A model system for the study of repair of DNA double-strand breaks in Saccharomyces cerevisiae

The efficiency of "LiCl transformation" in Saccharomyces cerevisiae haploid cells by an autonomously replicating pLL12 plasmid carrying yeast LEU2 and LYS2 genes is increased (by an order or more) when the plasmid is linearized by the restriction endonuclease XhoI cleavage of a unique site in LYS2 gene. Transformants were selected on the medium lacking leucine. This phenomenon has been shown to be a result of recombinational repair of double-strand breaks (DSB) of plasmid DNA stimulated by a restriction endonuclease. The kinetic data have shown the process of plasmid DNA DSB repair to consist of two phases. The completion of the first phase occurs during an hour and the second phase occurs in 14-18 hours. DNA double-strand gaps (the deleted sequences of plasmid LYS2 gene in DSB region) with maximal length of 2-2.5 kb are repaired with the same efficiency as DSB. The genetic control of the recombinational repair of plasmid DNA DSB has been studied.     

Factors influencing the sensitivity of two human bladder carcinoma cell lines to cis-diamminedichloroplatinum(II)

A two-fold difference in sensitivity to cis-diamminedichloroplatinum(II) (cisplatin), as judged by colony forming assays, has been demonstrated in two human bladder carcinoma continuous cell lines. Approximately twice as many DNA-DNA interstrand cross-links (ISL) and a 2-fold greater inhibition of DNA synthesis occurred in the more sensitive T24 cell line than in the RT112 cell line after exposure to the same concentrations of cisplatin. Equitoxic concentrations of cisplatin resulted in similar extents of ISL and inhibition of DNA synthesis in both cell lines. Although drug uptake was identical, twice as much cisplatin was bound to the DNA of T24 cells than RT112 cells. However after equitoxic concentrations of cisplatin the DNA from both cell lines was platinated to a similar extent. In addition, levels of glutathione (GSH), glutathione reductase (GR) and total glutathione-S-transferases (GST) were higher in the less sensitive RT112 cell line.     

Calmodulin isoforms in Arabidopsis encoded by multiple divergent mRNAs

Three new, unique cDNA sequences encoding isoforms of calmodulin (CaM) were isolated from an Arabidopsis cDNA library cloned in gt10. These sequences (ACaM-4, -5, and -6) represent members of the Arabidopsis CaM gene family distinct from the three DNA sequences previously reported. ACaM-4 and -6 encode full-length copies of CaM mRNAs of ca. 0.75 kb. The ACaM-5 sequence encodes a partial length copy of CaM mRNA that is lacking sequences encoding the amino-terminal 10 amino acids of mature CaM and the initiator methionine. The derived amino acid sequence of ACaM-5 is identical to the sequences encoded by two of the previously characterized ACaM cDNAs, and is identical to TCH-1 mRNA, whose accumulation was increased by touch stimulation. The polypeptides encoded by ACaM-4 and -6 differ from that encoded by ACaM-5 by six and two amino acid substititions, respectively. Most of the deduced amino acid sequence substitutions in the Arabidopsis CaM isoforms occurred in the fourth Ca²⁺-binding domain. Polymerase chain reaction amplification assays of ACaM-4, -5 and -6 mRNA sequences indicated that each accumulated in Arabidopsis leaf RNA fractions, but only ACaM-4 and -5 mRNAs were detected in silique total RNA. The six different CaM cDNA sequences each hybridize with unique Eco RI restriction fragments in genomic Southern blots of Arabidopsis DNA, indicating that these sequences were derived from distinct structural genes. Our results suggest that CaM isoforms in Arabidopsis may have evolved to optimize the interaction of this Ca²⁺-receptor protein with specific subsets of response elements.     

Ultrastructure of the primary gill lamellae of Scomber australasicus

The ultrastructure of the primary epithelium, and the efferent half of the subepithelium, of the primary gill lamellae of slimy mackerel ( Scomber australasicus ) is described. The following cells are identified and described: light nucleated epithelial cells (surface and basal), dark nucleated cells, mucous cells, acidophilic cells, type 1 cells, type 2 cells, type 3 cells and chloride cells in the epithelial region, and subepithelial cells A and B, fibroblasts, chondrocytes, cells of the wall of efferent blood vessel and some blood cells in the subepithelium.     

