Chromosome activities in cleaving mouse embryos to construct prenucleolar bodies orienting an intranuclear chromosome-chromosome configuration.

Most chromosomes of cleaving mouse embryos formed spherical structures called as prenucleolar bodies. Embryos stained with fluorescing dyes selective for AT-rich regions of DNA indicated that chromosome centromeres were organized by prenucleolar bodies and that chromosomes at pre-prophase of the cleavage division started to condense with their centromere regions aligned towards the peripheries of spheres. Embryo chromosomes as well as prenucleolar bodies were shown to bind a monoclonal antibody to spliceosomal snRNAs, which are contrasted to the nucleoli or chromosomes of somatic cells. More than 10 prenucleolar bodies were found in the pronuclei at the earliest stage after fertilization and 40 of them at most, diploid number of chromosomes of the mouse, in the nuclei of the 2-cell embryos. The number was decreased later in the cell cycles and at the 4-cell stage. Hence, every chromosome of cleaving mouse embryos is highly active in constructing prenucleolar bodies and behaves during the interphase until mitosis as a unit of the body; either as a single unit or as a member of the units fused. In other words, the prenucleolar bodies function as centers orienting chromosome-chromosome configuration in the fertilized eggs.

Fractal dimensions of ductal patterns in the parotid glands of normal subjects and patients with Sjögren syndrome.

RATIONALE AND OBJECTIVES: The sialographic ductal patterns of the parotid glands in patients with Sjögren syndrome were compared with those of normal patients by measuring the fractal dimensions. METHODS: Fractal dimensions were estimated using the modified pixel dilation method. RESULTS: The mean fractal dimension was 1.64 +/- 0.06 for the normal glands and 1.39 +/- 0.10 for the glands with Sjögren syndrome (P < .005). No correlation between the age or sex and fractal dimension was observed for both groups. In Sjögren syndrome, a significant difference in the fractal dimension was observed between the subgroup having punctuate fillings with a diameter less than 1 mm and the subgroup from 1 to 2 mm (P < .001). CONCLUSION: The fractal dimension is useful as a numeric grading of the complexity of the ductal pattern and the progression of parotid disease.

Restoration of proliferating cell nuclear antigen (PCNA) complex formation in xeroderma pigmentosum group A cells following cis-diamminedichloroplatinum (II)-treatment by cell fusion with normal cells.

We examined the role of the factor deficient in xeroderma pigmentosum group A (XP-A) cells in the formation of proliferating cell nuclear antigen (PCNA) complex with DNA in the DNA repair process in human fibroblasts following cis-diamminedichloroplatinum (CDDP)-treatment. Immunofluorescence staining after methanol fixation was used to detect the PCNA complex formation. When quiescent normal cells were PCNA-stained at 3 h after 100 microM CDDP treatment for 1 h, almost all nuclei of the cells showed a punctuated staining pattern. On the other hand, nuclei of XP-A cells were not stained. These results were the same with the findings following 10J/m2 of ultraviolet light (UV)-irradiation. The quantitative analysis of the PCNA immunofluorescence intensity of normal cells revealed that the mean intensity was increased by 4.8 times by the CDDP-treatment and 6.1 times by the UV-irradiation, compared with that of untreated cells. The intensities among nuclei ranged widely in both treatments. In contrast, the mean intensity was not increased in XP-A cells by the same treatments. However, when XP-A cells were fused with normal cells with polyethylene glycol (PEG) treatment, the nuclei of the XP-A cells showed positive PCNA-staining following CDDP-treatment or UV-irradiation in almost all cases. These results suggest that the PCNA complex formation may play a role in the DNA repair process after the step where the factor deficient in XP-A cells is involved following CDDP-treatment as well as following UV-irradiation.

Two types of proliferating cell nuclear antigen (PCNA) complex formation in quiescent normal and xeroderma pigmentosum group A fibroblasts following ultraviolet light (uv) irradiation.

