Genetic polymorphisms in transforming growth factor beta-1 (TGFB1) and childhood asthma and atopy

Transforming growth factor beta-1 (TGFB1) may influence asthma by modulating allergic airway inflammation and airway remodeling. The role of single nucleotide polymorphisms (SNPs) of TGFB1 in asthma remains inconclusive. We examined TGFB1 SNPs in relation to asthma risk and degree of atopy among 546 case-parent triads, consisting of asthmatics aged 4–17 years and their parents in Mexico City. Atopy to 24 aeroallergens was determined by skin prick tests. We genotyped five TGFB1 SNPs, including two known functional SNPs [C-509T (rs1800469), T869C (rs1982073)] and three others (rs7258445, rs1800472, rs8179181), using TaqMan and Masscode assays. We analyzed the data using log-linear and polytomous logistic methods. Three associated SNPs, including the two known functional SNPs, were statistically significantly related to asthma risk. Individuals carrying the T allele of C-509T had an increased risk of asthma [relative risk (RR) = 1.42, 95% confidence interval (CI) = 1.08–1.87 for one copy; RR (95%CI) = 1.95 (1.36–2.78) for two copies]. For T869C, the RRs (95%CI) were 1.47 (1.09–1.98) for one and 2.00 (1.38–2.90) for two copies of the C allele. Similar results were found for rs7258445. The haplotype containing all three risk alleles conferred an increased risk of asthma (RR = 1.48, 95% CI = 1.11–1.95 for one copy; RR = 1.77, 95% CI = 1.22–2.57 for two copies). These three SNPs were also related to the degree of atopy. This largest study to date of genetic variation in TGFB1 and asthma and atopy adds to increasing evidence for a role in these disorders.     

Influence of TGFBR2, TGFB3, DNMT1, and DNMT3A Knockdowns on CTGF,TGFBR2, and DNMT3A in Neonatal and Adult Human Dermal Fibroblasts Cell Lines

Dermal fibroblasts are responsible for the production of the extracellular matrix that undergoes significant changes during the skin aging process. These changes are partially controlled by the TGF-β signaling, which regulates tissue homeostasis dependently on several genes, including CTGF and DNA methyltransferases. To investigate the potential differences in the regulation of the TGF-β signaling and related molecular pathways at distinct developmental stages, we silenced the expression of TGFB1, TGFB3, TGFBR2, CTGF, DNMT1, and DNMT3A in the neonatal (HDF-N) and adult (HDF-A) human dermal fibroblasts using the RNAi method. Through Western blot, we analyzed the effects of the knockdowns of these genes on the level of the CTGF, TGFBR2, and DNMT3A proteins in both cell lines. In the in vitro assays, we observed that CTGF level was decreased after knockdown of DNMT1 in HDF-N but not in HDF-A. Similarly, the level of DNMT3A was decreased only in HDF-N after silencing of TGFBR2, TGFB3, or DNMT1. TGFBR2 level was lower in HDF-N after knockdown of TGFB3, DNMT1, or DNMT3A, but it was higher in HDF-A after TGFB1 silencing. The reduction of TGFBR2 after silencing of DNMT3A and vice versa in neonatal cells only suggests the developmental stage-specific interactions between these two genes. However, additional studies are needed to explain the dependencies between analyzed proteins.     

The cost of reproduction in the glaucous-winged gull

Summary Experimental enlargement of brood size in the glaucous-winged gull (Larus glaucescens) resulted in increased adult foraging time, decreased adult body weight at the end of the breeding season, and decreased over-winter adult survival. The decreased survival of breeding adults was associated with reduced body condition at the end of breeding (resulting from physiological costs of reproduction). Decreased survival was not due to an increased risk of injury or predation during the breeding season. Brood size did not directly affect the fecundity of surviving birds in the subsequent year. However, brood size may have an indirect effect on subsequent fecundity because the probability of mate loss increased among birds with large broods and the reproductive performance of birds with new mates was reduced. Based on estimates of life-time fitness calculated from fecundity and survivorship, birds with two- or three-chick broods (the normal brood size) have higher fitness than birds with one- or four-chick broods. However, the decreased fitness of birds with four-chick broods was slight, and probably not a sufficient explanation for the absence of natural four-chick broods in the glaucouswinged gull.     