The application of radiography to the study of fish nutrition

The measurement of individual food consumption rates of fish held in groups using radiography has enabled the development of a new approach to fish nutrition trials. In order to compare diets, groups of individually numbered fish are fed different experimental diets over extended periods of time (similar to standard nutrition trials) and food consumption rates are measured regularly over the course of the experiment. Analysis of covariance is then used to compare regression coefficients, obtained from mean consumption-growth relationships, from each diet. The advantages of the approach are several: (1) differences in appetite between fish fed different diets are monitored; (2) fewer fish are needed to establish consumption-growth curves over a large range of consumption rates; (3) measured food consumption rates, not ration levels, are used to calculate 'true' growth efficiencies; and (4) other factors, such as absorption efficiency, trypsin activity, the concentration of free amino acids in tissues and protein turnover can be measured for individual fish and related to differences in food consumption between fish in the same group. The approach has been used successfully with a variety of species to compare the growth response of groups fed two or more diets.     

A comparative binding of platinum anti-tumour compounds to plasma proteins in the rat (in vivo) and mouse (in vitro).

Plasma protein binding of 195mPt-labelled cisplatin, carboplatin and iproplatin has been studied in vivo in rat and in vitro in mouse, using both electrophoresis and trichloroacetic acid precipitation. After intravenous injection plasma clearance rates were biphasic for all 3 compounds, (t1/2 alpha, 13-17 min) but cisplatin was retained thereafter longer than the others. By 5 min, gel electrophoresis showed protein labelling with all 3 drugs but none involved low mol.wt. proteins (< 16 kDa). At 2 h a notable proportion of the protein bound platinum was associated with the latter components. There was a general resemblance between the distribution patterns of cisplatin and carboplatin whereas iproplatin showed a persistent retention of the label with time to higher mol. wt. proteins. From in vitro incubation with mouse plasma, rates of interaction respectively were cisplatin t1/2 alpha, 35 min, beta 8 h, carboplatin t1/2, 44 h and iproplatin t1/2, 104 h. By electrophoresis the protein bound fraction pattern (1 h) was again similar for cisplatin and carboplatin with virtually no binding to low mol. wt. proteins. After 24 h these were now involved to a high degree (40%). Iproplatin showed relatively marked binding to proteins of higher mol. wt. but no transfer with time to the low mol. wt. protein zone. A possible explanation is the need for in vivo metabolism for this compound as manifest in the rat. It is suggested that the significance of interaction with low mol. wt. proteins merits further investigation in relation to the antitumour and toxicological actions of these drugs.     

Taxol increases steady-state levels of lipopolysaccharide-inducible genes and protein-tyrosine phosphorylation in murine macrophages.

Taxol, a microtubule stabilizing agent, exhibits promise in the treatment of breast and ovarian tumors. Recently, this novel drug has been shown to activate murine macrophages to express TNF-alpha and to down-regulate TNF-alpha receptors, activities shared by bacterial LPS. Our study sought to determine if taxol could regulate gene expression in murine macrophages and to examine further the ability of taxol to generate an LPS-like signal. Toward this end, the ability of taxol to induce TNF-alpha mRNA and five other genes (IL-1 beta, IP-10, D3, D7, and D8) associated with LPS-activation of macrophages was examined by Northern blot analysis. Taxol alone (1-30 microM) induced murine C3H/OuJ macrophages to secrete bioactive TNF-alpha and express increased levels of each of the six genes under investigation. The magnitude and the kinetics of induction of each gene closely resembled that seen with Escherichia coli K235 LPS. Macrophages from LPS-hyporesponsive C3H/HeJ mice, however, failed to induce detectably any of the genes in response to taxol, despite being sensitive to the microtubule stabilizing effects of taxol as determined by immunofluorescence microscopy. The gene induction activity of taxol was in marked contrast to an alternative macrophage activator, heat killed Staphylococcus aureus, which induced a distinct gene profile in C3H/OuJ macrophages and which was equally active in C3H/OuJ and C3H/HeJ macrophages. These data are consistent with an ability of taxol to generate an LPS-like signal, possibly through a common signaling intermediate. As a first step toward identifying signal responses shared by taxol and LPS, we have shown that taxol, as shown previously for LPS, rapidly induces the tyrosine phosphorylation of a 41- and 42-kDa protein.