Early pregnancy diagnosis and embryonic and fetal development using real time B mode ultrasound in goats

Real time B mode ultrasound scanning was used for the diagnosis of early pregnancy and monitoring of embryonic and fetal development in goats. Estrus in 8 cyclic cross-bred does was synchronized using a single injection of PGF2α followed by mating with a buck. All the does were subjected to weekly transrectal (from day 14 of mating) and transabdominal ultrasonography (from day 28) up to the 4th month of gestation and once during the 5th month of gestation. Of the 8 animals, 6 does were confirmed pregnant. The embryonic vesicle was detected on day 21 and day 28 and the embryo proper on day 28, day 35 using the transrectal and transabdominal methods, respectively. Heart beat was observed as early as day 21 of gestation and it was recordable by day 28 using the transrectal approach. However, via transabdominal scanning the detection and recording of the heart beat was possible only on day 35 of gestation. Singles and twins were differentiated on day 35 and day 42 by the transrectal and transabdominal approaches, respectively. Placentomes as a circular ‘C’ shape structure was detectable on day 42 with an average diameter of 0.97 cm, using the rectal probe. Using the abdominal probe, placentomes were detectable on day 50 (average diameter of 1 cm of gestation). Skeletal structures such as the skull, rib cage and vertebral column were first viewed on day 56 in both approaches. A significant (P < 0.001) high correlation (r = 0.99) was recorded between the gestational age and head, thoracic and placentome parameters. It may concluded that the earliest identification of an embryonic vesicle (day 21) by transrectal scanning and confirmation of its viability by fetal heart beat (day 28) were the most reliable ultrasonographic features for early pregnancy diagnosis in goats. Transabdominal ultrasonographic measurements of placentome, head and thoracic diameter may be of use in the assessment of fetal age.     

Human chromatid cohesin component hRad21 is phosphorylated in M phase and associated with metaphase centromeres

Sister chromatids duplicated in S phase are connected with each other during G(2) and M phase until the onset of anaphase. This chromatid cohesion is essential for correct segregation of genetic material to daughter cells. Recently, understanding of the molecular mechanisms governing chromatid cohesion in yeast has been greatly advanced, whereas these processes in mammalian cells remain unclear. We report here biochemical and cytological analyses of human Rad21, a homologue of the yeast cohesin subunit, Scc1p/Mcd1p. hRad21 is a nuclear phosphorylated protein. Its abundance does not change during the cell cycle, and it becomes hyperyphosphorylated in M phase. Most hRad21 is not associated with chromatin when the nuclear envelope breakdown takes place in prophase. However, a detailed analysis of the spread chromosomes indicated that hRad21 remains associated with prometaphase-like chromosomes along their entire lengths. The mitotic chromatin-bound hRad21 becomes dissociated in a highly regulated manner because hRad21 remains specifically at the centromeres but disappears from the arm regions on metaphase-like chromosomes. Interestingly, hRad21 at the metaphase centromeres appears to be present at the inner pairing domain where the two sister chromatids are supposed to be in intimate contact. These results suggest that hRad21 has a critical role in chromatid cohesion in human mitotic cells.     

Altering fish embryos with aquaporin-3: an essential step toward successful cryopreservation

Fish populations are globally threatened by overharvesting and habitat degradation. The ability to bank fish embryos by cryopreservation could be crucial for preserving species diversity, for aquaculture (allowing circannual fish farming), and for managing fish models used in human biomedical research. However, no nonmammalian embryo has ever been successfully cryopreserved. For fish, low membrane permeability prevents cryoprotectants from entering the yolk to prevent cryodamage. Here, we present evidence of a membrane mechanism hindering cryopreservation of fish and propose a novel solution to this obstacle. Zebrafish (Danio rerio) embryos have rectifying membranes that allow water to leave but not to reenter readily. This feature may be an evolutionary trait that allows freshwater embryos to grow in hypoosmotic environments without osmoregulatory organs. However, this trait may also prevent successful fish embryo cryopreservation because both water and cryoprotectants must move into and out of cells. As a solution, we injected zebrafish embryos with mRNA for the aquaporin-3 water channel protein and demonstrated increased membrane permeability to water and to a cryoprotectant. Modeling indicates that sufficient cryoprotectant enters aquaporin-3-expressing zebrafish embryos to allow cryopreservation.     

