The effect of the dibenzylbutyrolactolic lignan (-)-cubebin on doxorubicin mutagenicity and recombinogenicity in wing somatic cells of Drosophila melanogaster

The dibenzylbutyrolactolic lignan (-)-cubebin was isolated from dry seeds of Piper cubeba L (Piperaceae). (-)-Cubebin possesses anti-inflammatory, analgesic and antimicrobial activities. Doxorubicin (DXR) is a topoisomerase-interactive agent that may induce single- and double-strand breaks, intercalate into the DNA and generate oxygen free radicals. Here, we examine the mutagenicity and recombinogenicity of different concentrations of (-)-cubebin alone or in combination with DXR using standard (ST) and high bioactivation (HB) crosses of the wing Somatic Mutation And Recombination Test in Drosophila melanogaster. The results from both crosses were rather similar. (-)-Cubebin alone did not induce mutation or recombination. At lower concentrations, (-)-cubebin statistically reduced the frequencies of DXR-induced mutant spots. At higher concentrations, however, (-)-cubebin was found to potentiate the effects of DXR, leading to either an increase in the production of mutant spots or a reduction, due to toxicity. These results suggest that depending on the concentration, (-)-cubebin may interact with the enzymatic system that catalyzes the metabolic detoxification of DXR, inhibiting the activity of mitochondria! complex 1 and thereby scavenging free radicals. Recombination was found to be the major effect of the treatments with DXR alone. The combined treatments reduced DXR mutagenicity but did not affect DXR recombinogenicity. (C) 2011 Elsevier Ltd. All rights reserved.

Evolution of a mate recognition system after hybridization between two Drosophila species

I investigated the genetic relationship between male and female components of the mate recognition system and how this relationship influenced the subsequent evolution of the two traits, in a series of replicate populations of interspecific hybrids. Thirty populations of hybrids between Drosophila serrata and Drosophila birchii were established and maintained for 24 generations. At the fifth generation after hybridization, the mating success of hybrid individuals with the D. serrata parent was determined. The genetic correlation between male and female components of the male recognition system, as a consequence of pleiotropy or tight physical linkage, was found to be significant but low (r = 0.388). This result suggested that pleiotropy may play only a minor role in the evolution of mate recognition in this system. At the twenty-fourth generation after hybridization, the mating success of the hybrids was again determined. The evolution of male and female components was investigated by analyzing the direction of evolution of each hybrid line with respect to its initial position in relation to the genetic regression. Male and female components appeared to converge on a single equilibrium point, rather than evolving along trajectories with slope equal to the genetic regression, toward a line of equilibria.

Pathway-specific targeting of GAB(A) a receptor subtypes to somatic and dendritic synapses in the central amygdala

Neurons in the central amygdala express two distinct types of ionotropic GABA receptor. One is the classical GABA(A) receptor that is blocked by low concentrations of bicuculline and positively modulated by benzodiazepines. The other is a novel type of ionotropic GABA receptor that is less sensitive to bicuculline but blocked by the GABA(C) receptor antagonist (1,2,5,6-tetrohydropyridine-4-yl) methylphosphinic acid (TPMPA) and by benzodiazepines. In this study, we examine the distribution of these two receptor types. Recordings of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) showed a wide variation in amplitude. Most events had amplitudes of 100 pA. Large-amplitude events also had rise times faster than small-amplitude events. Large-amplitude events were fully blocked by 10 muM bicuculline but unaffected by TPMPA. Small amplitude events were partially blocked by both bicuculline and TPMPA. Focal application of hypertonic sucrose to the soma evoked large-amplitude mIPSCs, whereas focal dendritic application of sucrose evoked small-amplitude mIPSCs. Thus inhibitory synapses on the dendrites of neurons in the central amygdala express both types of GABA receptor, but somatic synapses expressed purely GABA(A) receptors. Minimal stimulation revealed that inhibitory inputs arising from the laterally located intercalated cells innervate dendritic synapses, whereas inhibitory inputs of medial origin innervated somatic inhibitory synapses. These results show that different types of ionotropic GABA receptors are targeted to spatially and functionally distinct synapses. Thus benzodiazepines will have different modulatory effects on different inhibitory pathways in the central amygdala.

