Telomere and Telomerase Biology

Telomeres are the physical ends of eukaryotic linear chromosomes. Telomeres form special structures that cap chromosome ends to prevent degradation by nucleolytic attack and to distinguish chromosome termini from DNA double-strand breaks. With few exceptions, telomeres are composed primarily of repetitive DNA associated with proteins that interact specifically with double- or single-stranded telomeric DNA or with each other, forming highly ordered and dynamic complexes involved in telomere maintenance and length regulation. In proliferative cells and unicellular organisms, telomeric DNA is replicated by the actions of telomerase, a specialized reverse transcriptase. In the absence of telomerase, some cells employ a recombination-based DNA replication pathway known as alternative lengthening of telomeres. However, mammalian somatic cells that naturally lack telomerase activity show telomere shortening with increasing age leading to cell cycle arrest and senescence. In another way, mutations or deletions of telomerase components can lead to inherited genetic disorders, and the depletion of telomeric proteins can elicit the action of distinct kinases-dependent DNA damage response, culminating in chromosomal abnormalities that are incompatible with life. In addition to the intricate network formed by the interrelationships among telomeric proteins, long noncoding RNAs that arise from subtelomeric regions, named telomeric repeat-containing RNA, are also implicated in telomerase regulation and telomere maintenance. The goal for the next years is to increase our knowledge about the mechanisms that regulate telomere homeostasis and the means by which their absence or defect can elicit telomere dysfunction, which generally results in gross genomic instability and genetic diseases.

Colostro e leite de éguas: análise mirobiológica e contagem de células somáticas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Testis morphology and dichotomic spermatogenesis in the sugarcane borer Diatraea saccharalis (F.) (Lepidoptera: Crambidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The basement membrane and the sex establishment in the juvenile hermaphroditism during gonadal differentiation of the Gymnocorymbus ternetzi (Teleostei: Characiformes: Characidae)

Although there are several studies on morphogenesis in Teleostei, until now there is no research describing the role of the basement membrane in the establishment of the germinal epithelium during gonadal differentiation in Characiformes. In attempt to study these events that result in the formation of ovarian and testicular structures, gonads of Gymnocorymbus ternetzi were prepared for light microscopy. During gonadal development in G. ternetzi, all individuals first developed ovarian tissue. The undifferentiated gonad was formed by somatic cells (SC) and primordial germ cells (PGCs). After successive mitosis, the PGCs became oogonia, which entered into meiosis originating oocytes. An interstitial tissue developed. In half of the individuals, presumptive female, prefollicle cells synthesized a basement membrane around oocyte forming a follicle. Along the ventral region of the ovary, the tissue invaginated to form the ovigerous lamellae, bordered by the germinal epithelium. Stroma developed and the follicle complexes were formed. The gonadal aromatase was detected in interstitial cells in the early steps of the gonadal differentiation in both sexes. In another half of the individuals, presumptive male, there was no synthesis of basement membrane. The interstitium was invaded by numerous granulocytes. Pre-Leydig cells proliferated. Apoptotic oocytes were observed and afterward degenerated. Spermatogonia appeared near the degenerating oocytes and associated to SCs, forming testicular tubules. Germinal epithelium developed and the basement membrane was synthesized. Concomitantly, there was decrease of the gonadal aromatase and increase in the 3β-HSD enzyme expression. Thus, the testis was organized on an ovary previously developed, constituting an indirect gonochoristic differentiation.

Capacitação de produtores rurais para a melhoria da qualidade do leite cru produzido na região de Araçatuba - SP

This paper reports the results of an extension project carried out at Unesp/Veterinary Medicine course in Araçatuba city with milk farmers of the region. The aim of the project was to supply information to farmers concerning to good quality milk production and, at the same time, to follow up the evolution of milk quality parameters, according to the current Brazilian legislation. Every 45 days, approximately, lectures were presented to milk farmers in Araçatuba city region, approaching chemical and microbiological composition of milk, prevention and detection of mastitis, hygiene proceedings in milking and conservation of milk, cleaning and sanitization operations of facilities and equipments and prevention of adulteration. During the intervals between lectures, milk samples were collected from collective milk cooling tanks and analyzed for microbiological, hygienic and physicochemical tests. The main inadequacies in milk quality were high total bacteria and somatic cells counts, low solids contents and water addition. These problems did not proceed to betterment during the project time. So, it was concluded that the time for instruction of farmers was not enough for a progress in the quality of the milk produced in the region, pointing out the need in continuing this kind of work.

