Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast

The Schizosaccharomyces pombe pfh1⁺ gene (PIF1 homolog) encodes an essential enzyme that has both DNA helicase and ATPase activities and is implicated in lagging strand DNA processing. Mutations in the pfh1⁺ gene suppress a temperature-sensitive allele of cdc24⁺, which encodes a protein that functions with Schizosaccharomyces pombe Dna2 in Okazaki fragment processing. In this study, we describe the enzymatic properties of the Pfh1 helicase and the genetic interactions between pfh1 and cdc24, dna2, cdc27 or pol 3, all of which are involved in the Okazaki fragment metabolism. We show that a full-length Pfh1 fusion protein is active as a monomer. The helicase activity of Pfh1 displaced only short (<30 bp) duplex DNA regions efficiently in a highly distributive manner and was markedly stimulated by the presence of a replication-fork-like structure in the substrate. The temperature-sensitive phenotype of a dna2-C2 or a cdc24-M38 mutant was suppressed by pfh1-R20 (a cold-sensitive mutant allele of pfh1) and overexpression of wild-type pfh1⁺ abolished the ability of the pfh1 mutant alleles to suppress dna2-C2 and cdc24-M38. Purified Pfh1-R20 mutant protein displayed significantly reduced ATPase and helicase activities. These results indicate that the simultaneous loss-of-function mutations of pfh1⁺ and dna2⁺ (or cdc24⁺) are essential to restore the growth defect. Our genetic data indicate that the Pfh1 DNA helicase acts in concert with Cdc24 and Dna2 to process single-stranded DNA flaps generated in vivo by pol δ-mediated lagging strand displacement DNA synthesis.     

A monoclonal antibody distinguishes growth cartilage from other types of cartilage: a new probe for osteogenic cartilage

Summary Monoclonal antibodies (mAbs) were raised by injection of a homogenate of cultured growth cartilage (GC) cells from young rabbit ribs. These mAbs were examined by immunohistochemical staining for their reactivity to paraffin sections of rabbit tissues. The results showed that an mAb reacted preferentially with late hypertrophic and calcified costal GC zones. The mAb also reacted with hypertrophic GC adjacent to bone that existed in sternum and femur, but not to other cartilages, including resting cartilage, articular cartilage, auricular cartilage, nasal cartilage, tracheal cartilage and meniscus cartilage, or with other tissues, including tendon, skin, muscles, lung, liver, heart, thymus, spleen, eye and gut. It reacted with a wider area of the GC zone when the sections were decalcified, although its reactivity with the extended area was much less intensive than that with late hypertrophic and calcified GC zones. On treatment of the sections with bacterial collagenase, neither the reactive area nor its intensity were changed, while when treated with trypsin the reactivity was lost.These results suggest the existence of a certain molecule which distinguishes GC (osteogenic cartilage) from other (non-osteogenic) cartilage. This mAb is a useful probe for distinguishing osteogenic cartilage from non-osteogenic cartilage, and for studying differentiation steps of cartilage cells in endochondral bone formation. The mAb can also be used as a probe for clinical and stored specimens because it reacts with decalcified and paraffin-embedded human specimens.     

SGIP1alpha is an endocytic protein that directly interacts with phospholipids and Eps15

SGIP1 has been shown to be an endophilin-interacting protein that regulates energy balance, but its function is not fully understood. Here, we identified its splicing variant of SGIP1 and named it SGIP1alpha. SGIP1alpha bound to phosphatidylserine and phosphoinositides and deformed the plasma membrane and liposomes into narrow tubules, suggesting the involvement in vesicle formation during endocytosis. SGIP1alpha furthermore bound to Eps15, an important adaptor protein of clathrin-mediated endocytic machinery. SGIP1alpha was colocalized with Eps15 and the AP-2 complex. Upon epidermal growth factor (EGF) stimulation, SGIP1alpha was colocalized with EGF at the plasma membrane, indicating the localization of SGIP1alpha at clathrin-coated pits/vesicles. SGIP1alpha overexpression reduced transferrin and EGF endocytosis. SGIP1alpha knockdown reduced transferrin endocytosis but not EGF endocytosis; this difference may be due to the presence of redundant pathways in EGF endocytosis. These results suggest that SGIP1alpha plays an essential role in clathrin-mediated endocytosis by interacting with phospholipids and Eps15.     

