Microtubule-organizing center formation at telomeres induces meiotic telomere clustering

During meiosis, telomeres cluster and promote homologous chromosome pairing. Telomere clustering requires the interaction of telomeres with the nuclear membrane proteins SUN (Sad1/UNC-84) and KASH (Klarsicht/ANC-1/Syne homology). The mechanism by which telomeres gather remains elusive. In this paper, we show that telomere clustering in fission yeast depends on microtubules and the microtubule motors, cytoplasmic dynein, and kinesins. Furthermore, the γ-tubulin complex (γ-TuC) is recruited to SUN- and KASH-localized telomeres to form a novel microtubule-organizing center that we termed the “telocentrosome.” Telocentrosome formation depends on the γ-TuC regulator Mto1 and on the KASH protein Kms1, and depletion of either Mto1 or Kms1 caused severe telomere clustering defects. In addition, the dynein light chain (DLC) contributes to telocentrosome formation, and simultaneous depletion of DLC and dynein also caused severe clustering defects. Thus, the telocentrosome is essential for telomere clustering. We propose that telomere-localized SUN and KASH induce telocentrosome formation and that subsequent microtubule motor-dependent aggregation of telocentrosomes via the telocentrosome-nucleated microtubules causes telomere clustering.     

MAIGO5 Functions in Protein Export from Golgi-Associated Endoplasmic Reticulum Exit Sites in Arabidopsis

Plant cells face unique challenges to efficiently export cargo from the endoplasmic reticulum (ER) to mobile Golgi stacks. Coat protein complex II (COPII) components, which include two heterodimers of Secretory23/24 (Sec23/24) and Sec13/31, facilitate selective cargo export from the ER; however, little is known about the mechanisms that regulate their recruitment to the ER membrane, especially in plants. Here, we report a protein transport mutant of Arabidopsis thaliana, named maigo5 (mag5), which abnormally accumulates precursor forms of storage proteins in seeds. mag5-1 has a deletion in the putative ortholog of the Saccharomyces cerevisiae and Homo sapiens Sec16, which encodes a critical component of ER exit sites (ERESs). mag mutants developed abnormal structures (MAG bodies) within the ER and exhibited compromised ER export. A functional MAG5/SEC16A–green fluorescent protein fusion localized at Golgi-associated cup-shaped ERESs and cycled on and off these sites at a slower rate than the COPII coat. MAG5/SEC16A interacted with SEC13 and SEC31; however, in the absence of MAG5/SEC16A, recruitment of the COPII coat to ERESs was accelerated. Our results identify a key component of ER export in plants by demonstrating that MAG5/SEC16A is required for protein export at ERESs that are associated with mobile Golgi stacks, where it regulates COPII coat turnover.     

FTIR study of light-dependent activation and DNA repair processes of (6-4) photolyase

The UV component of sunlight threatens all life on the earth by damaging DNA. The photolyase (PHR) DNA repair proteins maintain genetic integrity by harnessing blue light to restore intact bases from the major UV-induced photoproducts, cyclobutane pyrimidine dimers (CPD), and (6-4) photoproducts ((6-4) PPs). The (6-4) PHR must catalyze not only covalent bond cleavage between two pyrmidine bases but also hydroxyl or amino group transfer from the 5'- to 3'-pyrimidine base, requiring a more complex mechanism than that postulated for CPD PHR. In this paper, we apply Fourier transform infrared (FTIR) spectroscopy to (6-4) PHR and report difference FTIR spectra that correspond to its photoactivation, substrate binding, and light-dependent DNA repair processes. The presence of DNA carrying a single (6-4) PP uniquely influences vibrations of the protein backbone and a protonated carboxylic acid, whereas photoactivation produces IR spectral changes for the FAD cofactor and the surrounding protein. Difference FTIR spectra for the light-dependent DNA damage repair reaction directly show significant DNA structural changes in the (6-4) lesion and the neighboring phosphate group. Time-dependent illumination of samples with different enzyme:substrate stoichiometries successfully distinguished signals characteristic of structural changes in the protein and the DNA resulting from binding and catalysis.     

A novel low-temperature-active β-glucosidase from symbiotic <Emphasis Type="Italic">Serratia</Emphasis> sp. TN49 reveals four essential positions for substrate accommodation

To maximize the production of flag-tagged cartilage oligomeric matrix protein angiopoietin-1 (FCA1) from Chinese hamster ovary (CHO) cells, the effects of culture pH and temperature on cell growth and FCA1 production were investigated. Cells were cultivated in a bioreactor at different culture pH (6.7, 6.9, 7.2, and 7.5) and temperatures (33 and 37 °C). Lowering the culture temperature suppressed cell growth while allowing maintenance of high cell viability for a longer culture period. The specific FCA1 productivity (q _FCA1) was increased at low culture temperature. Accordingly, the highest FCA1 concentration was obtained at pH 7.2 and 33 °C, and was approximately 4.0-fold higher than that at pH 7.2 and 37 °C. However, aggregates and a monomeric form of FCA1, which are undesirable due to reduced biological activity or immunogenicity, were significant at pH 7.2 and 33 °C. It was also found that the expression pattern of FCA1 was affected more significantly by culture pH than by the culture temperature. FCA1 aggregation dramatically decreased at culture pH 7.5 regardless of the culture temperature. Furthermore, the monomeric form of FCA1 was not observed. Taken together, optimization of culture temperature and culture pH (33 °C and pH 7.5) significantly improves the production of biologically active FCA1 with tetrameric or pentameric forms from CHO cells.     

