Lipoplex size determines lipofection efficiency with or without serum

In order to identify factors affecting cationic Iiposome-mediated gene transfer, the relationships were examined among cationic liposome/DNA complex (lipoplex)-cell interactions, lipoplex size and lipoplex-mediated transfection (lipofection) efficiency. It was found that lipofection efficiency was determined mainly by lipoplex size, but not by the extent of lipoplex-cell interactions including binding, uptake or fusion. In addition, it was found that serum affected mainly lipoplex size, but not lipoplex-cell interactions, which effect was the major reason behind the inhibitory effect of serum on lipofection efficiency. It was concluded that, in the presence or absence of serum, lipoplex size is a major factor determining Iipofection efficiency. Moreover, in the presence or absence of serum, lipoplex size was found to affect lipofection efficiency by controlling the size of the intracellular vesicles containing lipoplexes after internalization, but not by affecting lipoplex-cell interactions. In addition, large lipoplex particles showed, in general, higher lipofection efficiency than small particles. These results imply that, by controlling lipoplex size, an efficient lipid delivery system may be achieved for in vitro and in vivo gene therapy.     

Novel glycoproteins inhibiting the binding of colorectal cancer cells to E-selectin

Novel glycoproteins carrying sialyl-LeA (SLeA) antigens (SL-GP) were isolated from ascites fluid from a patient with colorectal cancer by immunoaffinity chromatography. Their characteristics, including binding capacity to E-selectin, were investigated. SL-GP showed a typical mucin type amino acid composition in which Ser, Thr and Pro together accounted for greater than 50% of the total amino acid residues. A large amount of carbohydrate (about 80%) was present in SL-GP. The number of O-glycans carrying SLeA antigens comprised about 9% of the total number of O-glycosidic chains. SL-GP could bind to IL-1 treated HUVEC, and the binding was inhibited by anti-E-selectin and anti-SLeA monoclonal antibodies. The binding of colorectal cancer cells, LS 180, to HUVEC was assayed in the presence of SL-GP, oligosaccharides prepared from SL-GP and human milk SLeA hexasaccharide. SL-GP inhibited the binding most effectively, whereas equivalent amounts of the SL-GP oligosaccharides and milk SLeA hexasaccharide inhibited it only slightly. These results constitute direct evidence that a unique arrangement of SLeA antigens on the polypeptide chain, probably a cluster, is essential for the binding to E-selectin.     

Grit,a GTPase-activating protein for the Rho family,regulates neurite extension through association with the TrkA receptor and N-Shc and CrkL/Crk adapter molecules

Neurotrophins are key regulators of the fate and shape of neuronal cells and act as guidance cues for growth cones by remodeling the actin cytoskeleton. Actin dynamics is controlled by Rho GTPases. We identified a novel Rho GTPase-activating protein (Grit) for Rho/Rac/Cdc42 small GTPases. Grit was abundant in neuronal cells and directly interacted with TrkA, a high-affinity receptor for nerve growth factor (NGF). Another pool of Grit was recruited to the activated receptor tyrosine kinase through its binding to N-Shc and CrkL/Crk, adapter molecules downstream of activated receptor tyrosine kinases. Overexpression of the TrkA-binding region of Grit inhibited NGF-induced neurite elongation. Further, we found some tendency for neurite promotion in full-length Grit-overexpressing PC12 cells upon NGF stimulation. These results suggest that Grit, a novel TrkA-interacting protein, regulates neurite outgrowth by modulating the Rho family of small GTPases.     

Eukaryotic phylotypes in aquatic moss pillars inhabiting a freshwater lake in East Antarctica, based on 18S rRNA gene analysis

Aquatic mosses of Leptobryum species form unique tower-like pillars of vegetation termed “moss pillars” in Antarctic lakes. Moss pillars have distinct redox-affected sections: oxidative exteriors and reductive interiors. We have proposed that a “pillar” is a community and habitat of functionally interdependent organisms and may represent a mini-biosphere. Batteries of 16S rRNA genotypes, or phylotypes, of eubacteria and cyanobacteria, but no archaea, have been identified in moss pillars. However, detailed identification or phylogenetic analyses of the moss and their associated eukaryotic microbiota have not been performed. This study analyzed near-full-length 18S rRNA gene sequences obtained from two whole moss pillars. In total, 28 PCR clone libraries from two whole moss pillars were constructed, and 96 clones from each library (total 2,688 clones) were randomly selected and sequenced. Molecular phylogenetic analysis revealed that the phylotype belonging to Bryophyta, considered to be derived from moss, was closely related (99.9?%) to the 18S rRNA gene sequence from Leptobryum pyriforme. Unexpectedly, phylotypes belonging to a novel clade of fungi dominated (approximately 27–75?%) the moss pillar libraries. This suggests that fungi may contribute to carbon cycling in the moss pillar as parasites or decomposers. In addition, phylotypes related to ciliates and tardigrades were subdominant in the exterior, while the phylotype of the ameba-like, single-celled eukaryote, Cercomonas (Cercozoa), was detected only in the interior. These features were shared by both moss pillars. The 18S rRNA gene-based profiles demonstrated that redox-related factors may control distribution of some eukaryotic microbes in a whole moss pillar.     