CDC91L1 (PIG-U) is a newly discovered oncogene in human bladder cancer

Genomic amplification at 20q11-13 is a common event in human cancers. We isolated a germline translocation breakpoint at 20q11 from a bladder cancer patient. We identified CDC91L1, the gene encoding CDC91L1 (also called phosphatidylinositol glycan class U (PIG-U), a transamidase complex unit in the glycosylphosphatidylinositol (GPI) anchoring pathway), as the only gene whose expression was affected by the translocation. CDC91L1 was amplified and overexpressed in about one-third of bladder cancer cell lines and primary tumors, as well as in oncogenic uroepithelial cells transformed with human papillomavirus (HPV) E7. Forced overexpression of CDC91L1 malignantly transformed NIH3T3 cells in vitro and in vivo. Overexpression of CDC91L1 also resulted in upregulation of the urokinase receptor (uPAR), a GPI-anchored protein, and in turn increased STAT-3 phosphorylation in bladder cancer cells. Our findings suggest that CDC91L1 is an oncogene in bladder cancer, and implicate the GPI anchoring system as a potential oncogenic pathway and therapeutic target in human cancers.     

Lunar spawning in Siganus canaliculatus

A major spawning of the seagrass rabbitfish Siganus canaliculatus occurred 4 days after the new moon in both May and June 1993, and 7 days after the new moon in 1994. The gonadosomatic index ( I _G) and serum vitellogenin (VTG) levels fluctuated according to the lunar cycle; IG and VTG levels showed peaks at around the new moon and the waning moon, respectively, suggesting that spawning of this species is synchronized with the lunar cycle. Vitellogenic oocytes appeared on day 2 after the first spawning and were fully mature on day 30. When a greater percentage of the most advanced oocytes attained the tertiary yolk stage, they formed a batch and separated from the adjacent group of smaller pre-vitellogenic oocytes, indicating that S. canaliculatus is a multiple spawner with an ovary belonging to the group-synchronous type of oocyte development. Batch fecundity, assessed using batches of oocytes at and after the tertiary yolk stage, ranged from c. 0·52 to 2·56 million eggs. The relationship between batch fecundity (F) and fork length (1) can be represented as F=0·0536854L^5·07292.     

The NF90/NF45 complex participates in DNA break repair via nonhomologous end joining

Nuclear factor 90 (NF90), an RNA-binding protein implicated in the regulation of gene expression, exists as a heterodimeric complex with NF45. We previously reported that depletion of the NF90/NF45 complex results in a multinucleated phenotype. Time-lapse microscopy revealed that binucleated cells arise by incomplete abscission of progeny cells followed by fusion. Multinucleate cells arose through aberrant division of binucleated cells and displayed abnormal metaphase plates and anaphase chromatin bridges suggestive of DNA repair defects. NF90 and NF45 are known to interact with the DNA-dependent protein kinase (DNA-PK), which is involved in telomere maintenance and DNA repair by nonhomologous end joining (NHEJ). We hypothesized that NF90 modulates the activity of DNA-PK. In an in vitro NHEJ assay system, DNA end joining was reduced by NF90/NF45 immunodepletion or by RNA digestion to an extent similar to that for catalytic subunit DNA-PKcs immunodepletion. In vivo, NF90/NF45-depleted cells displayed increased γ-histone 2A.X foci, indicative of an accumulation of double-strand DNA breaks (DSBs), and increased sensitivity to ionizing radiation consistent with decreased DSB repair. Further, NF90/NF45 knockdown reduced end-joining activity in vivo. These results identify the NF90/NF45 complex as a regulator of DNA damage repair mediated by DNA-PK and suggest that structured RNA may modulate this process.     

Nuclear Transport of the Major Capsid Protein Is Essential for Adeno-Associated Virus Capsid Formation

Adeno-associated virus capsids are composed of three proteins, VP1, VP2, and VP3. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but the functional roles of each protein are still not well understood. By analyzing a series of deletion mutants of VP2, we identified a region necessary for nuclear transfer of VP2 and found that the efficiency of nuclear localization of the capsid proteins and the efficiency of virus-like particle (VLP) formation correlated well. To confirm the importance of the nuclear localization of the capsid proteins, we fused the nuclear localization signal of simian virus 40 large T antigen to VP3 protein. We show that this fusion protein could form VLP, indicating that the VP2-specific region located on the N-terminal side of the protein is not structurally required. This finding suggests that VP3 has sufficient information for VLP formation and that VP2 is necessary only for nuclear transfer of the capsid proteins.     

Cyclic tetrapeptides with –SS– bridging between amino acid side chains for potent histone deacetylases’ inhibition

Cyclic depsipeptide FK228 with an intramolecular disulfide bond is a potent inhibitor of histone deacetylases (HDAC). FK228 is stable in blood because of its prodrug function, whose –SS– bond is reduced within the cell. Here, cyclic peptides with –SS– bridges between a variety of amino acids were synthesized and assayed for HDAC inhibition. Cyclic peptide 3, cyclo(-l-amino acid-l-amino acid-l-Val-d-Pro-), with an –SS– bridge between the first and second amino acids, was found to be a potent HDAC inhibitor. Cyclic peptide 7, cyclo(-l-amino acid-d-amino acid-l-Val-d-Pro-), with an –SS– bridge between the first and second amino acids, was also a potent HDAC inhibitor.