Mutagenicity of 2-amino-N6-hydroxyadenine at the tk locus in L5178Y strains differing in repair capabilities and karyotype

The cytotoxicity and mutagenicity of 2-amino-N6-hydroxyadenine (AHA) were measured in strains of L5178Y differing in repair capabilities and karyotype. Strain LY-R83 is monosomic for chromosome 11 and is therefore hemizygous for the tk gene, while strains LY-R16 and LY-S1 are TK+/- heterozygotes. Both strain LY-R83 and LY-R16 are sensitive to UV light and are presumed to be deficient in the excision of pyrimidine dimers as shown for the parental strain, LY-R (Hagen et al., 1988; Szumiel et al., 1988). Strain LY-S1 is sensitive to the cytotoxic effects of ionizing radiation and is presumed to be defective in the repair of radiation-induced DNA double-strand breaks, as shown for the parental strain, LY-S (Evans et al., 1987a; Wlodek and Hittelman, 1987). The sensitivities of the three strains to the cytotoxic effects of AHA were similar. After a 4-hour treatment with AHA at 37 degrees C, the D37 for all three strains was approximately 35 ng/ml. The AHA-induced mutant frequency was similar for the hemizygous TK+ strain LY-R83 and the heterozygous TK +/- strain LY-R16, but was slightly higher for strain LY-S1 than for either LY-R strain at an AHA concentration of 100 ng/ml. The proportion of AHA-induced LY-S1 TK -/- mutants forming colonies with diameters less than 0.3 mm was much lower than following treatment with X radiation (24% vs. 61% for AHA and X radiation, respectively). These results indicate that the vast majority of AHA-induced TK -/- mutants harbor single gene mutations. AHA did not result in cyanide-insensitive oxygen uptake, and treatment with this compound did not induce a significant number of DNA single-strand breaks, DNA alkali labile lesions, or DNA degradation in either strain. However, two hours after AHA removal, DNA single-strand breaks and/or alkali-labile lesions, possibly due to the occurrence of DNA repair, were apparent in the DNA of both strain LY-R16 and strain LY-S1.     

The effect of dose rate on X-radiation-induced mutant frequency and the nature of DNA lesions in mouse lymphoma L5178Y cells

The induction of mutants at the heterozygous tk locus by X radiation was found to be dose-rate dependent in L5178Y-R16 (LY-R16) cells, but very little dose-rate dependence was observed in the case of strain L5178Y-S1 (LY-S1), which is deficient in the repair of DNA double-strand breaks. Induction of mutants by X radiation at the hemizygous hprt locus was dose-rate independent for both strains. These results are in agreement with the hypothesis that the majority of X-radiation-induced TK-/- mutants harbor multilocus deletions caused by the interaction of damaged DNA sites. Repair of DNA lesions during low-dose-rate X irradiation would be expected to reduce the probability of lesion interaction. The results suggest that in contrast to the TK-/- mutants, the majority of mutations at the hprt locus in these strains of L5178Y cells are caused by single lesions subject to dose-rate-independent repair. The vast majority of the TK-/- mutants of strain LY-R16 showed loss of the entire active tk allele, whether the mutants arose spontaneously or were induced by high-dose-rate or low-dose-rate X irradiation. The proportion of TK-/- mutants with multilocus deletions (in which the products of both the tk gene and the closely linked gk gene were inactivated) was higher in the repair-deficient strain LY-S1 than in strain LY-R16. However, even though the mutant frequency decreased with dose rate, the proportion of mutants showing inactivation of both the tk and gk genes increased with a decrease in dose rate. The reason for these apparently conflicting results concerning the effect of DNA repair on the induction of extended lesions is under investigation.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Neuroprotective strategies for the treatment of inherited photoreceptor degeneration

Photoreceptor degeneration is the hallmark of several groups of inherited neurodegenerative diseases causing blindness in humans. These diseases are a major cause of visual handicap and to date no satisfactory treatment is available. Here, we briefly review different approaches for the treatment of photoreceptor degeneration, to then focus on neuroprotection. Up to date, translation of experimental neuroprotection into a clinical setting has faced major obstacles, which are in part due to an incomplete understanding of the regulation of pro-survival as well as neurodegenerative mechanisms. Previous approaches were often based on the hypothesis that photoreceptor cell death was governed by a single, apoptotic cell death mechanism. This perception has turned out too simple as recent work has demonstrated that photoreceptor cell death is governed by non-apoptotic mechanisms as well. Moreover, there is evidence, that several different destructive processes are executed in parallel. Briefly reviewing the complexity of degenerative mechanisms, this review discusses relevant pathways, options to target signaling cascades, final common denominators of cell death, and the interplay of events executing cell death. In particular, we focus on cGMP-signaling, epigenetic and proteolytic processes and the corresponding enzymatic activities that were recently shown to be causally related to retinal degeneration. Finally, we illustrate how a better understanding of destructive mechanisms may enable identification and validation of novel targets for neuroprotection, and allow development of next generation neuroprotective treatments as well as combination therapy.     

