Sensitivity to X-irradiation in relation to cell size of CHO cells synchronized in early G1

Abstract. A population of line CHO Chinese hamster cells was synchronized by mitotic selection and allowed to enter early G₁, after which the largest and smallest cells in the population were sorted, irradiated, and their viability determined. Despite sizeable differences in volume, metabolic capability and cell cycle progression rates, an equivalent level of survival was obtained for the two populations, indicating that the factors responsible for the volume, metabolic and progression heterogeneity do not contribute greatly to radiation sensitivity.     

Application of pyronin Y(G) in cytochemistry of nucleic acids

Chinese hamster ovary (CHO) cells or isolated nuclei were stained with pyronin Y(PY) and analyzed by absorption or fluorescence microscopy, as well as by flow cytometry. Specificity of the staining reaction was assayed by testing sensitivity of the stainable material to RNase or DNase. The colored complexes detected by light absorption in fixed cells stained with PY are nonfluorescent and are most likely the products of condensation of single-stranded (ss) RNA by PY; the poly(rA) and poly(rA,rG) are the most sensitive to condensation. The products of PY interaction with double-stranded (ds) nucleic acids are fluorescent and can be detected in cells by cytofluorometry. PY used alone stains both DNA and RNA, and the staining capabilities of these nucleic acids vary depending upon the PY concentration at equilibrium; at a concentration above 330 microM, the RNA stainability decreases, perhaps due to its denaturation and condensation caused by the dye. In the presence of Hoechst 33342, PY can specifically stain RNA in fixed cells or isolated cell nuclei. Because only complexes of PY with ds RNA are fluorescent, this dye can be used as a probe of RNA conformation, e.g., to monitor denaturation of RNA in situ. The RNA stainability of mitotic cells is about 25% lower than that of cells in G2 phase, which indicates that during mitosis proportionately less cellular RNA is in the ds conformation. The advantages and limitations of the two cytochemical methods for DNA/RNA detection, one based on the use of Hoechst 33342 and PY, and another employing the metachromatic properties of acridine orange, are compared.     

Solid tumor preparation for clinical application of flow cytometry

Intense interest in advanced squamous cell cancers of the head and neck (SCC of H&N) has resulted from the recent progress made in tumor responses with chemotherapy and radiotherapy. Unfortunately, the response patterns and clinical outcome of such patients are not adequately predicted on an individual patient basis using clinical parameters or conventional morphology. The study of flow cytometrically determined cellular parameters in such tumors is therefore of interest, but is hindered by inadequate tumor preparative technology. The previous article (10) in this journal describes the use of a murine SCC tumor, LC12, which was employed for comparative testing and determination of optimum techniques of preparation for this tumor. This report describes the application of these techniques to 144 specimens of human SCC of H&N. The mean total yield for these specimens is 7.4 X 10(7) cells/g of tissue. The mean viable enzymatic yield (3.3 X 10(7) cells/g) was higher than the mean viable mechanical yield (2.0 X 10(7) cells/g) except when lymph nodes were the source of the specimen (5.4 X 10(7) cells/g). The mean dye exclusion viability from enzymatically dissociated specimens were above 90%. Significant aneuploidal subpopulation losses were evident in mechanically dissociated and enucleated specimens. 65% of the enzymatically dissociated specimens were successfully cultured with a mean cloning efficiency of 2.1 X 10(-3). Preparative techniques derived from comparative testing with a murine standard tumor have been successfully applied to 144 specimens of SCC of H&N with resultant high yields and excellent viability. Technical problems detected during the preliminary testing with LC12 were confirmed in the human tumors.     

Macrophage-induced cytostasis: kinetic analysis of bromodeoxyuridine-pulsed cells