Relationship between gonadal steroid hormones and vulvar bleeding in southern tamandua, Tamandua tetradactyla

This study aimed at demonstrating the profiles of circulating gonadal steroid hormones during the estrous cycle and pregnancy in a southern tamandua (Tamandua tetradactyla). Additionally, this study clarified the relationship between vulvar bleeding and hormonal changes. The concentrations of serum progesterone (P(4)) and estradiol-17β (E(2)) were determined by enzyme immunoassays. Serum P(4) and E(2) concentrations changed cyclically and the estrous cycle length (± SD) based on the E(2) cycles was 44.3 ± 4.5 days. Vulvar bleeding started to be seen at the decreasing of P(4). The cycle length for vulvar bleeding was 43.3 ± 4.2 days. Interval from the first day of bleeding to the peak of E(2) concentration was 23.1 ± 3.1 days. Serum P(4) during pregnancy remained high and E(2) increased 8 weeks after conception and remained high until parturition. The female delivered normally after a 165 day-pregnancy period and reared the offspring well. Approximately 3 weeks after parturition, serum E(2) and P(4) cycles resumed. Visual bleeding may be useful as a real-time indicator for understanding the ovarian cycle of southern tamanduas, and estrus could be expected approximately 3 weeks after the first bleeding.     

Exercise-induced left bundle branch block and subsequent mechanical left ventricular dyssynchrony--resolved with pharmacological therapy

A 53-year-old man with depressed ejection fraction (EF) of 35% and QRS width of 88 ms at rest was admitted to our institution with a complaint of exertional chest discomfort and dyspnea. During treadmill exercise, left bundle-branch block (LBBB) with a QRS width of 152 ms occurred at a heart rate of 100 bpm. During LBBB, the patient showed significant mechanical dyssynchrony as evidenced by a two-dimensional speckle tracking radial strain of 260 ms (≥ 130 ms), defined as the time difference between anterior-septum and posterior wall. Five-month after carvedilol and candesartan administration, EF had improved to 49% and LBBB did not occur until a heart rate of 126 bpm was attained during treadmill exercise. It appears that pharmacological therapy may be useful for patients with heart failure and exercise-induced LBBB.     

Quantitative and qualitative urinary cellular patterns correlate with progression of murine glomerulonephritis

The kidney is a nonregenerative organ composed of numerous functional nephrons and collecting ducts (CDs). Glomerular and tubulointerstitial damages decrease the number of functional nephrons and cause anatomical and physiological alterations resulting in renal dysfunction. It has recently been reported that nephron constituent cells are dropped into the urine in several pathological conditions associated with renal functional deterioration. We investigated the quantitative and qualitative urinary cellular patterns in a murine glomerulonephritis model and elucidated the correlation between cellular patterns and renal pathology.Urinary cytology and renal histopathology were analyzed in BXSB/MpJ (BXSB; glomerulonephritis model) and C57BL/6 (B6; control) mice. Urinary cytology revealed that the number of urinary cells in BXSB mice changed according to the histometric score of glomerulonephritis and urinary albumin; however, no correlation was detected for the levels of blood urea nitrogen and creatinine. The expression of specific markers for podocytes, distal tubules (DTs), and CDs was detected in BXSB urine. Cells immunopositive for Wilms tumor 1 (podocyte marker) and interleukin-1 family, member 6 (damaged DT and CD marker) in the kidney significantly decreased and increased in BXSB versus B6, respectively. In the PCR array analysis of inflammatory cytokines and chemokines, Il10, Cxcl2, C3, and Il1rn showed relatively higher expression in BXSB kidneys than in B6 kidneys. In particular, the highest expression of C3 mRNA was detected in the urine from BXSB mice. Furthermore, C3 protein and mRNA were localized in the epithelia of damaged nephrons.These findings suggest that epithelial cells of the glomerulus, DT, and CD are dropped into the urine, and that these patterns are associated with renal pathology progression. We conclude that evaluation of urinary cellular patterns plays a key role in the early, noninvasive diagnosis of renal disease.     