Establishment and characterization of cultured epithelial cells lacking expression of ZO-1

In well polarized epithelial cells, closely related ZO-1 and ZO-2 are thought to function as scaffold proteins at tight junctions (TJs). In epithelial cells at the initial phase of polarization, these proteins are recruited to cadherin-based spotlike adherens junctions (AJs). As a first step to clarify the function of ZO-1, we successfully generated mouse epithelial cell clones lacking ZO-1 expression (ZO-1-/- cells) by homologous recombination. Unexpectedly, in confluent cultures, ZO-1-/- cells were highly polarized with well organized AJs/TJs, which were indistinguishable from those in ZO-1+/+ cells by electron microscopy. In good agreement, by immunofluorescence microscopy, most TJ proteins including claudins and occludin appeared to be normally concentrated at TJs of ZO-1-/- cells with the exception that a ZO-1 deficiency significantly up- or down-regulated the recruitment of ZO-2 and cingulin, another TJ scaffold protein, respectively, to TJs. When the polarization of ZO-1-/- cells was initiated by a Ca2+ switch, the initial AJ formation did not appear to be affected; however, the subsequent TJ formation (recruitment of claudins/occludin to junctions and barrier establishment) was markedly retarded. This retardation as well as the disappearance of cingulin were rescued completely by exogenous ZO-1 but not by ZO-2 expression. Quantitative evaluation of ZO-1/ZO-2 expression levels led to the conclusion that ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization but that they are not functionally identical. Finally, we discussed advantageous aspects of the gene knock-out system with cultured epithelial cells in epithelial cell biology.     

Using food network unfolding to evaluate food–web complexity in terms of biodiversity: theory and applications

Food–web complexity often hinders disentangling functionally relevant aspects of food–web structure and its relationships to biodiversity. Here, we present a theoretical framework to evaluate food–web complexity in terms of biodiversity. Food network unfolding is a theoretical method to transform a complex food web into a linear food chain based on ecosystem processes. Based on this method, we can define three biodiversity indices, horizontal diversity (D_H), vertical diversity (D_V) and range diversity (D_R), which are associated with the species diversity within each trophic level, diversity of trophic levels, and diversity in resource use, respectively. These indices are related to Shannon's diversity index (H′), where H′ = D_H + D_V ? D_R. Application of the framework to three riverine macroinvertebrate communities revealed that D indices, calculated from biomass and stable isotope features, captured well the anthropogenic, seasonal, or other within‐site changes in food–web structures that could not be captured with H′ alone.     

SUPERWOMAN1 and DROOPING LEAF genes control floral organ identity in rice

We analyzed recessive mutants of two homeotic genes in rice, SUPERWOMAN1 (SPW1) and DROOPING LEAF (DL). The homeotic mutation spw1 transforms stamens and lodicules into carpels and palea-like organs, respectively. Two spw1 alleles, spw1-1 and spw1-2, show the same floral phenotype and did not affect vegetative development. We show that SPW1 is a rice APETALA3 homolog, OsMADS16. In contrast, two strong alleles of the dl locus, drooping leaf-superman1 (dl-sup1) and drooping leaf-superman2 (dl-sup2), cause the complete transformation of the gynoecium into stamens. In these strong mutants, many ectopic stamens are formed in the region where the gynoecium is produced in the wild-type flower and they are arranged in a non-whorled, alternate pattern. The intermediate allele dl-1 (T65), results in an increase in the number of stamens and stigmas, and carpels occasionally show staminoid characteristics. In the weakest mutant, dl-2, most of the flowers are normal. All four dl alleles cause midrib-less drooping leaves. The flower of the double mutant, spw1 dl-sup, produces incompletely differentiated organs indefinitely after palea-like organs are produced in the position where lodicules are formed in the wild-type flower. These incompletely differentiated organs are neither stamens nor carpels, but have partial floral identity. Based on genetic and molecular results, we postulate a model of stamen and carpel specification in rice, with DL as a novel gene controlling carpel identity and acting mutually and antagonistically to the class B gene, SPW1.     

pH Condition in temperature shift cultivation enhances cell longevity and specific hMab productivity in CHO culture