The essential requirement for Runx1 in the development of the sternum

Runx1 is highly expressed in chondroprogenitor and osteoprogenitor cells and in vitro experiments suggest that Runx1 is important in the early stages of osteoblast and chondrocyte differentiation. However, because Runx1 knockout mice are early embryonic lethal due to failure of hematopoiesis, the role of Runx1 in skeletogenesis remains unclear. We studied the role of Runx1 in skeletal development using a Runx1 reversible knockout mouse model. By crossing with Tie2-Cre deletor mice, Runx1 expression was selectively rescued in the endothelial and hematopoietic systems but not in the skeleton. Although Runx1^Re/Re embryos survived until birth and had a generally normal skeleton, the development of mineralization in the sternum and some skull elements was significantly disrupted. In contrast to wild-type embryos, the sternum of E17.5 Runx1^Re/Re embryos showed high levels of Sox-9 and collagen type II expression and lack of development of hypertrophic chondrocytes. In situ hybridization analysis demonstrated that, in contrast to the vertebrae and long bones, the sternum of wild-type embryos expresses high levels of Runx1, but not Runx2, the master regulator of skeletogenesis. Thus, although Runx1 is not essential for major skeletal development, it does play an essential role in the development of the sternum and some skull elements.     

The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system

GPR54 is a G-protein-coupled receptor that displays a high percentage of identity in the transmembrane domains with the galanin receptors. The ligand for GPR54 has been identified as a peptide derived from the KiSS-1 gene. KiSS-1 has been shown to have anti-metastatic effects, suggesting that KiSS-1 or its receptor represents a potential therapeutic target. To further our understanding of the physiological function of this receptor, we have generated a mutant mouse line with a targeted disruption of the GPR54 receptor (GPR54 -/-). The analysis of the GPR54 mutant mice revealed developmental abnormalities of both male and female genitalia and histopathological changes in tissues which normally contain sexually dimorphic features. These data suggest a role for GPR54/KiSS-1 in normal sexual development, and indicate that study of the GPR54 mutant mice may provide valuable insights into human reproductive syndromes.     

The N-terminal domain of NPC1L1 protein binds cholesterol and plays essential roles in cholesterol uptake

Niemann-Pick C1-like 1 (NPC1L1) is a multitransmembrane protein playing a crucial role in dietary and biliary cholesterol absorption. Cholesterol promotes the formation and endocytosis of NPC1L1-flotillin-cholesterol membrane microdomains, which is an early step in cholesterol uptake. How cholesterol is sensed in this step is unknown. Here, we find that the N-terminal domain (NTD) of NPC1L1 binds cholesterol. Mutation of residue Leu-216 in NPC1L1-NTD eliminates cholesterol binding, decreases the formation of NPC1L1-flotillin-cholesterol membrane microdomains, and prevents NPC1L1-mediated cholesterol uptake in culture cells and mice livers. NPC1L1-NTD specifically binds cholesterol but not plant sterols, which may account for the selective cholesterol absorption in intestine. Furthermore, 25- or 27-hydroxycholesterol competes with cholesterol to bind NPC1L1-NTD and inhibits the cholesterol induced endocytosis of NPC1L1. Together, these results demonstrate that plasma membrane-localized NPC1L1 binds exogenous cholesterol via its NTD, and facilitates the formation of NPC1L1-flotillin-cholesterol membrane microdomains that are then internalized into cells through the clathrin-AP2 pathway. Our study uncovers the mechanism of cholesterol sensing by NPC1L1 and proposes a mechanism for selective cholesterol absorption.     

海南睑虎繁殖生物学特征初步观察

关于睑虎属(Goniurosaurus)物种的研究主要集中在新种描述、分类分布以及系统进化等方面,繁殖生物学资料基本空白.本课题组于2017年9月至2019年8月对海南吊罗山国家级自然保护区内海南睑虎(G.hainanensis)的繁殖生物学特征进行了调查研究.结果发现,海南睑虎的繁殖期为3～10月,共观察到雌性个体1...     

The Role of Calcium in the Fertilization Process in Flowering Plants

The fertilization process in flowering plants in a broad sense includes a progamic phase preceding the phase of double fertilization. To our present knowledge, calcium as a second messenger in the signal transduction plays important roles in all the links of this process. The present review attempts to highlight the recent advances in this research field, including: calcium in relation to in vitro pollen tube growth (distribution of calcium in pollen tube tip; regulation of pollen tube growth by calcium; calcium oscillation in pollen tube); distribution of calcium in pistil and its relation to in vivo pollen tube growth (calcium in relation to pollen-pistil recognition; pollen tube growth in pistil; pollen tube entry into embryo sac and the discharge and transportation of sperms); and calcium in relation to sperm-egg fusion and egg cell activation. In conclusion the author summarizes into several main view points, and gives recommendation for further researches on this topic.     