Language Differences in the Brain Network for Reading in Naturalistic Story Reading and Lexical Decision

Differences in how writing systems represent language raise important questions about whether there could be a universal functional architecture for reading across languages. In order to study potential language differences in the neural networks that support reading skill, we collected fMRI data from readers of alphabetic (English) and morpho-syllabic (Chinese) writing systems during two reading tasks. In one, participants read short stories under conditions that approximate natural reading, and in the other, participants decided whether individual stimuli were real words or not. Prior work comparing these two writing systems has overwhelmingly used meta-linguistic tasks, generally supporting the conclusion that the reading system is organized differently for skilled readers of Chinese and English. We observed that language differences in the reading network were greatly dependent on task. In lexical decision, a pattern consistent with prior research was observed in which the Middle Frontal Gyrus (MFG) and right Fusiform Gyrus (rFFG) were more active for Chinese than for English, whereas the posterior temporal sulcus was more active for English than for Chinese. We found a very different pattern of language effects in a naturalistic reading paradigm, during which significant differences were only observed in visual regions not typically considered specific to the reading network, and the middle temporal gyrus, which is thought to be important for direct mapping of orthography to semantics. Indeed, in areas that are often discussed as supporting distinct cognitive or linguistic functions between the two languages, we observed interaction. Specifically, language differences were most pronounced in MFG and rFFG during the lexical decision task, whereas no language differences were observed in these areas during silent reading of text for comprehension.     

Effects of low dose rate (0.003-0.025 Gy/h) chronic X-irradiation on radioresistant and radiosensitive L5178Y mouse lymphoma cells

Cultures of radioresistant (LY-R) and radiosensitive (LY-S) strains of L5178Y mouse lymphoma cells were exposed continuously to X-rays delivered at dose rates ranging from 0.003 to 0.025 Gy/h for up to 35 days. Populations of both strains proliferated actively during the exposure, but the growth rates were reduced in a dose rate-dependent manner. The reduction of growth rate occurred for strain LY-S earlier during the exposure and at lower dose rates than for strain LY-R. The survival (as measured by colony forming ability) of strain LY-R was affected only slightly at all dose rates applied. For strain LY-S, a decrease in the surviving fraction was observed in the initial part of the exposure. This decrease was followed by a plateau and eventually by an increase, in some cases to values close to the control level. The increase in the surviving fraction indicated that the radioresistance of the exposed LY-S cells had increased. This pattern was particularly clear for dose rates greater than 0.014 Gy/h. The pre-irradiated cells exhibited radioresistance when exposed to acute X-radiation after termination of the chronic exposure. The increase in radiation resistance was stable for at least 70 days after termination of the protracted exposure. These results show that mutagenic and/or selective phenomena leading to an increase in radiation resistance of mammalian cells can be caused by protracted exposures to X-rays at dose rates permitting active proliferation.     

The Oomycete <Emphasis Type="Italic">Pythium oligandrum</Emphasis> Can Suppress and Kill the Causative Agents of Dermatophytoses

Pythium oligandrum (Oomycota) is known for its strong mycoparasitism against more than 50 fungal and oomycete species. However, the ability of this oomycete to suppress and kill the causal agents of dermatophytoses is yet to be studied. We provide a complex study of the interactions between P. oligandrum and dermatophytes representing all species dominating in the developed countries. We assessed its biocidal potential by performing growth tests, on both solid and liquid cultivation media and by conducting a pilot clinical study. In addition, we studied the molecular background of mycoparasitism using expression profiles of genes responsible for the attack on the side of P. oligandrum and the stress response on the side of Microsporum canis. We showed that dermatophytes are efficiently suppressed or killed by P. oligandrum in the artificial conditions of cultivations media between 48 and 72 h after first contact. Significant intra- and interspecies variability was noted. Of the 69 patients included in the acute regimen study, symptoms were completely eliminated in 79% of the patients suffering from foot odour, hyperhidrosis disappeared in 67% of cases, clinical signs of dermatomycoses could no longer be observed in 83% of patients, and 15% of persons were relieved of symptoms of onychomycosis. Our investigations provide clear evidence that the oomycete is able to recognize and kill dermatophytes using recognition mechanisms that resemble those described in oomycetes attacking fungi infecting plants, albeit with some notable differences.     

Early evolution of metazoan serine/threonine and tyrosine kinases: identification of selected kinases in marine sponges

The phylum Porifera (sponges) was the first to diverge from the common ancestor of the Metazoa. In this study, six cDNAs coding for protein- serine/threonine kinases (PS/TKs) are presented; they have been isolated from libraries obtained from the demosponges Geodia cydonium and Suberites domuncula and from the calcareous sponge Sycon raphanus. Sequence alignments of the catalytic domains revealed that two major families of PS/TK, the "conventional" (Ca(2+)-dependent) protein kinase C (PKC), the cPKC subfamily, as well as the "novel" (Ca(2+)- independent) PKC (nPKC), form two separate clusters. In each cluster, the sequence from S. raphanus diverges first. To approach the question about the origin of protein-tyrosine kinases (PTK), which are found only in Metazoa, we analyzed two additional PS/TKs which have been cloned from S. domuncula: the stress-responsive protein kinase (KRSvSD) and the protein-kinase-C-related kinase (PRKvSD). The construction of the phylogenetic tree, comprising the eight PS/TKs and the PTK cloned previously from G. cydonium, revealed that the PTK derived from the branch including the KRSvSD kinase. These data facilitate the first molecular approach to elucidate the origin of metazoan PTK within the PS/TK superfamily.      

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.