Cytogenic Evidence for the Specific Distinction of an Alaskan Marmot, Marmota broweri Hall and Gilmore (Mammalia: Sciuridae)

Cytogenetic studies based upon somatic cells (bone marrow) have disclosed that the marmot hitherto designated Marmota caligata broweri Hall and Gilmore, occurring in the Brooks Range of Arctic Alaska, differs from M. c. caligata (Eschscholtz) in number of chromosomes (2n=36 as compared with 2n=42 in M. caligata) and in proportions of chromosomal types. Typical karyograms for the two species are presented. It is concluded that the Brooks Range marmot is specifically distinct from M. caligata, the applicable name being Marmota broweri Hall and Gilmore. Also determined were diploid chromosome numbers for two other Nearctic species of marmots, M. flaviventris (Audubon and Bachman), with 42, and M. olympus (Merriam), with 40. It is suggested that M. broweri survived the last (Wisconsin) glaciations in the amphi-Beringian refugium, and that its closest affinities may be with one of the Eurasian species of Marmota.

Quality and quantity of milk produced by Saanen goats submitted to stress during three sequential days

The experiment aimed to study the effect of physiological stress on cortisol levels, quality and quantity of milk through punctual administration of ACTH. Twelve Saanen goats were divided in two experimental groups: ACTH group (0,5 mu g of ACTH/Kg.L.W); Placebo group (placebo solution). Milk production, and percentages of protein, fat, lactose and SCC (somatic cells counting) of the milk were analyzed before, during and after the administration of ACTH/placebo. Simultaneously to the ACTH/placebo administration and during three sequential days, blood was collected to evaluate cortisol concentrations. At times -30 and zero, both groups presented basal concentrations of cortisol. The increase of cortisol contents was significant at times 60 (group ACTH: 59.00 +/- 5.70 and groups placebo: 5.23 +/- 1.37ng/mL) and 120 (group ACTH: 47.96 +/- 9.72 and group placebo: 4.38 +/- 1,14ng/mL) since the cortisol content was higher on the ACTH group. The values returned to the basal level at 300 minutes. Concerning milk production, no differences were found between ACTH and placebo groups. Milk, protein, fat, lactose and SCC did not distinguish one group from another. The results indicated that the physiological stress induced during three days was not harmful to milk production and milk quality of Saanen goats.

In Vitro Development and Cell Allocation After Aggregation of Syngeneic Wild Type and Fluorescence-Expressing Bovine Cloned Embryos

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the first third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may influence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells. Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the. 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student's test (P < 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were similar between one another. Cloned GFP embryos had lower in vitro development to the blastocyst stage than WT embryos, which had more TCN than parthenote or aggregated chimeric WT/GFP embryos. Aggregated GFP embryos had fewer cells than the other embryo groups. Discussion: The in vitro development of GFP cloned embryos was lower than WT embryos, with no effects on cell allocation in resulting blastocysts. Differences in blastocyst rate between groups were likely due to lower GFP-expressing cell viability, as GFP donor cells were at high population cell doublings when used for cloning. On a per embryo basis, embryo aggregation on Day 1 resulted in blastocyst development similar to non-aggregated embryos on Day 7, with no differences in cell proportion between groups. The use of GFP-expressing cells was proven a promising strategy for the study of cell allocation during embryo development, which may assist in the elucidation of mechanisms of abnormalities after in vitro embryo manipulations, leading to the development of improved protocols for the in vitro production (IVP) of bovine embryos.

Nuclear Transfer with Apoptotic Bovine Fibroblasts: Can Programmed Cell Death Be Reprogrammed?