Physiological roles of group X-secreted phospholipase A2 in reproduction, gastrointestinal phospholipid digestion, and neuronal function

Although the secreted phospholipase A(2) (sPLA(2)) family has been generally thought to participate in pathologic events such as inflammation and atherosclerosis, relatively high and constitutive expression of group X sPLA(2) (sPLA(2)-X) in restricted sites such as reproductive organs, the gastrointestinal tract, and peripheral neurons raises a question as to the roles played by this enzyme in the physiology of reproduction, digestion, and the nervous system. Herein we used mice with gene disruption or transgenic overexpression of sPLA(2)-X to clarify the homeostatic functions of this enzyme at these locations. Our results suggest that sPLA(2)-X regulates 1) the fertility of spermatozoa, not oocytes, beyond the step of flagellar motility, 2) gastrointestinal phospholipid digestion, perturbation of which is eventually linked to delayed onset of a lean phenotype with reduced adiposity, decreased plasma leptin, and improved muscle insulin tolerance, and 3) neuritogenesis of dorsal root ganglia and the duration of peripheral pain nociception. Thus, besides its inflammatory action proposed previously, sPLA(2)-X participates in physiologic processes including male fertility, gastrointestinal phospholipid digestion linked to adiposity, and neuronal outgrowth and sensing.     

The essential roles of Mps1 in spermatogenesis and fertility in mice

Monopolar spindle 1 (MPS1), which plays a critical role in somatic mitosis, has also been revealed to be essential for meiosis I in oocytes. Spermatogenesis is an important process involving successive mitosis and meiosis, but the function of MPS1 in spermatogenesis remains unclear. Here, we generated Mps1 conditional knockout mice and found that Ddx4-cre-driven loss of Mps1 in male mice resulted in depletion of undifferentiated spermatogonial cells and subsequently of differentiated spermatogonia and spermatocytes. In addition, Stra8-cre-driven ablation of Mps1 in male mice led to germ cell loss and fertility reduction. Spermatocytes lacking Mps1 have blocked at the zygotene-to-pachytene transition in the prophase of meiosis I, which may be due to decreased H2B ubiquitination level mediated by MDM2. And the expression of many meiotic genes was decreased, while that of apoptotic genes was increased. Moreover, we also detected increased apoptosis in spermatocytes with Mps1 knockout, which may have been the reason why germ cells were lost. Taken together, our findings indicate that MPS1 is required for mitosis of gonocytes and spermatogonia, differentiation of undifferentiated spermatogonia, and progression of meiosis I in spermatocytes.Subject terms: Cell division, Spermatogenesis     

MiR-17-92 cluster regulates cell proliferation and collagen synthesis by targeting TGFB pathway in mouse palatal mesenchymal cells

Elongation and elevation of palatal shelves, mainly caused by proliferation and extra-cellular matrix synthesis of palatal mesenchymal cells (PMCs), are essential for normal palatal development. Transforming growth factor beta (TGFB) pathway could induce proliferation inhibition and collagen synthesis in PMCs. Recent studies found that miRNA-17-92 (miR-17-92) cluster, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a, expressed in the 1st bronchial arch of mouse embryos during the period of palatal shelf elongation and elevation, and directly targeted TGFB pathway in cancer cell lines. Whether miR-17-92 cluster expresses and targets TGFB pathway in PMCs has not yet been studied. Using quantitative real-time RT-PCR, we found that miR-17-92 expressed in PMCs and decreased from embryonic day (E) 12 to E14 in palatal shelves. MTT assay and Western blot showed that miR-17-92 inhibited TGFB1 induced proliferation inhibition and collagen synthesis in PMCs by decreasing TGFBR2, SMAD2, and SMAD4 protein level. Further luciferase assay showed that miR-17 and miR-20a directly targeted 3′UTR of TGFBR2, and that miR-18a directly targeted 3′UTR of SMAD2 and SMAD4. We thus conclude that miR-17-92 cluster could inhibit TGFB pathway induced proliferation inhibition and collagen synthesis in PMCs by directly targeting TGFBR2, SMAD2, and SMAD4.     

水貂睾丸退化期间TGFB家族生长因子及其受体基因表达量的变化

为了探究水貂季节性睾丸退化的分子机制,本研究利用HE染色技术对不同退化时期的水貂睾丸组织进行了组织学研究,同时利用荧光定量PCR技术对不同退化时期的水貂睾丸组织中TGFB家族生长因子及其受体基因表达量变化进行了研究。结果发现在水貂睾丸退化期间,其直径逐渐缩短到原来的1/2,重量逐渐降低到原来的1/8。HE染色结果发现,4~7月水貂睾丸经历了严重退化,而且曲细精管的管腔逐渐消失,同时曲细精管的直径减小到其最大尺寸的1/3。荧光定量PCR结果发现TGFb3、INHBA、TGFBR1、ACVR2A、ACVR2B、SMAD2、SMAD4基因相对表达量在水貂睾丸退化期间都呈下降趋势。本研究表明TGFB信号通路可能在水貂睾丸退化期间发挥了重要功能。     

Evidence against essential roles for subdomain 1 of actin in actomyosin sliding movements