In situ hybridization detection of homeobox genes reveals distinct expression patterns in oral squamous cell carcinomas

Aims: To analyse the expression of three homeobox genes (HOXA7, PITX1 and PRRX1) in oral squanous cell carcinomas (OSCC) and the relationship of such expression to certain distinct histopathological features of OSCC and in comparison to adjacent non-neoplastic epithelium (NT). Methods and results: Digoxigenin-labelled riboprobes that are specific for each homeobox gene were generated and in situ hybridization was carried out on frozen sections. In NT samples, HOXA7 and PITX1 transcripts were found more frequently in all epithelial layers, while PRRX1 was expressed in the basal layer. With OSCC samples, expression of the three genes was associated with all histological features. However, the HOXA7 and PITX1 signals were more intense in sheets and nests and PRRX1 in small nests and isolated cells. Conclusion: HOXA7, PIXT1 and PRRX1 homeobox genes have different patterns of expression in OSCC depending on its histological features.

Differential display and suppression subtractive hybridization analysis of the pulp of ripening banana

The fruit of banana undergoes several important physico-chemical changes during ripening. Analysis of gene expression would permit identification of important genes and regulatory elements involved in this process. Therefore, transcript profiling of preclimacteric and climacteric fruit was performed using differential display and Suppression subtractive hybridization. Our analyses resulted in the isolation of 12 differentially expressed cDNAs, which were confirmed by dot-blots and northern blots. Among the sequences identified were sequences homologous to plant aquaporins, adenine nucleotide translocator, immunophilin, legumin-like proteins, deoxyguanosine kinase and omega-3 fatty acid desaturase. Some of these cDNAs correspond to newly isolated genes involved in changes related to the respiratory climacteric, or stress-defense responses. Functional characterization of ripening-associated genes could provide information useful in controlling biochemical pathways that would have an impact on banana quality and shelf life. (C) 2009 Elsevier B.V. All rights reserved.

Transcriptional Response of Peripheral Lymphocytes to Early Fibrosarcoma: A Model System for Cancer Detection Based on Hybridization Signatures

Since circulating leukocytes, mainly B and T cells, continuously maintain vigilant and comprehensive immune surveillance, these cells could be used as reporters for signs of infection or other pathologies, including cancer. Activated lymphocyte clones trigger a sensitive transcriptional response, which could be identified by gene expression profiling. To assess this hypothesis, we conducted microarray analysis of the gene expression profile of lymphocytes isolated from immunocompetent BALB/c mice subcutaneously injected with different numbers of tumorigenic B61 fibrosarcoma cells. Flow cytometry demonstrated that the number of circulating T (CD3(+)CD4(+) or CD3(+)CD8(+)) or B (CD19(+)) cells did not change. However, the lymphocytes isolated from tumor cell-injected animals expressed a unique transcriptional profile that was identifiable before the development of a palpable tumor mass. This finding demonstrates that the transcriptional response appears before alterations in the main lymphocyte subsets and that the gene expression profile of peripheral lymphocytes can serve as a sensitive and accurate method for the early detection of cancer. Exp Biol Med 234:802-812, 2009

Distinct patterns of somatic alterations in a lymphoblastoid and a tumor genome derived from the same individual

Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein-protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation.

A study of adrenocortical tumors in children and adolescents by a comparative genomic hybridization technique

Adrenocortical tumors (ACT) are rare neoplasms of the adrenal glands accounting for 0.2% of all pediatric cancers. However, the incidence of ACT in South Brazilian children is 10 to 15 times greater than the worldwide incidence. Comparative genomic hybridization studies have revealed the presence of a high degree of chromosomal instability in ACT. We evaluated 16 ACT, 8 of them carcinomas and 8 adenomas. The presence of changes in DNA copy numbers was determined by comparative genomic hybridization, and the findings were validated by real-time polymerase chain reaction on the basis of IGF-II gene expression. The adenomas showed a mean of 19.7 imbalances per case, with the most frequent gain and loss being 4p15.1-p15.3 and 20p11.2-p13.2, respectively. The carcinomas presented with a mean of 35.5 imbalances per case, with the more frequent gain being 2q14.1-q24.3 and the more frequent losses being 3q21-q26.2, 20q12-qter, and 22q11.2-q13.3. The most frequent imbalances in both adenomas and carcinomas were gains of 1p21-p31.2, 2p12-p21 and loss of 20p11.2-p12. The expression of IGF-II mRNA (11p15.5) was higher in samples that presented with a gain of this region. It has been established that great genomic instability exists in pediatric ACT.