Cell death by apoptosis is considered to be irreversible. However, reports have indicated that its reversibility is possible if the cells have not yet reached the "point of no return.'' In order to add new information about this topic, we used cells at different moments of apoptotic process as nuclear donors in somatic cell nuclear transfer (SCNT) in order to test if programmed cell death can be reversed. Adult bovine fibroblasts were treated with 10 mu M of staurosporine (STP) for 3 h and analyzed for phosphatidylserine externalization (Annexin assay) and presence of active caspase-9. Annexin-positive (Anx +) and Caspase-9-positive (Casp-9 +) cells were isolated by FACS and immediately transferred into enucleated in vitro matured bovine oocytes. After STP treatment, 89.9% of cells were Anx + (4.6% in control cells; p < 0.01) and 24.9% were Casp-9 + (2.4% in control cells; p < 0.01). Fusion and cleavage were not affected by the use apoptotic cells (p > 0.05). Also, the use of Anx + cells did not affect blastocyst production compared to control (26.4% vs. 22.9%, respectively; p > 0.05). However, blastocyst formation was affected by the use of Casp-9 + cells (12.3%; p < 0.05). These findings contribute to the idea of that apoptosis is reversible only at early stages. Additionally, we hypothesize that the "point of no return'' for apoptosis may be located around activation of Caspase-9.

Validation of models for predicting milk urea nitrogen concentrations, estimating dry matter intake by the NRC (2001)

The objective of this study was to validate three different models for predicting milk urea nitrogen using field conditions, attempting to evaluate the nutritional adequacy diets for dairy cows and prediction of nitrogen excreted to the environment. Observations (4,749) from 855 cows were used. Milk yield, body weight (BW), days in milk and parity were recorded on the milk sampling days. Milk was sampled monthly, for analysis of milk urea nitrogen (MUN), fat, protein, lactose and total solids concentration and somatic cells count. Individual dry matter intake was estimated using the NRC (2001). The three models studied were derived from a first one to predict urinary nitrogen (UN). Model 1 was MUN = UN/12.54, model 2 was MUN = UN/17.6 and model 3 was MUN = UN/(0.0259 × BW), adjusted by body weight effect. To evaluate models, they were tested for accuracy, precision and robustness. Despite being more accurate (mean bias = 0.94 mg/dL), model 2 was less precise (residual error = 4.50 mg/dL) than model 3 (mean bias = 1.41 and residual error = 4.11 mg/dL), while model 1 was the least accurate (mean bias = 6.94 mg/dL) and the least precise (residual error = 5.40 mg/dL). They were not robust, because they were influenced by almost all the variables studied. The three models for predicting milk urea nitrogen were different with respect to accuracy, precision and robustness.

DNA methylation, histone modification and the transcription factor dE2F1 in Drosophila

Drosophila melanogaster enthält eine geringe Menge an 5-methyl-Cytosin. Die von mir untersuchte männliche Keimbahn von Drosophila weist jedoch keine nachweisbaren Mengen an DNA-Methylierung auf. Eine künstliche Expression der murinen de novo Methyltransferasen, DNMT3A und DNMT3B1, in den Fliegenhoden, führte nicht zu der erwarteten Methylierungszunahme und hatte keinen Effekt auf die Fruchtbarkeit der Männchen. Auch die gewebespezifische Expression unter der Verwendung des UAS/GAL4-Systems zeigte keine phenotypischen Veränderungen. Hingegen fanden wir auf Protein-Ebene des Chromatins von D. melanogaster und D. hydei spezifische Modifikationsmuster der Histone H3 und H4 in der Keimbahn, wie auch in den somatischen Zellen des Hodenschlauches. Die Modifikationsmuster der beiden Zelltypen unterscheiden sich grundlegend und weichen zudem von dem für Eu- und Heterochromatin erwarteten ab, was auf eine größere Komplexität des „Histon-Codes“ als angenommen hindeutet. Folglich liegt die epigenetische Information in Drosophila wahrscheinlich anstatt auf DNA- auf Protein-Ebene, wodurch Genexpression über die Chromatinstruktur reguliert wird. Es wurde gezeigt, dass der Transkriptionsfaktor E2F, der eine Schlüsselfunktion im Zellzyklus hat, durch unterschiedliche Transkripte offenbar quantitativ reguliert wird. Unsere Nachforschungen ergaben, dass die drei E2F1 Genprodukte in Drosophila neben ihrer Zellspezifität auch in unterschiedlichen Expressionsniveaus auftreten, was die Annahme einer quantitativen Expression unterstützt. Die verschiedenen Funktionen der multiplen Gene in Säugern, könnten so funktionell kompensiert werden. Die durch die Expression dreier dE2F1-Transkripte vermutete Synthese verschiedener Proteine konnte nicht bewiesen werden.