Srs2 Plays a Critical Role in Reversible G2 Arrest upon Chronic and Low Doses of UV Irradiation via Two Distinct Homologous Recombination-Dependent Mechanisms in Postreplication Repair-Deficient Cells

Differential posttranslational modification of proliferating cell nuclear antigen (PCNA) by ubiquitin or SUMO plays an important role in coordinating the processes of DNA replication and DNA damage tolerance. Previously it was shown that the loss of RAD6-dependent error-free postreplication repair (PRR) results in DNA damage checkpoint-mediated G₂ arrest in cells exposed to chronic low-dose UV radiation (CLUV), whereas wild-type and nucleotide excision repair-deficient cells are largely unaffected. In this study, we report that suppression of homologous recombination (HR) in PRR-deficient cells by Srs2 and PCNA sumoylation is required for checkpoint activation and checkpoint maintenance during CLUV irradiation. Cyclin-dependent kinase (CDK1)-dependent phosphorylation of Srs2 did not influence checkpoint-mediated G₂ arrest or maintenance in PRR-deficient cells but was critical for HR-dependent checkpoint recovery following release from CLUV exposure. These results indicate that Srs2 plays an important role in checkpoint-mediated reversible G₂ arrest in PRR-deficient cells via two separate HR-dependent mechanisms. The first (required to suppress HR during PRR) is regulated by PCNA sumoylation, whereas the second (required for HR-dependent recovery following CLUV exposure) is regulated by CDK1-dependent phosphorylation.DNA damage occurs frequently in all organisms as a consequence of both endogenous metabolic processes and exogenous DNA-damaging agents. In nature, the steady-state level of DNA damage is usually very low. However, chronic low-level DNA damage can lead to age-related genome instability as a consequence of the accumulation of DNA damage (12, 27). Increasing evidence implicates DNA damage-related replication stress in genome instability (7, 21). Replication stress occurs when an active fork encounters DNA lesions or proteins tightly bound to DNA. These obstacles pose a threat to the integrity of the replication fork and are thus a potential source of genome instability, which can contribute to tumorigenesis and aging in humans (4, 11). Confronted with this risk, cells have developed fundamental DNA damage response mechanisms in order to faithfully complete DNA replication (8).In budding yeast Saccharomyces cerevisiae, the Rad6-dependent postreplication repair (PRR) pathway is subdivided into three subpathways, which allow replication to resume by bypassing the lesion without repairing the damage (3, 22, 33). Translesion synthesis (TLS) pathways dependent on the DNA polymerases eta and zeta promote error-free or mutagenic bypass depending on the DNA lesion and are activated upon monoubiquitination of proliferating cell nuclear antigen (PCNA) at Lys164 (K164) (5, 16, 37). The Rad5 (E3) and Ubc13 (E2)/Mms2 (E2 variant)-dependent pathway promotes error-free bypass by template switching and is activated by polyubiquitination of PCNA via a Lys63-linked ubiquitin chain (16, 38, 41). It remains mechanistically unclear how polyubiquitinated PCNA promotes template switching at the molecular level. In addition to its ubiquitin E3 activity, Rad5 also has a helicase domain and was recently shown to unwind and reanneal fork structures in vitro (6). This led to the proposal that Rad5 helicase activity is required at replication forks to promote fork regression and subsequent template switching. It is possible that PCNA polyubiquitination acts to facilitate Rad5-dependent template switching by inhibiting monoubiquitination-dependent TLS activity and/or by recruiting alternative proteins to the fork.In addition to modification by ubiquitin, PCNA can also be sumoylated on Lys164 by the SUMO E3 ligase Siz1 (16). A second sumoylation site, Lys127, is also targeted by an alternative SUMO E3 ligase, Siz2, albeit with lower efficiency (16, 30). PCNA SUMO modification results in recruitment of the Srs2 helicase and subsequent inhibition of Rad51-dependent recombination events (29, 32). The modification can therefore allow the replicative bypass of lesions by promoting the RAD6 pathway. Srs2 is known to act as an antirecombinase by eliminating recombination intermediates. This can occur independently of PCNA sumoylation, and when srs2Δ cells are UV irradiated or other antirecombinases, such as Sgs1, are concomitantly deleted, toxic recombination structures accumulate (1, 10). Such genetic data are consistent with the ability of Srs2 to disassemble the Rad51 nucleoprotein filaments formed on single-stranded DNA (ssDNA) in vitro (20, 40). In addition to directly inhibiting homologous recombination (HR), Srs2 is also involved in regulating HR outcomes to not produce crossover recombinants in the mitotic cell cycle (18, 34, 35).The UV spectrum present in sunlight is a primary environmental cause of exogenous DNA damage. Sunlight is a potent and ubiquitous carcinogen responsible for much of the skin cancer in humans (17). In the natural environment, organisms are exposed to chronic low-dose UV light (CLUV), as opposed to the acute high doses commonly used in laboratory experiments. Hence, understanding the cellular response to CLUV exposure is an important approach complementary to the more traditional laboratory approaches for clarifying the biological significance of specific DNA damage response pathways. A recently developed experimental assay for the analysis of CLUV-induced DNA damage responses was used to show that the PCNA polyubiquitination-dependent error-free PRR pathway plays a critical role in tolerance of CLUV exposure by preventing the generation of excessive ssDNA when replication forks arrest, thus suppressing counterproductive checkpoint activation (13).Mutants of SRS2 were first isolated by their ability to suppress the radiation sensitivity of rad6 and rad18 mutants (defective in PRR) by a mechanism that requires a functional HR pathway (23, 36). In this study, we analyzed the function of Srs2 in CLUV-exposed PRR-deficient cells. We established that Srs2 acts in conjunction with SUMO-modified PCNA to lower the threshold for checkpoint activation and maintenance by suppressing the function of HR in rad18Δ cells exposed to CLUV. We also showed that Srs2 is separately involved in an HR-dependent recovery process following cessation of CLUV exposure and that this second role for Srs2, unlike its primary role in checkpoint activation and maintenance, is regulated by CDK1-dependent phosphorylation. Thus, Srs2 is involved in both CLUV-induced checkpoint-mediated arrest and recovery from CLUV exposure in PRR-deficient cells, and these two functions, while both involving HR, are separable and thus independent.     