We examined the relationship between the formation of proliferating cell nuclear antigen (PCNA) complex with DNA and nucleotide excision repair in human fibroblasts following ultraviolet light (uv) irradiation. PCNA complex formation was detected by the immunofluorescence method after methanol fixation and nucleotide excision repair activity was detected as the unscheduled DNA synthesis (UDS) by autoradiography labeled with [3H]thymidine. Quiescent normal cells showed a strong punctuated pattern of PCNA staining 5 min to 3 h and UDS 3 h after 10 J/m2 of uv irradiation, but they no longer showed PCNA staining and UDS 24 h after irradiation. In contrast, xeroderma pigmentosum group A (XP-A) cells, which lack UDS activity, did not show PCNA staining up to 30 min after irradiation; however, unexpectedly, they were stained 3 h and even 24 h after irradiation with their staining pattern being different from that in normal cells. Namely, the fluorescence spots in XP-A cells were larger in size and much smaller in number than those in normal cells. When XP-A cells were fused with normal cells with polyethylene glycol treatment, nuclei of XP-A cells showed a PCNA staining pattern similar to that of normal cells at 30 min, which was no longer detected 24 h after irradiation. These results suggest that there exist two types of PCNA complex formation, nucleotide excision repair-related and -unrelated, in human fibroblasts following uv irradiation.

Induction of proliferating cell nuclear antigen (PCNA) complex formation in quiescent fibroblasts from a xeroderma pigmentosum patient.

Accumulated evidence indicates that proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and forms tight association with DNA replication sites during DNA replication or DNA repair synthesis. In this study, such PCNA complex formation was investigated by the indirect immunofluorescence method, using both normal human fibroblasts and those derived from a xeroderma pigmentosum group A (XP-A) patient. XP-A fibroblasts in both proliferating and quiescent states did not show any differences from normal fibroblasts in the properties of PCNA-staining in the untreated conditions. The PCNA complex formation was induced in quiescent normal fibroblasts by both ultraviolet light (UV)- and X-irradiation, whereas in XP-A fibroblasts it was induced by X-irradiation, but not by UV-irradiation. However, PCNA complex was induced in quiescent XP-A fibroblasts by UV-irradiation when the cells had previously incorporated 5-bromodeoxyuridine (BrdU). These observations indicate a close correlation of PCNA complex formation and unscheduled DNA synthesis (UDS). Thus, it was concluded that PCNA complex formation was commonly induced in at least three conditions to produce UDS in spite of different types of DNA damages and DNA repair mechanisms.

A method for determination of fractal dimensions of sialographic images.

A method for measuring the fractal dimension of radiographic images was devised by the modified pixel dilation (PD) method, and its accuracy was examined by two types of image analyzers: one controlled by a minicomputer and based on the hexagonal array of the pixels, and the other controlled by a personal computer and based on the square array of the pixels. It was shown that the fractal dimensions measured by using the image analyzers for three curves (straight line, Koch curve, and Sierpinski gasket) were deviated by less than 2.1% from the theoretical dimension. The time required for the measurements fell within 10 seconds by the minicomputer-controlled image analyzer, and within 12 minutes by the personal computer-controlled image analyzer, which contrasts to the 30 minutes or more required for measuring fractal dimensions by other available methods. Analysis of the ductal patterns in sialograms by this method indicated that the fractal dimensions were significantly different between normal parotids and parotids with Sjögren syndrome. The modified PD method can be used for measuring the fractal dimension of ductal patterns in sialograms. The measurements may serve as numerical gradings of the progression of disease.

Dental radiographic signs characteristic to Hallermann-Streiff syndrome.

A review of the literature and examination of a personally observed case led to the conclusion that anterior displacement of the temporomandibular joints provides a useful diagnostic criterion for the Hallermann-Streiff syndrome.

Factors involved in the high radiographic sensitivity of E-speed films.

A microscopic and densitometric analysis of the high speed and fog density of E-speed film was conducted. The grain sizes of silver bromide (AgBr) and developed silver metals were measured, and the optical densities of the film were compared by means of various development procedures. Silver bromide grains were found to be large and accounted for the high speed of the film. Development caused chains of silver metal clusters to form, which contributed to the larger fog density as well as the higher speed.

