromosome exposed to the fluorochromes and staining conditions commonly used for flow cytometry. We find that the electropherograms of the protein of metaphase chromosomes which have been exposed to various fluorochromes are indistinguishable from controls. The analysis of proteins from individual chromosomes sorted by flow cytometry can now be undertaken.

Vitamin C preferential toxicity for malignant melanoma cells.

Vitamin C has been suggested and disputed as an anti-cancer agent. For cells in culture, no preferential effect against any type of cancer has yet been demonstrated. Our aim here is to show that vitamin C is selectively toxic to at least one type of malignant cell--a melanoma--at concentrations that might be attained in humans. Copper ions react with ascorbate and generate free radicals in solution. Ascorbate when combined with copper rapidly reduces the viscosity of DNA solutions and has exhibited some carcinostatic effects on transplanted sarcoma 180 tumours in mice. We reasoned that the elevated copper concentration in melanoma could result in a more selective toxicity for ascorbate.

Chromatin freeze fracture electron microscopy: a comparative study of core particles, chromatin, metaphase chromosomes, and nuclei.

Chromatin gels, metaphase chromosomes, and intact nuclei were studied by freeze fracturing followed by electron microscopy. The results complement and extend those obtained by classical electron microscopy techniques as they are obtained without fixation or dehydration. The freeze fracturing technique permits a determination of the hydrated diameters of nucleosomes in chromatin and in nuclei to be 13 nm by comparing to simultaneously studied test objects. Nucleosomes in chromatin fibers are closely spaced but are discrete particles in all conditions studied. In the presence of divalent ions, most chromatin in solution, chromosomes, and nuclei is organized into fibers whose thickness is larger than 40 nm. The images are not at all compatible with a super bead organization of the nucleofilament. Freeze fractures of intact nuclei provides information on the distribution of chromatin in a hydrated unfixed state. The images suggest that most of the chromatin is localized in large domains in contact with the inner nuclear membrane.

Isolated metaphase chromosomes. II. Proteins of Chinese hamster chromosomes.

The proteins on metaphase chromosomes theoretically may be distributed ubiquitously throughout the karyotype, may be present uniquely on individual chromosomes or classes of chromosomes, or may exist in any combination of the above. Separation of chromosomes according to size using sucrose velocity gradients in high capacity zonal centrifuge rotors allows sufficient fractionation of the genome to indicate the distribution of proteins within the karyotype. Flow cytometric analysis and direct microscopic analysis were used to evaluate qualitatively the types of chromosomes present in the fractions obtained. This report is the first quantitative evidence that some of the chromosomal proteins are not distributed ubiquitously on all of the chromosomes of the karyotype.

Simultaneous observation of quinacrine bands and silver grains on radiolabeled metaphase chromosomes.

A procedure is described for quinacrine banding of radiolabeled metaphase chromosomes for autoradiography. The chromosomes can be labeled either in vivo or by in situ hybridization. The banding procedure involves treating the slides with RNase and formamide and staining in quinacrine. The slides are then processed for autoradiography. After development of the photoemulsion, the chromosomes can be karyotyped with UV light by their fluorescent banding patterns and the silver grains overlaying the chromosomes can be demonstrated by the addition of tungsten light. It is possible by careful manipulation of the visible light to simultaneously observe both fluorescent bands and silver grains. This technique should significantly increase the accuracy of chromosome identification after autoradiography and decrease the time and effort required for such analysis.

Scanning-electron microscopy of chromosome aberrations.

This study is the first report of scanning-electron microscopy of isolated and purified metaphase chromosomes containing drug-induced aberrations. The technique reported allows high resolution topological examination of chromosomal aberrations which may pass undetected with conventional techniques.

Binding to chromosomes of herpes simplex-related antigens in biochemically transformed cells.

Association of herpes simplex virus (HSV)-related antigens with chromosomes was demonstrated in human and mouse cells biochemically transformed by HSV that had been irradiated with ultraviolet light. This was accomplished by using peroxidase-anti-peroxidase immunological staining with rabbit antisera that had high neutralizing titers against both HSV-specific thymidine kinase activity and virus infectivity. Antisera-against HSV did not react with chromosomes of uninfected cells nor did normal sera react with any of the constitutents of biochemically transformed cells. Methanol/acetic acid treatment of biochemically transformed cells eliminated their nuclear staining for HSV-related antigens. In vitro binding of HSV-related antigens to chromosomes was demonstrated by incubating soluble antigens from high salt extracts of HSV-infected cells with methanol/acetic acid-fixed chromosomes of biochemically transformed or uninfected cells, followed by exposure to antiserum against HSV and peroxidase-anti-peroxidase staining. There was no staining when soluble extracts from uninfected cells were substituted for those from HSV-infected cells. The results show that cells biochemically transformed and lytically infected by HSV, respectively, contain antigens, which like the Epstein-Barr virus-associated nuclear antigen (EBNA), bind to chromosomes in vivo and in vitro.

Bandign isolated metaphase chromosomes by a sequential flurescent G/Q technique.

G/Q-banding is a new, rapid, fluorescent technique for banding isolated chromosomes that incorporates characteristics of both G- and Q- banding. G-bands, while easily characterized, are often inconsistent when using isolated chromosomes, and Q-bands, while reliable, fade rapidly under UV exposure, making prolonged observation and photography difficult. G/Q-banding combines these techniques to sequentially utilize quinacrine staining over Giemsa banding to produce slow-fading fluorescent G/Q-bands. The background fluorescence in G/Q preparations fades quickly under continued UV exposure, while the chromosomes remain brightly banded and can be observed and photographed for at least five minutes. G/Q-banding was extended to whole cell chromosome spreads and produced results identical to those obtained with isolated chromosomes. Whole cell karyotypes indicate that G/Q-bands generally correspond to Q-bands. Advantages of G/Q-banding as a unique and universal technique over current double-staining procedures are discussed.

Isolated metaphase chromosomes: scanning electron microscopic appearance of salt-extracted chromosomes.

Scanning electron microscopy of salt extracted, isolated metaphase chromosomes has been used to further the understanding of the role of proteins and DNA in chromosome architecture. Metaphase chromosome surfaces consist of numerous compact microconvules with an average diameter of 520 +/- 78 A which result from several orders of supercoiling. Treatment of isolated chromosomes with various salt concentrations ranging from 0.4 to 2.0 M NaCl revealed a longitudinal array of fibres within the chromatids as well as regions of coalesced nucleoprotein. The results demonstrate sensitivity of chromosome structure based on chromatin supercoiling, to progressive extraction of chromosomal proteins and DNA over the range of salt concentrations examined.