Reliability of short comparative genomic hybridization in fibroblasts and blastomeres for a comprehensive aneuploidy screening: first clinical application

BACKGROUND: Comparative genomic hybridization (CGH) is a valuable alternative to fluorescence in situ hybridization (FISH) for preimplantation genetic screening (PGS) because it allows full karyotype analysis. However, this approach requires the cryopreservation of biopsied embryos until results are available. The aim of this study is to reduce the hybridization period of CGH, in order to make this short-CGH technique suitable for PGS of Day-3 embryos, avoiding the cryopreservation step. METHODS: Thirty-two fibroblasts from six aneuploid cell lines (Coriell) and 48 blastomeres from 10 Day-4 embryos, discarded after PGS by FISH with 9 probes (9-chr-FISH), were analysed by short-CGH. A reanalysis by the standard 72 h-CGH and FISH using telomeric probes was performed when no concordant results between short-CGH and FISH diagnosis were observed. The short-CGH was subsequently applied in a clinical case of advanced maternal age. RESULTS: In 100% of the fibroblasts analysed, the characteristic aneuploidies of each cell line were detected by short-CGH. The results of the 48 blastomeres screened by short-CGH were supported by both 72 h-CGH results and FISH reanalysis. The chromosomes most frequently involved in aneuploidy were 22 and 16, but aneuploidies for the other chromosomes, excepting 1, 10 and 13, were also detected. Forty-one of the 94 aneuploid events observed (43.6%) corresponded to chromosomes which are not analysed by 9-chr-FISH. CONCLUSIONS: We have performed a preliminary validation of the short-CGH technique, including one clinical case, suggesting this approach may be applied to Day-3 aneuploidy analysis, thereby avoiding embryo cryopreservation and perhaps helping to improve implantation rate after PGS.

Apoptosis and necrosis of polymorphonuclear leukocytes in goat milk with high and low somatic cell counts

The purpose of the present trial was to compare the percentages of necrotic and apoptotic polymorphonuclear leukocytes (PMNL) in goat milk with low and high somatic cell count (SCC). Twenty eight milk samples were collected from 20 lactating goats, determined to be negative in bacteriological examination, and divided in three groups, according to their SCC: samples with SCC lower than 500 x 10(3) cells/mL; between 500 and 1500 x 10(3) cells/mL; and higher than 1500 x 10(3) cells/m L. SCC was performed in an automatic somatic cell counter. Apoptosis and necrosis were quantified using dual-color flow cytometry with fluorescein labeled annexin-V and propidium iodide (PI). Results of the present study showed a significant positive correlation between the percentage of the viable PMNL and milk SCC(r = 0.495, P=0.008), as well as a significant negative correlation between apoptotic PMNL and milk SCC(r = -0.486, P = 0.009). Results also pointed out lower PMNL viability rates due to higher apoptosis rates in milk samples with SCC lower than 5 x 10(5) cells/mL. (C) 2011 Elsevier B.V. All rights reserved.

Microbial and sensory changes throughout the ripening of Prato cheese made from milk with different levels of somatic cells

