Polyester Wadding for Specimen Orientation During Embedding in Methacrylates

Polyester fibers are not dissolved by either glycol methacrylate or methyl methacrylate. Commercial polyester wadding is consequently an advantageous material to use in getting precise orientation of tissue specimens during embedding in methacrylate.     

Permanent Preservation of Whole Alizarin Red S Skeletons by Clearing and Embedding in Polyester Resins

A one-step clearing and embedding procedure for alizarin red S stained skeletons is described. Embryos are fixed in formalin, skinned and eviscerated. After staining in a 10 mg/liter solution of alizarin red S in 5% aqueous KOH, specimens are dehydrated in a graded series of acetone-polyester monomer solutions. Finally, the specimens are embedded at room temperature in the polyester resin. A special reusable metallic mold is described for embedment of large fetuses. Specimens previously cleared in glycerol can be processed with this method.     

A Comparison of Internal Standards for Plant Cytophotometry

Plant cell nuclei were compared with chicken erythrocyte nuclei for use as internal standards for microspectrophotometry. The amount of DNA per nucleus and the coefficient of variation for measurement of individual nuclei were determined for cells from dormant embryos of Pinus taeda and Pinus coulteri, from onion root tips and from chicken erythrocytes. The chicken erythrocytes had the least variability and thus were best suited for use as a standard. Onion root tips were least suitable, with a coefficient of variation 2 1/2 times that of erythrocytes. Although onion root tips have been used as an internal standard in other studies, their mitotic activity, in contrast with the nonreplication of DNA of mature erythrocytes, is reflected in a broad distribution of nuclei with values in the 2C-4C range. Coulter pine mature embryos were at the 3C level, whether dry or hydrated, while loblolly pine embryos were in the 2C state. This confirms previous reports. The coefficient of variability for the pine embryo cells was 1 1/2 times that of erythrocytes for nonhydrated seeds and twice the erythrocyte value for hydrated seeds. The larger 2C values for pine (26 pg for P. taeda and 17 pg for P. coulteri) are closer to values expected for many plant species than the 3 pg level of the chicken erythrocytes. Dormant P. taeda embryo cells (2C) are suggested as an alternative where the experimental material has large DNA values and/or chicken erythrocytes are difficult to procure. Large sample size is recommended for the plant materials if they are to be used as internal standards in Feulgen cytophotometry.     

Complete complementary DNA of rat tyrosine aminotransferase messenger RNA. Deduction of the primary structure of the enzyme

The primary structure of rat tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase; EC 2.6.1.5), a liver-specific enzyme involved in gluconeogenesis, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 2362 nucleotides long (excluding the poly(A) tail) and codes for a polypeptide of 454 amino acids with a molecular weight of 50634. Unambiguous identification was obtained by comparison of this sequence with the amino acid sequences of several peptides obtained from the purified enzyme.     

DNA analysis and sorting of rat testis cells using two-parameter flow cytometry

By use of two-parameter flow cytometry of rat testis cell suspensions stained with mithramycin for DNA (the peak amplitude of the fluorescence signal versus total fluorescence intensity integrated over time), eight cell compartments could be distinguished without pre-enrichment of the samples. Cells in these compartments were identified by sorting and subsequent microscopic examination.     

Flow cytometric analysis and sorting of human endometrial cells after immunocytochemical labeling for cytokeratin using a monoclonal antibody

Endometrial cells in suspension were stained with propidium iodide and a monoclonal antibody against a cytokeratin intermediate filament protein specific for glandular and columnar cells (RGE 53). In this way columnar epithelial cells of the normal endometrium and of adenocarcinomas can be distinguished and separated by flow cytometry from non-epithelial cells (fibroblasts and inflammatory cells) and squamous epithelial cells, all of which are negative for RGE 53. This makes it possible to analyse and also sort pure fractions of this particular tissue type for further studies. The use of propidium iodide allows simultaneous DNA flow cytometry of these columnar epithelial cells. Therefore, the use of antibodies to cytokeratin in combination with propidium iodide can be of help in analyzing and sorting pure fractions of both normal and malignant cells. This allows a more refined examination of complex cell mixtures using flow cytometry.     

Reactions of the UVRABC excision nuclease with DNA damaged by diamminedichloroplatinum(II).

Mutants of Escherichia coli, which are blocked in excision repair (uvrA6, uvrB5, or uvrC34) are exceptionally sensitive to the antitumor drug cis-Pt(II)(NH3)2Cl2 (cis-DDP) but not the trans isomer. Plasmid DNA, damaged by either the cis or trans compound and treated with the UVRABC excision nuclease was cut as shown by conversion of supercoiled DNA to relaxed forms. All three protein products of the uvrA, uvrB, and uvrC genes were required for incision. End-labeled fragments damaged with cis-DDP and reacted with the UVRABC nuclease were cut at the 8th phosphodiester bond 5' and at the 4th phosphodiester bond 3' to adjacent GG's. DNA treated with trans-DDP was not cut appreciably at adjacent GG's by the repair enzyme as subsequent analysis of reaction products after enzyme digestion gave a pattern similar to those obtained with control untreated fragments. The results indicate that the UVRABC nuclease may promote cell survival by the removal of adjacent GG's which are crosslinked by cis-Pt(II)(NH3)2Cl2.     

Characterization of mitoses with sister chromatid differentiation (SCD) and consequences for the analysis of proliferation kinetics and sister chromatid exchanges in asynchronously growing cells

Summary Labeling cells with bromodeoxyuridine (BrdU) permits the differentiation of mitoses of the first, second, and third generation after the addition of BrdU. The term second mitoses is used for those cells which have incorporated BrdU for two-S-phases and which exhibit sister chromatid differentiation (SCD). However, SCD can also be obtained if the cell was in S-phase at the time of BrdU-addition and had already replicated part of its DNA. Such cells with incomplete BrdU-substitution in the first S-phase can only be differentiated from completely substituted ones by the quality of the SCD and are usually also grouped as second mitoses in the evaluation of experiments. Due to the heterogeneity of the evaluated second mitoses, the determination of proliferation delay and the incidence of sister chromatid exchange-induction can depend on the time of chromosome preparation.     

Effect of visual cues from female on the postejaculatory operant behavior of male rat

Bar-press response reinforced by the contact with estrous female were investigated in male rats in two experimental situations, when the visual communication with female was prevented or when it was permitted. After the instrumental responses directly preceded by ejaculations, depending on the increasing duration of response latency, three categories of male behavior were observed: avoiding of the contact with female, precopulatory behavior, or copulatory behavior. The enabling the visual communication with a female caused the significant increase of occurrence of copulatory behavior and significant decrease of contact-avoiding behavior, whereas the precopulatory behavior showed unsignificant increase. It is suggested that when prevented to see the female male rats can perform the instrumental response even when the sexual arousal is to low for precopulatory behavior. Such unrewarded responses lead to the appearance of frustration. On the contrary, when the female is visible the instrumental response is delayed up to the moment when the visual cues coming from female can evoke at least precopulatory behavior.