Canonical Wnt signaling in the visceral muscle is required for left-right asymmetric development of the Drosophila midgut

Many animals develop left-right (LR) asymmetry in their internal organs. The mechanisms of LR asymmetric development are evolutionarily divergent, and are poorly understood in invertebrates. Therefore, we studied the genetic pathway of LR asymmetric development in Drosophila. Drosophila has several organs that show directional and stereotypic LR asymmetry, including the embryonic gut, which is the first organ to develop LR asymmetry during Drosophila development. In this study, we found that genes encoding components of the Wnt-signaling pathway are required for LR asymmetric development of the anterior part of the embryonic midgut (AMG). frizzled 2 (fz2) and Wnt4, which encode a receptor and ligand of Wnt signaling, respectively, were required for the LR asymmetric development of the AMG. arrow (arr), an ortholog of the mammalian gene encoding low-density lipoprotein receptor-related protein 5/6, which is a co-receptor of the Wnt-signaling pathway, was also essential for LR asymmetric development of the AMG. These results are the first demonstration that Wnt signaling contributes to LR asymmetric development in invertebrates, as it does in vertebrates. The AMG consists of visceral muscle and an epithelial tube. Our genetic analyses revealed that Wnt signaling in the visceral muscle but not the epithelium of the midgut is required for the AMG to develop its normal laterality. Furthermore, fz2 and Wnt4 were expressed in the visceral muscles of the midgut. Consistent with these results, we observed that the LR asymmetric rearrangement of the visceral muscle cells, the first visible asymmetry of the developing AMG, did not occur in embryos lacking Wnt4 expression. Our results also suggest that canonical Wnt/β-catenin signaling, but not non-canonical Wnt signaling, is responsible for the LR asymmetric development of the AMG. Canonical Wnt/β-catenin signaling is reported to have important roles in LR asymmetric development in zebrafish. Thus, the contribution of canonical Wnt/β-catenin signaling to LR asymmetric development may be an evolutionarily conserved feature between vertebrates and invertebrates.     

Molecular and biochemical characterization of a novel intracellular low-temperature-active xylanase

A 990 bp full-length gene (xynAHJ2) encoding a 329- residue polypeptide (XynAHJ2) with a calculated mass of 38.4 kDa was cloned from Bacillus sp. HJ2 harbored in a saline soil. XynAHJ2 showed no signal peptide, distinct amino acid stretches of glycoside hydrolase (GH) family 10 intracellular endoxylanases, and the highest amino acid sequence identity of 65.3% with the identified GH 10 intracellular mesophilic endoxylanase iM-KRICT PX1-Ps from Paenibacillus sp. HPL-001 (ACJ06666). The recombinant enzyme (rXynAHJ2) was expressed in Escherichia coli and displayed the typical characteristics of low-temperatureactive enzyme (exhibiting optimum activity at 35 degrees C, 62% at 20 degrees C, and 38% at 10 degrees C; thermolability at > or =45 degrees C). Compared with the reported GH 10 low-temperature-active endoxylanases, which are all extracellular, rXynAHJ2 showed low amino acid sequence identities (<45%), low homology (different phylogenetic cluster), and difference of structure (decreased amount of total accessible surface area and exposed nonpolar accessible surface area). Compared with the reported GH 10 intracellular endoxylanases, which are all mesophilic and thermophilic, rXynAHJ2 has decreased numbers of arginine residues and salt bridges, and showed resistance to Ni2+, Ca2+, or EDTA at 10 mM final concentration. The above mechanism of structural adaptation for low-temperature activity of intracellular endoxylanase rXynAHJ2 is different from that of GH 10 extracellular low-temperature-active endoxylanases. This is the first report of the molecular and biochemical characterizations of a novel intracellular low-temperatureactive xylanase.     

Eukaryotic class II cyclobutane pyrimidine dimer photolyase structure reveals basis for improved ultraviolet tolerance in plants

Ozone depletion increases terrestrial solar ultraviolet B (UV-B; 280–315 nm) radiation, intensifying the risks plants face from DNA damage, especially covalent cyclobutane pyrimidine dimers (CPD). Without efficient repair, UV-B destroys genetic integrity, but plant breeding creates rice cultivars with more robust photolyase (PHR) DNA repair activity as an environmental adaptation. So improved strains of Oryza sativa (rice), the staple food for Asia, have expanded rice cultivation worldwide. Efficient light-driven PHR enzymes restore normal pyrimidines to UV-damaged DNA by using blue light via flavin adenine dinucleotide to break pyrimidine dimers. Eukaryotes duplicated the photolyase gene, producing PHRs that gained functions and adopted activities that are distinct from those of prokaryotic PHRs yet are incompletely understood. Many multicellular organisms have two types of PHR: (6-4) PHR, which structurally resembles bacterial CPD PHRs but recognizes different substrates, and Class II CPD PHR, which is remarkably dissimilar in sequence from bacterial PHRs despite their common substrate. To understand the enigmatic DNA repair mechanisms of PHRs in eukaryotic cells, we determined the first crystal structure of a eukaryotic Class II CPD PHR from the rice cultivar Sasanishiki. Our 1.7 Å resolution PHR structure reveals structure-activity relationships in Class II PHRs and tuning for enhanced UV tolerance in plants. Structural comparisons with prokaryotic Class I CPD PHRs identified differences in the binding site for UV-damaged DNA substrate. Convergent evolution of both flavin hydrogen bonding and a Trp electron transfer pathway establish these as critical functional features for PHRs. These results provide a paradigm for light-dependent DNA repair in higher organisms.