The objective of this research was to evaluate the effects of 2 levels of raw milk somatic cell count (SCC) on the composition of Prato cheese and on the microbiological and sensory changes of Prato cheese throughout ripening. Two groups of dairy cows were selected to obtain low-SCC (<200,000 cells/mL) and high-SCC (>700,000 cells/mL) milks, which were used to manufacture 2 vats of cheese. The pasteurized milk was evaluated according to the pH, total solids, fat, total protein, lactose, standard plate count, coliforms at 45 degrees C, and Salmonella spp. The cheese composition was evaluated 2 d after manufacture. Lactic acid bacteria, psychrotrophic bacteria, and yeast and mold counts were carried out after 3, 9, 16, 32, and 51 d of storage. Salmonella spp., Listeria monocytogenes, and coagulase-positive Staphylococcus counts were carried out after 3, 32, and 51 d of storage. A 2 x 5 factorial design with 4 replications was performed. Sensory evaluation of the cheeses from low- and high-SCC milks was carried out for overall acceptance by using a 9-point hedonic scale after 8, 22, 35, 50, and 63 d of storage. The somatic cell levels used did not affect the total protein and salt: moisture contents of the cheeses. The pH and moisture content were higher and the clotting time was longer for cheeses from high-SCC milk. Both cheeses presented the absence of Salmonella spp. and L. monocytogenes, and the coagulase-positive Staphylococcus count was below 1 x 10(2) cfu/g throughout the storage time. The lactic acid bacteria count decreased significantly during the storage time for the cheeses from both low- and high-SCC milks, but at a faster rate for the cheese from high-SCC milk. Cheeses from high-SCC milk presented lower psychrotrophic bacteria counts and higher yeast and mold counts than cheeses from low- SCC milk. Cheeses from low- SCC milk showed better overall acceptance by the consumers. The lower overall acceptance of the cheeses from high-SCC milk may be associated with texture and flavor defects, probably caused by the higher proteolysis of these cheeses.

Coordinated regulation of follicle development by germ and somatic cells

The continuum of folliculogenesis begins in the fetal ovary with the differentiation of the oogonia and their isolation within the primordial follicles. Primordial follicle activation is an enigmatic process, whereby some follicles enter the growing pool to become primary follicles, thereby embarking on an irreversible progression towards ovulation or atresia. This process is under the coordinated regulation of factors from the oocyte itself, as well as from the somatic cells of the ovary, in particular the theca and granulosa cells, which are structural components of the follicle. These two influences provide the principal stimuli for the growth of the follicle to the late preantral or early antral stage of development. The endocrine effects of the gonadotrophins FSH and LH are essential to the continued progression of the follicle and most atresia can be attributed to the failure to receive or process the gonadotrophin signals. The peri-ovulatory state has received intensive investigation recently, demonstrating a coordinated role for gonadotrophins, steroids, epidermal growth factor family proteins and prostaglandins. Thus, a complex programme of coordinated interaction of governing elements from both germ and somatic cell sources is required for successful follicle development.

Evaluation of somatic cell count thresholds to detect subclinical mastitis in Gyr cows

The objectives of this study were (1) to determine the sensitivity (Se) and specificity (Sp) of somatic cell count (SCC) thresholds to identify subclinical mastitis in Gyr cows caused by major and minor pathogens; (2) to study the effects of month of sampling, rear or front mammary quarters, herd, intramammary infection (IMI), and bacterial species on SCC at quarter level; and (3) to describe the prevalence of IMI in Gyr cows in commercial dairy herds. In total, 221 lactating Gyr cows from 3 commercial dairy farms were selected. Milk samples were collected from individual quarters once a month for 1 yr from all lactating cows for SCC and bacteriological analysis. Mammary quarters were considered the experimental units and the SCC results were log(10)-transformed. Four SCC thresholds (100, 200, 300 and 400 x 10(3) cells/mL) were used to determine Se and Sp to identify infected mammary quarters. The overall prevalence of IMI in quarter milk samples of Gyr cows was 49.8%, and the prevalence of minor pathogens was higher (31.9%) than that of major pathogens (17.8%). Quarter samples with microbial isolation presented higher SCC compared with negative samples. Sensitivity and Sp of selected SCC thresholds varied according to the group of pathogen (major and minor) involved in the IMI definition. Sensitivity increased and Sp decreased when mammary quarters with only major pathogens isolation were considered positive. The use of a single SCC analysis to classify quarters as uninfected or infected in Gyr cows may not be a useful test for this breed because Se and Sp of SCC at the studied thresholds were low. The occurrence of IMI and the bacterial species are the main factors responsible for SCC variation in mammary quarters of Gyr cows. Milk samples with major pathogens isolation elicited higher SCC than those with minor pathogens.