Controlling cell proliferation during cell culturing is an effective way to improve the production yield in mammalian cell culture. We examined the effect of temperature shifts (TS) under pH control conditions in Chinese hamster ovary cells. When we shifted the culture temperature from 37 °C to 31 °C before a stationary phase at pH 6.8 (TS/pH 6.8), cell viability remained high, and the final human monoclonal antibody (hMab) concentration increased to 2.3 times that in the culture remaining at 37 °C. However, there were no significant effects on the cell viability or production yield with the same TS at pH 7.0 (TS/pH 7.0). The average specific hMab productivity and mRNA level of TS/pH 7.0 were the same as that of TS/pH 6.8. The control of cell growth by the TS or the addition of rapamycin was effective in the maintenance of cell viability, but there was no significant increase of the average specific hMab productivity in the culture where cell proliferation was controlled with rapamycin. The hMab mRNA concentration decreased to 55%–65% at a 37 °C culture with the addition of actinomycin D. In contrast, actinomycin D did not affect the mRNA level in the TS culture. This result suggested that the increase in the mRNA level in the TS condition was caused by an increase in mRNA stability. In this study, we show that TS can produce two unrelated effects: a prolongation of cell longevity and an improvement in mRNA stability.     

9HODE stimulates cell proliferation and extracellular matrix synthesis in human mesangial cells via PPARgamma

Plasma oxidized low-density lipoprotein (OX-LDL) levels are elevated in patients with renal diseases, including diabetic nephropathy. We examined effects of OX-LDL on cell proliferation and extracellular matrix (ECM) production by using normal human mesangial cells. Furthermore, we examined possible involvement of peroxisome proliferator-activated receptor gamma (PPARgamma). Mesangial cell proliferation with OX-LDL, 9-hydroxy-10,12-octadecadienoic acid (9HODE), and 13-hydroxy-9,11-octadecadienoic acid (13HODE), the major components of OX-LDL, were determined by 5-bromo-2'-deoxyuridine (BrdU) or 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) incorporation. The effect of OX-LDL on mesangial cell proliferation with PD98059 pretreatment was determined by BrdU incorporation. Type IV collagen, fibronectin, and PPARgamma expression with OX-LDL or 9HODE or 13HODE was determined by Western blotting. Type IV collagen expression with antisense oligonucleotide against PPARgamma pretreatment was also determined by Western blotting. The effect of PD98059 pretreatment on PPARgamma expression was determined by Western blotting. In mesangial cells exposed to isolated OX-LDL from human plasma, BrdU incorporation was increased, and this increase was deleted by PD98059. Type IV collagen expression was significantly increased by OX-LDL. 9HODE and 13HODE increased BrdU and MTT incorporation into mesangial cells and also increased expressions of Type IV collagen and fibronection, the major components of ECM. PPARgamma expression in mesangial cells was stimulated by 9HODE. The reduction of PPARgamma synthesis by pretreatment of antisense oligonucleotide against PPARgamma remarkably attenuated Type IV collagen synthesis induced by 9HODE. PPARgamma expression induced by 9HODE was also reduced by PD98059 pretreatment. These findings demonstrate that 9HODE, the major component of OX-LDL, stimulates cell proliferation and ECM production of human mesangial cells. In addition, the stimulatory effects are, at least in part, mediated by PPARgamma, which may exist in downstream of ERK1/2 pathway.     

Regulation of the initiation step of DNA replication by cyclin-dependent kinases

Cyclin-dependent kinases (CDKs) play a central role in the regulation of cell cycle progression in eukaryotes. The onset of S phase, the initiation of chromosomal DNA replication, is a major cell cycle event that is regulated by CDKs. Eukaryotic chromosomal DNA replication is highly regulated and occurs as a two-step reaction. The first reaction, known as licensing, is essential for DNA replication by making cell replication competent and occurs in G1 phase. Once cells enter S phase, licensed chromosomes initiate DNA replication through the action of two conserved protein kinases, S phase-specific CDK and Cdc7-Dbf4 (or Dbf4-dependent kinase). Our understanding of the regulatory mechanisms of DNA replication in model eukaryotes has advanced considerably in the past decade. In this review, we overview the regulation of DNA replication in the eukaryotic cell cycle, focusing specifically on how CDKs regulate the initiation step of DNA replication.     

Analyses of group III secreted phospholipase A2 transgenic mice reveal potential participation of this enzyme in plasma lipoprotein modification, macrophage foam cell formation, and atherosclerosis

Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.