Sialographic images of pathological changes in the mouse parotid gland.

In order to correlate pathological changes of mouse parotid glands with their sialographic images, we conducted studies on mice with known pathological changes: mice with Stensen's duct ligated, NZB mice with a systemic auto-immune disease, and aged mice. The sialographic images were found to be specific for the pathological changes: The glands after ligation of Stensen's duct were characterized by dilated, large excretory ducts with a reduced system of peripheral ducts; the glands of NZB mice showed lobular leakage of the contrast medium from small excretory ducts; and the glands of aged mice showed a great reduction in the ductal system. It is concluded that sialography yields useful information on the pathological changes of the ductal systems in the mouse parotid gland.

Postnatal development of the duct system in the mouse parotid gland.

The morphology of the parotid gland in adult mice is mouse strain-specific. C57BL/6 and C3H/He strains of mouse are representatives of two types of the morphology identified previously. The postnatal development of such morphologic differences was investigated by sialography of excised glands of these strains of mouse. It was observed that the mouse strain-dependent morphological characteristics were already present at birth, except for the branching pattern of the peripheral duct system, which became differentiated at 3 weeks of age. These results indicate that the C3H/He mouse-specific branching pattern of the peripheral ducts reflects the profile of matured secretory units and ducts, and that the C57BL/6 mouse-specific pattern resembles that of an immature C3H/He mouse.

Modulation in protein phosphorylation during G2 phase of the cell cycle in two-cell mouse embryos.

The protein phosphorylation activities in extracts were assayed for 2-cell mouse embryos at three stages of the G2 phase of the cell cycle. The 2-cell embryos were unique in having a prolonged G2 phase and so easily staged at early G2 (EG2), middle G2 (MG2) and late G2 (LG2) by timing the embryo isolation from pregnant mice. The embryo extracts were used both as sources of protein kinases and their substrates. The phosphoproteins of the extracts were labelled with [gamma-32P]ATP and separated by electrophoresis on SDS-polyacrylamide gels. The present study revealed that protein phosphorylation increased 3-6-fold during the progression of 2-cell embryos from EG2 to LG2 and the level of protein phosphorylation at any stages was greatly decreased by the presence of cAMP. Thus, the protein phosphorylation system of 2-cell mouse embryos seems to differ from those reported systems in mammals in its negative dependence on cAMP.

Radiosensitivity variation during the cell cycle in pronuclear mouse embryos in vitro.

The radiosensitivity of pronuclear mouse (B6D2 F2 X ICR) embyros has been measured in vitro as a function of time during the cell cycle. This was done by measuring the dose of X-rays (LD50) required to prevent development of 50% of the pronuclear embryos to the blastocyst stage in 5 days of culture. The LD50 was found to vary from 1 to 2 Gy during the period from G1 to the first cleavage. The cell cycle in the pronuclear embryo was analysed by [3H]thymidine autoradiography. Compared with earlier studies on two-cell mouse embryo radiosensitivity, the pronuclear embryos appear to be more sensitive to radiation than the two-cell embryos. If, however, one considers the radiation sensitivity on a blastomere basis, the pronuclear embryos are not different in their radiation sensitivity from the two-cell embryos. Thus, during the early cleavage stages of mice, radiosensitivity is mainly governed by the content of cells of various cell cycle ages in the embryo.

Changes in the activity of antifungals in mixed cultures of bacteria and yeasts.

When Candida cells were grown in mixed cultures with Escherichia coli, Salmonella typhimurium, or Pseudomonas aeruginosa, their sensitivity to antifungal agents affecting sterol metabolism or functioning (polyenes and miconazole) was increased by several log units. This phenomenon was overcome by the addition to the culture medium of sugars utilized either by the bacteria or the yeast. Antifungals without direct effect on sterol metabolism (5-fluorocytosine, methylparaben, and 4-hydroxyquinazoline) were not more active in mixed than in pure cultures.