DNA-Reparatur-assoziierte genetische und epigenetische Faktoren für die Zweittumorentstehung nach Tumoren im Kindes- und Jugendalter

Die Ursachen der Zweittumorentwicklung bei Personen, die eine Krebserkrankung in der Kindheit überlebten, sind weitgehend unklar. Strahlenexposition oder Chemotherapie führen in normalen somatischen Zellen zu DNA-Schäden, welche bei fehlerhafter Reparatur eine Karzinogenese auslösen können. Es ist denkbar, dass genetische Unterschiede z. B. in den Signalwegen der Zellzykluskontrolle und der DNA-Reparatur nach therapieinduzierten DNA-Schäden eine entscheidende Rolle bei der Zweittumorentwicklung spielen. Im Rahmen dieser Arbeit wurden 20 Personen, die eine Krebserkrankung in der Kindheit überlebten und einen unabhängigen Zweittumor entwickelten, mit 20 gematchten Kontrollpersonen ohne Zweittumorentwicklung verglichen. Die primären Fibroblasten der Patienten wurden auf somatische, genetische und/oder epigenetische Unterschiede in DNA-Reparaturnetzwerken untersucht. Die biologisch relevantesten Ergebnisse lieferten Proteinuntersuchungen mittels Antikörper-Microarrays. Hierbei wurde eine konstitutiv erniedrigte Menge an RAD9A und einigen anderen DNA-Reparatur-Proteinen (BRCA1, DDIT3, MSH6, p53, RAD51) in den Zweittumorpatienten im Vergleich zu den Eintumorpatienten festgestellt. Nach einer DNA-Schädigung durch 1 Gray Bestrahlung erhöhte sich die RAD9A-Proteinmenge, wobei die Zweittumorpatienten eine geringere Induktion als die Eintumorpatienten zeigten. Bei der Quantifizierung der mRNA-Expression mittels RTq-PCR wurde ein niedrigerer RAD9A-mRNA-Level sowohl in den unbehandelten und als auch in den 1 Gray bestrahlten Zellen der Zweittumorpatienten festgestellt. SNP-Array und Methylierungsanalysen konnten keine Auffälligkeiten im RAD9A-Lokus nachweisen. Diese Ergebnisse unterstützen die Hypothese, dass Modulationen von RAD9A und anderen Zellzyklusarrest- und DNA-Reparaturproteinen zum Risiko einer Zweittumorentwicklung in Kinderkrebspatienten beitragen. Bei einem diskordanten monozygoten Zwillingspaar wurde in ca. 20% der Zellen des Zweittumorzwillings eine Hypermethylierung des Tumorsuppressorgens BRCA1 festgestellt, die mit einer konstitutiv erniedrigten BRCA1-Proteinexpression einhergeht und einen möglichen Krebsrisikofaktor darstellt. Die partielle Deletion des Gens RSPO3, die wahrscheinlich als somatisches Zellmosaik beim Zweittumorzwilling vorliegt, korreliert mit einer niedrigeren RSPO3-mRNA-Expression und ist vermutlich auch mit einer erhöhten Krebsprädisposition assoziiert.

Imetelstat (GRN163L)--telomerase-based cancer therapy

Telomeres and telomerase play essential roles in the regulation of the lifespan of human cells. While normal human somatic cells do not or only transiently express telomerase and therefore shorten their telomeres with each cell division, most human cancer cells typically express high levels of telomerase and show unlimited cell proliferation. High telomerase expression allows cells to proliferate and expand long-term and therefore supports tumor growth. Owing to the high expression and its role, telomerase has become an attractive diagnostic and therapeutic cancer target. Imetelstat (GRN163L) is a potent and specific telomerase inhibitor and so far the only drug of its class in clinical trials. Here, we report on the structure and the mechanism of action of imetelstat as well as about the preclinical and clinical data and future prospects using imetelstat in cancer therapy.

Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland

Different pathogens, such as Escherichia coli and Staphylococcus aureus, can be responsible for different outcomes of mastitis; that is, acute and severe or chronic and subclinical. These differences in the disease could be related to different mammary responses to the pathogens. The objective of this study was to determine if intramammary challenge with the endotoxins lipopolysaccharide (LPS), from E. coli, and lipoteichoic acid (LTA), from Staph. aureus, induce different immune responses in vivo in milk cells and mammary tissue. To provide a reference level for comparing the challenge and to show the different stimulation of the mammary immune system on a quantitatively similar level, dosages of LPS and LTA were chosen that induced an increase of somatic cells in milk to similar maxima. One udder quarter in each of 21 lactating dairy cows was challenged with 0.2 mug of LPS or 20 mug of LTA. From these quarters and from respective control quarters, milk cells or tissue biopsies were obtained at 0, 6, and 12h relative to the challenge to measure mRNA expression of tumor necrosis factor-alpha (TNFalpha), IL-1beta, IL-8, lactoferrin, and RANTES (regulated upon activation, normal T-cell expressed and secreted). Furthermore, if no biopsies were performed, hourly milk samples were taken for measurement of somatic cell count, lactate dehydrogenase (LDH), and TNFalpha. Somatic cell count increased in all treatments to similar maxima with LPS and LTA treatments. Concentrations of TNFalpha in milk increased with LPS but not with LTA. The activity of LDH in milk increased in both treatments and was more pronounced with LPS than with LTA. The mRNA expression of TNFalpha, IL-1beta, IL-8, and RANTES showed increases in milk cells, and LPS was a stronger inducer than LTA. Lactoferrin mRNA expression decreased in milk cells with LPS and LTA treatments. The measured factors did not change in either treatment in mammary tissue. Challenge of udder quarters with dosages of LPS and LTA that induce similar increases in SCC stimulate the appearance of different immune factor patterns. This dissimilar response to LPS and LTA may partly explain the different course and intensity of mastitis after infection with E. coli and Staph. aureus, respectively.

Differential expression of IGF-I mRNA and peptide in the male and female gonad during early development of a bony fish, the tilapia Oreochromis niloticus

Insulin-like growth factor I (IGF-I) plays a key role in the complex system that regulates bony fish growth, differentiation, and reproduction. The major source of circulating IGF-I is liver, but IGF-I-producing cells also occur in other organs, including the gonads. Because no data are available on the potential production sites of IGF-I in gonad development, developmental stages of monosex breedings of male and female tilapia from 0 day postfertilization (DPF) to 90 DPF were investigated for the production sites of IGF-I at the peptide (immunohistochemistry) and mRNA (in situ hybridization) level. IGF-I mRNA first appeared in somatic cells of the male and female gonad anlage at 7 DPF followed by IGF-I peptide around 9-10 DPF. Gonad anlagen were detected from 7 DPF. Starting at 7 DPF, IGF-I peptide but no IGF-I mRNA was observed in male and female primordial germ cells (PGCs) provided that IGF-I mRNA was not under the detection level, this observation may suggest that IGF-I originates from the somatic cells and is transferred to the PGCs or is of maternal origin. While in female germ cells IGF-I mRNA and peptide appeared at 29 DPF, in male germ cells both were detected as late as at 51-53 DPF. It is assumed that the production of IGF-I in the germ cells is linked to the onset of meiosis that in tilapia ovary starts at around 28 DPF and in testes at around 52-53 DPF. In adult testis, IGF-I mRNA and peptide occurred in the majority of spermatogonia and spermatocytes as well as in Leydig cells, the latter indicating a role of IGF-I in the synthesis of male sex steroids. In adult ovary, IGF-I mRNA and IGF-I peptide were always present in small and previtellogenic oocytes but only IGF-I peptide infrequently occurred in oocytes at the later stages. IGF-I expression appeared in numerous granulosa and some theca cells of follicles at the lipid stage and persisted in follicles with mature oocytes. The results suggest a crucial role of local IGF-I in the formation, differentiation and function of tilapia gonads.