Role of the kinesin-2 family protein, KIF3, during mitosis

During mitosis, kinesin and dynein motor proteins play critical roles in the equal segregation of chromosomes between two daughter cells. Kinesin-2 is composed of two microtubule-based motor subunits, KIF3A/3B, and a kinesin-associated protein known as KAP3, which links KIF3A/3B to cargo that is carried to cellular organelles along microtubules in interphase cells. We have shown here that the kinesin-2 complex is localized with components of the mitotic apparatus such as spindle microtubules and centrosomes. Furthermore, we found that expression of a mutant KIF3B, which is able to associate with KIF3A but not KAP3 in NIH3T3 cells, caused chromosomal aneuploidy and abnormal spindle formation. Our data suggested that the kinesin-2 complex plays an important role not only in interphase but also in mitosis.     

The assembly and maintenance of heterochromatin initiated by transgene repeats are independent of the RNA interference pathway in mammalian cells

A role for the RNA interference (RNAi) pathway in the establishment of heterochromatin is now well accepted for various organisms. Less is known about its relevance and precise role in mammalian cells. We previously showed that tandem insertion of a 1,000-copy inducible transgene into the genome of baby hamster kidney (BHK) cells initiated the formation of an extremely condensed chromatin locus. Here, we characterized the inactive transgenic locus as heterochromatin, since it was associated with heterochromatin protein 1 (HP1), histone H3 trimethylated at lysine 9, and cytosine methylation in CpG dinucleotides. Northern blot analysis did not detect any transgene-derived small RNAs. RNAi-mediated Dicer knockdown did not disrupt the heterochromatic transgenic locus or up-regulate transgene expression. Moreover, neither Dicer knockdown nor overexpression of transgene-directed small interfering RNAs altered the bidirectional transition of the transgenic locus between the heterochromatic and euchromatic states. Interestingly, tethering of HP1 to the transgenic locus effectively induced transgene silencing and chromatin condensation in a Dicer-independent manner, suggesting a role for HP1 in maintaining the heterochromatic locus. Our results suggest that the RNAi pathway is not required for the assembly and maintenance of noncentromeric heterochromatin initiated by tandem transgene repeats in mammalian cells.     

'Click chemistry' on polysaccharides: a convenient, general, and monitorable approach to develop (1-->3)-beta-D-glucans with various functional appendages

(1-->3)-beta-D-Glucans having various functional appendages (lactoside, ferrocene, pyrene, and porphyrin) can be prepared in an convenient, quantitative, and regioselective manner through regioselective bromination-azidation of curdlan to afford 6-azido-6-deoxycurdlan followed by chemoselective [3+2]-cycloadditions with various functional modules bearing a terminal alkyne group. The ability to monitor reaction conversions is an additional advantage of this synthetic approach over the conventional direct modifications on polysaccharides; the reaction can be readily monitored based on the intensity of azido peaks in the in situ attenuated total reflection infrared spectra.     

Serotype‐dependent expression patterns of stabilized lipopolysaccharide aggregates in Aggregatibacter actinomycetemcomitans strains

Above a critical concentration, amphiphilic lipopolysaccharide (LPS) molecules in an aqueous environment form aggregate structures, probably because of interactions involving hydrophobic bonds. Ionic bonds involving divalent cations stabilize these aggregate structures, making them resistant to breakdown by detergents. The aim of this study was to examine expression patterns of stabilized LPS aggregates in Aggregatibacter actinomycetemcomitans, a microorganism that causes periodontitis. A. actinomycetemcomitans strains of various serotypes and truncated LPS mutants were prepared for this study. Following treatment with a two‐phase separation system using the detergent Triton X‐114, crude LPS extracts of the study strains were separated into detergent‐phase LPS (DP‐LPS) and aqueous‐phase LPS (AP‐LPS). Repeated treatment of the aqueous phase with the two‐phase separation system produced only a slight decrease in AP‐LPS, suggesting that AP‐LPS was resistant to the detergent and thus distinguishable from DP‐LPS. The presence of divalent cations increased the yield of AP‐LPS. AP‐LPS expression patterns were serotype‐dependent; serotypes b and f showing early expression, and serotypes a and c late expression. In addition, highly truncated LPS from a waaD (rfaD) mutant were unable to generate AP‐LPS, suggesting involvement of the LPS structure in the generation of AP‐LPS. The two‐phase separation was able to distinguish two types of LPS with different physical states at the supramolecular structure level. Hence, AP‐LPS likely represents stabilized LPS aggregates, whereas DP‐LPS might be derived from non‐stabilized aggregates. Furthermore, time‐dependent expression of stabilized LPS aggregates was found to be serotype‐dependent in A. actinomycetemcomitans.     

Suppressing effect of gamma-irradiated weevils on progeny production in the West Indian sweetpotato weevil Euscepes postfasciatus (Coleoptera: Curculionidae)

The West Indian sweet potato weevil Euscepes postfasciatus (Fairmaire) (Coleoptera: Curculionidae) is a major pest of sweet potato Ipomoea batatas (L.) in the tropical and subtropical regions. The sterile insect technique (SIT) could be used as one of the most effective methods for suppression or eradication of the weevil. The effectiveness of SIT depends on the ability of the released sterile males to mate with and inseminate wild females. However, the effect of sterile weevils on the fitness of E. postfasciatus has not been evaluated on natural density. Here, we investigated the effect of gamma-irradiated weevil density on the number of weevil progeny. When irradiated weevils were released in numbers equal to those of non-irradiated weevils, the number of progeny was reduced by half of that in the control treatment, and it remained at this state for 2?weeks. Our results show that irradiated weevils ensure adequate and efficient suppression of wild weevils. We conclude that the SIT programs will be employed as effective eradication method for E. postfasciatus.     