We have engineered acto-S1chimera proteins carrying the entire actin inserted in loop 2 of the motor domain of Dictyostelium myosin II with 24 or 18 residue-linkers (CP24 and CP18, respectively). These proteins were capable of self-polymerization as well as copolymerization with skeletal actin and exhibited rigor-like structures. The MgATPase rate of CP24-skeletal actin copolymer was 1.06 s(-1), which is slightly less than the V(max) of Dictyostelium S1. Homopolymer filaments of skeletal actin, CP24, and CP18 moved at 4.7+/-0.6, 2.9+/-0.6, and 4.1+/-0.8 microm/s (mean+/-SD), respectively, on coverslips coated with skeletal myosin at 27 degrees C. Statistically thermodynamic considerations suggest that the S1 portion of chimera protein mostly resides on subdomain 1 (SD-1) of the actin portion even in the presence of ATP. This and the fact that filaments of CP18 with shorter linkers moved faster than CP24 filaments suggest that SD-1 might not be as essential as conventionally presumed for actomyosin sliding interactions.     

Recruitment of Orc6l, a dormant maternal mRNA in mouse oocytes, is essential for DNA replication in 1-cell embryos

Mouse oocytes acquire the ability to replicate DNA during meiotic maturation, presumably to ensure that DNA replication does not occur precociously between MI and MII and only after fertilization. Acquisition of DNA replication competence requires protein synthesis, but the identity of the proteins required for DNA replication is poorly described. In Xenopus, the only component missing for DNA replication competence is CDC6, which is synthesized from a dormant maternal mRNA recruited during oocyte maturation, and a similar situation also occurs during mouse oocyte maturation. We report that ORC6L is another component required for acquisition of DNA replication competence that is absent in mouse oocytes. The dormant maternal Orc6l mRNA is recruited during maturation via a CPE present in its 3′ UTR. RNAi-mediated ablation of maternal Orc6l mRNA prevents the maturation-associated increase in ORC6L protein and inhibits DNA replication in 1-cell embryos. These results suggest that mammalian oocytes have more complex mechanisms to establish DNA replication competence when compared to their Xenopus counterparts.     

Mouse knock-out of IOP1 protein reveals its essential role in mammalian cytosolic iron-sulfur protein biogenesis

Iron-sulfur proteins play an essential role in a variety of biologic processes and exist in multiple cellular compartments. The biogenesis of these proteins has been the subject of extensive investigation, and particular focus has been placed on the pathways that assemble iron-sulfur clusters in the different cellular compartments. Iron-only hydrogenase-like protein 1 (IOP1; also known as nuclear prelamin A recognition factor like protein, or NARFL) is a human protein that is homologous to Nar1, a protein in Saccharomyces cerevisiae that, in turn, is an essential component of the cytosolic iron-sulfur protein assembly pathway in yeast. Previous siRNA-induced knockdown studies using mammalian cells point to a similar role for IOP1 in mammals. In the present studies, we pursued this further by knocking out Iop1 in Mus musculus. We find that Iop1 knock-out results in embryonic lethality before embryonic day 10.5. Acute, inducible global knock-out of Iop1 in adult mice results in lethality and significantly diminished activity of cytosolic aconitase, an iron-sulfur protein, in liver extracts. Inducible knock-out of Iop1 in mouse embryonic fibroblasts results in diminished activity of cytosolic but not mitochondrial aconitase and loss of cell viability. Therefore, just as with knock-out of Nar1 in yeast, we find that knock-out of Iop1/Narfl in mice results in lethality and defective cytosolic iron-sulfur cluster assembly. The findings demonstrate an essential role for IOP1 in this pathway.     

Contributions of growth, stasis, and reproduction to fitness in brooding and broadcast spawning marine bivalves

Reproductive modes in marine invertebrates can be generally grouped into two types: those brooding larvae and those broadcast-spawning gametes into the water. We asked if these different life-history strategies differ based on how contribution to fitness is partitioned between growth, stasis, and reproduction. To investigate this question, we used published demographic data on ten diverse species of marine bivalves. We parameterized simple matrix-population models and calculated the sums of elasticities to growth, stasis, and reproduction parameters and plotted the results on triangular axes. We also assessed whether contribution patterns were correlated with reproductive mode and tropical, temperate, or polar environments. We found that some of the broadcast spawners fell in the region of the plot with high elasticities for stasis and that some of the brooders fell in the region of the plot with higher growth and reproduction elasticities than stasis ones. However, instead of a sharp dichotomy, we found a continuum in contributions of stasis parameters with long-lived brooders and short-lived broadcast spawners in the same region of the plot. There was no clear pattern of reproductive mode associated with any particular environment, but we think these preliminary results are intriguing and that further work on comparative demography of marine invertebrates is warranted. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.