Spermatogonia-specific proteins expressed in prepubertal buffalo (Bubalus bubalis) testis and their utilization for isolation and in vitro cultivation of spermatogonia

Buffalo is an economically important livestock species in Asia. Little is known about male germ line technology owing to lack of sufficient understanding regarding expression of germ- and somatic-cell specific-proteins in the testis. In this study, we identified UCHL-1 (PGP 9.5) and lectin- Dolichos biflorus agglutinin (DBA) as specific markers for spermatogonia in buffalo testis. Expression of germ-cell and pluripotency-specific proteins such as DDX4 (VASA) and POU5F1 (OCT3/4) were also present in spermatogonia. Interestingly, the expression of somatic cell-specific proteins such as VIMENTIN and GATA4 were also detected in germ cells. Using two-step enzymatic digestion followed by differential plating and Percoll density-gradient centrifugation, an approximately 55% spermatogonia-enriched cell population could be obtained from the prepubertal buffalo testis. Isolated spermatogonia could survive and proliferate in vitro in DMEM/F12 medium containing 10% fetal bovine serum in the absence of any specific growth factors for a week. Cultured spermatogonia showed DBA affinity and expressed DDX4 and POU5F1. These results may help to establish a long-term culture system for buffalo spermatogonia.     

On the origin of Acochlidia and other enigmatic euthyneuran gastropods,with implications for the systematics of Heterobranchia

Background  

A robust phylogenetic hypothesis of euthyneuran gastropods, as a basis to reconstructing their evolutionary history, is still hindered by several groups of aberrant, more or less worm-like slugs with unclear phylogenetic relationships. As a traditional "order" in the Opisthobranchia, the Acochlidia have a long history of controversial placements, among others influenced by convergent adaptation to the mainly meiofaunal habitats. The present study includes six out of seven acochlidian families in a comprehensive euthyneuran taxon sampling with special focus on minute, aberrant slugs. Since there is no fossil record of tiny, shell-less gastropods, a molecular clock was used to estimate divergence times within Euthyneura.     

Effects of population density, sex morph, and tree size on reproduction in a heterodichogamous maple, Acer mono, in a temperate forest of Japan

The effects of local population density, sex morph [protogynous (PG) or protandrous (PA)], and individual tree size on the demographic processes of seed production were investigated in a heterodichogamous maple, Acer mono Maxim. var. Marmoratum (Nichols.) Hara f. dissectum, in a temperate forest of Japan. As the distance from conspecific reproductive adults increased, the percentage of immature seed fall and empty seeds increased significantly, indicating higher pollination success along with local population density. Although the difference was not distinct, pollination success was affected by the local population density of the reciprocal sex morph rather than that of both sex morphs. The trees at higher local population density sites suffered higher seed mortality due to predation and decay, and tended to produce smaller seeds. Thus, the impacts of local population density operated both positively and negatively on reproduction. As a factor of individual traits, tree size scarcely affected any demographic processes. On the other hand, sex morph did affect pollination success. Trees of PG type had lower immature seed fall than those of PA type, suggesting that the former has higher efficiency of pollen acceptance than the latter. The results on seed demography presented here partly support previous suggestions that heterodichogamous plants exhibit reciprocal cross-pollination and gender specialization as reproductive traits.