Reduced BMP Signaling Results in Hindlimb Fusion with Lethal Pelvic/Urogenital Organ Aplasia: A New Mouse Model of Sirenomelia

Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of phenotypes observed. Here, we provide new evidences that reduced BMP (Bone Morphogenetic Protein) signaling disrupts caudal body formation in mice and phenocopies sirenomelia. Bmp4 is strongly expressed in the developing caudal body structures including the peri-cloacal region and hindlimb field. In order to address the function of Bmp4 in caudal body formation, we utilized a conditional Bmp4 mouse allele (Bmp4^flox/flox) and the Isl1 (Islet1)-Cre mouse line. Isl1-Cre is expressed in the peri-cloacal region and the developing hindimb field. Isl1Cre;Bmp4^flox/flox conditional mutant mice displayed sirenomelia phenotypes including hindlimb fusion and pelvic/urogenital organ dysgenesis. Genetic lineage analyses indicate that Isl1-expressing cells contribute to both the aPCM (anterior Peri-Cloacal Mesenchyme) and the hindlimb bud. We show Bmp4 is essential for the aPCM formation independently with Shh signaling. Furthermore, we show Bmp4 is a major BMP ligand for caudal body formation as shown by compound genetic analyses of Bmp4 and Bmp7. Taken together, this study reveals coordinated development of caudal body structures including pelvic/urogenital organs and hindlimb orchestrated by BMP signaling in Isl1-expressing cells. Our study offers new insights into the pathogenesis of sirenomelia.     

ATM-dependent phosphorylation of the checkpoint clamp regulates repair pathways and maintains genomic stability

Upon genotoxic stress and during normal S phase, ATM phosphorylates the checkpoint clamp protein Rad9 in a manner that depends on Ser272. Ser272 is the only known ATM-dependent phosphorylation site in human Rad9. However, Ser272 phosphorylation is not required for survival or checkpoint activation after DNA damage. The physiological function of Ser272 remains elusive. Here, we show that ATM-dependent Rad9^Ser272 phosphorylation requires the MRN complex and controls repair pathways. Furthermore, the mutant cells accumulate large numbers of chromosome breaks and induce gross chromosomal rearrangements. Our findings establish a new and unexpected role for ATM: it phosphorylates the checkpoint clamp in order to control repair pathways, thereby maintaining genomic integrity during unperturbed cell cycle and upon DNA damage.     

Low-dose lipopolysaccharide pretreatment suppresses choroidal neovascularization via IL-10 induction

Recent studies have suggested that some kinds of microbial infection may have a crucial role in the development of many diseases such as autoimmune diseases and certain types of cancer. It has been reported that some chronic infections, such as Chlamydia pneumoniae, and immunological dysfunctions are associated with age-related macular degeneration (AMD), a leading cause of blindness. To evaluate the association between systemic low-level inflammation induced by infection and AMD pathogenesis, we investigated whether intraperitoneal injection of lipopolysaccharide (LPS) can modulate the development of laser-induced choroidal neovascularization (CNV), a key feature of AMD. Contrary to our expectations, the sizes of CNV in mice with LPS pretreatment were approximately 65% smaller than those of the control mice. After LPS pretreatment, serum IL-10 concentration and IL-10 gene expression in peritoneal macrophages and in the posterior part of the eye increased. Peritoneal injection of anti-IL10 antibody reduced CNV suppression by LPS pretreatment. Moreover, adoptive transfer of the resident peritoneal macrophages from LPS-treated mice into control littermates resulted in an approximately 26% reduction in the size of CNV compared with PBS-treated mice. We concluded that CNV formation was suppressed by low-dose LPS pretreatment via IL-10 production by macrophages.     

Incorporation of ZP1 into perivitelline membrane after in vivo treatment with exogenous ZP1 in Japanese quail (Coturnix japonica)

In birds, the egg envelope surrounding the oocyte prior to ovulation is called the perivitelline membrane and it plays important roles in fertilization. In a previous study we demonstrated that one of the components of the perivitelline membrane, ZP3, which is secreted from the ovarian granulosa cells, specifically interacts with ZP1, another constituent that is synthesized in the liver of Japanese quail. In the present study, we investigated whether ZP1 injected exogenously into the blood possesses the ability to reconstruct the perivitelline membrane of Japanese quail. When ZP1 purified from the serum of laying quail was injected into other female birds, the signal of this exogenous ZP1 was detected in the perivitelline membrane. In addition, we revealed, by means of ligand blot analysis, that serum ZP1 interacts with both ZP1 and ZP3 of the perivitelline membrane. By contrast, when ZP1 derived from the perivitelline membrane was administered, it failed to become incorporated into the perivitelline membrane. Interestingly, serum ZP1 recovered from other Galliformes, including chicken and guinea fowl, could be incorporated into the quail perivitelline membrane, but the degree of interaction between quail ZP3 and ZP1 of the vitelline membrane of laid eggs from chicken and guinea fowl appeared to be weak. These results demonstrate that exogenous ZP1 purified from the serum, but not ZP1 from the perivitelline membrane, can become incorporated into the perivitelline membrane upon injection into other types of female birds. To our knowledge, this is the first demonstration that the egg envelope component, when exogenously administered to animals, can reconstruct the egg envelope in vivo.