The effect of tumoricidal macrophages on the cell cycle progression of six different cell lines was studied using an anti-bromodeoxyuridine (BrdUrd) monoclonal antibody to follow the traverse of BrdUrd-labeled cells. Exponentially growing cultured mammalian cells, from six different cell lines, were prepulsed with BrdUrd before exposure to tumoricidal macrophages. The cultured cells were then analyzed as a function of time for DNA content (by propidium iodide staining) and for BrdUrd incorporation (using a fluoresceinisothiocyanate [FITC]-conjugated anti-BrdUrd monoclonal antibody). The position of the cells in cycle and the progression of the BrdUrd-labeled cohort was followed using flow cytometry. The cell lines examined were: Colon 26, BALB/c-3T3, ST3T3 (a spontaneously transformed, tumorigenic clone of 3T3), WCHE5 (a clone of whole Chinese hamster embryo cells), RIF (a radiation-induced fibrosarcoma), and A101D (a human melanoma). The bivariate distributions showed that for all six cell lines the BrdUrd-labeled cohort in the control cultures progressed around the cell cycle during the first 12 h of culture, as the cells exponentially increased. In contrast, when each cell line was incubated with tumoricidal macrophages, the BrdUrd-labeled cohort did not progress through cell cycle but remained in S phase throughout the 12-h culture period. There was also no evidence for progression of cells out of G1. The data show that cells were arrested in every phase of cell cycle. This study suggests that cytostasis, as manifested by the termination of progression in all phases of the cell cycle, is a universal phenomenon induced by tumoricidal macrophages.     

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

An extensive review on antifungal approaches in the treatment of mucormycosis

During the period of COVID-19, the occurrences of mucormycosis in immunocompromised patients have increased significantly. Mucormycosis (black fungus) is a rare and rapidly progressing fungal infection associated with high mortality and morbidity in India as well as globally. The causative agents for this infection are collectively called mucoromycetes which are the members of the order Mucorales. The diagnosis of the infection needs to be performed as soon as the occurrence of clinical symptoms which differs with types of Mucorales infection. Imaging techniques magnetic resonance imaging or computed tomography scan, culture testing, and microscopy are the approaches for the diagnosis. After the diagnosis of the infection is confirmed, rapid action is needed for the treatment in the form of antifungal therapy or surgery depending upon the severity of the infection. Delaying in treatment declines the chances of survival. In antifungal therapy, there are two approaches first-line therapy (monotherapy) and combination therapy. Amphotericin B ( 1 ) and isavuconazole ( 2 ) are the drugs of choice for first-line therapy in the treatment of mucormycosis. Salvage therapy with posaconazole ( 3 ) and deferasirox ( 4 ) is another approach for patients who are not responsible for any other therapy. Adjunctive therapy is also used in the treatment of mucormycosis along with first-line therapy, which involves hyperbaric oxygen and cytokine therapy. There are some drugs like VT-1161 ( 5 ) and APX001A ( 6 ), Colistin, SCH 42427, and PC1244 that are under clinical trials. Despite all these approaches, none can be 100% successful in giving results. Therefore, new medications with favorable or little side effects are required for the treatment of mucormycosis.     

Changes in cell nuclei during S phase: progressive chromatin condensation and altered expression of the proliferation-associated nuclear proteins Ki-67, cyclin (PCNA), p105, and p34.

Using multiparameter flow cytometry we have measured the nuclear DNA content of exponentially growing HL-60 cells in conjunction with protein content, nuclear forward light scatter, DNA in situ sensitivity to denaturation, DNA accessibility to 7-aminoactinomycin D (7-AMD), and content of the proliferation-associated proteins: cyclin (PCNA), p105, p34, and Ki-67. Multivariate analysis of the data made it possible to correlate changes in each parameter with the degree of cell advancement through S phase (amount of replicated DNA). A decrease of the protein/DNA ratio, lowered DNA accessibility to 7-AMD, increased sensitivity of DNA to denaturation, and increased ability of isolated nuclei to scatter light all paralleled cell progression through S phase. These changes indicate that during S phase chromatin progressively condenses and suggest that the condensation is associated with the efflux of nonhistone proteins from the nucleus. The increase in the content of the antigen detected by the Ki-67 antibody was observed to exceed the increase in DNA content during S phase and the rate of the Ki-67/DNA increase was higher during the second half of S phase. Thus, this protein appears to be primarily synthesized during S, especially late in S phase, and is degraded in G1. The ratio of cyclin (PCNA)/DNA remained rather constant whereas the contents of p105 and p34 proteins, when expressed per unit of DNA, both decreased during S phase. The data indicate that significant changes in structure and composition of chromatin take place during S phase and suggest that the composition of chromatin associated with the nonreplicated DNA is different compared to chromatin associated with the newly replicated DNA.     

Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210

The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.