Two Carotenoid Oxygenases Contribute to Mammalian Provitamin A Metabolism

Mammalian genomes encode two provitamin A-converting enzymes as follows: the β-carotene-15,15′-oxygenase (BCO1) and the β-carotene-9′,10′-oxygenase (BCO2). Symmetric cleavage by BCO1 yields retinoids (β-15′-apocarotenoids, C₂₀), whereas eccentric cleavage by BCO2 produces long-chain (>C₂₀) apocarotenoids. Here, we used genetic and biochemical approaches to clarify the contribution of these enzymes to provitamin A metabolism. We subjected wild type, Bco1^−/−, Bco2^−/−, and Bco1^−/−Bco2^−/− double knock-out mice to a controlled diet providing β-carotene as the sole source for apocarotenoid production. This study revealed that BCO1 is critical for retinoid homeostasis. Genetic disruption of BCO1 resulted in β-carotene accumulation and vitamin A deficiency accompanied by a BCO2-dependent production of minor amounts of β-apo-10′-carotenol (APO10ol). We found that APO10ol can be esterified and transported by the same proteins as vitamin A but with a lower affinity and slower reaction kinetics. In wild type mice, APO10ol was converted to retinoids by BCO1. We also show that a stepwise cleavage by BCO2 and BCO1 with APO10ol as an intermediate could provide a mechanism to tailor asymmetric carotenoids such as β-cryptoxanthin for vitamin A production. In conclusion, our study provides evidence that mammals employ both carotenoid oxygenases to synthesize retinoids from provitamin A carotenoids.     

Fenretinide inhibits vitamin A formation from β-carotene and regulates carotenoid levels in mice

N-[4-hydroxyphenyl]retinamide, commonly known as fenretinide, a synthetic retinoid with pleiotropic benefits for human health, is currently utilized in clinical trials for cancer, cystic fibrosis, and COVID-19. However, fenretinide reduces plasma vitamin A levels by interacting with retinol-binding protein 4 (RBP4), which often results in reversible night blindness in patients. Cell culture and in vitro studies show that fenretinide binds and inhibits the activity of β-carotene oxygenase 1 (BCO1), the enzyme responsible for endogenous vitamin A formation. Whether fenretinide inhibits vitamin A synthesis in mammals, however, remains unknown. The goal of this study was to determine if the inhibition of BCO1 by fenretinide affects vitamin A formation in mice fed β-carotene. Our results show that wild-type mice treated with fenretinide for ten days had a reduction in tissue vitamin A stores accompanied by a two-fold increase in β-carotene in plasma (P < 0.01) and several tissues. These effects persisted in RBP4-deficient mice and were independent of changes in intestinal β-carotene absorption, suggesting that fenretinide inhibits vitamin A synthesis in mice. Using Bco1^?/? and Bco2^?/? mice we also show that fenretinide regulates intestinal carotenoid and vitamin E uptake by activating vitamin A signaling during short-term vitamin A deficiency. This study provides a deeper understanding of the impact of fenretinide on vitamin A, carotenoid, and vitamin E homeostasis, which is crucial for the pharmacological utilization of this retinoid.     

The retinol signaling pathway in mouse pluripotent P19 cells

atRA (all-trans-retinoic acid), the active metabolite of retinol (vitamin A), is essential for embryogenesis and maintenance of cellular phenotype in adults. Chemicals that interfere with the metabolism of retinol to atRA, therefore, are a human health concern. During development of a screen for disruptors of this signaling pathway, we investigated whether the mouse pluripotent P19 cell metabolizes retinol to atRA and thus can be used in a cell-based screen for disruptors of the pathway. We found that retinol induced the identical pattern of homeobox gene expression as atRA and its precursor, retinal. Retinol was 160-fold less potent than atRA as an inducer, however. In spite of its lower potency, increased Hoxa1 gene expression was detected 30 min after retinol exposure and increased 40-fold by 2 h. Rdh10 and Aldh1a2/Raldh2, which together convert retinol to atRA in the embryo, were the predominant alcohol and aldehyde dehydrogenases expressed in P19 cells. The cell expressed high mRNA levels of retinol binding proteins, Rbp1 and Rbp4, and the 13,14-dihydroretinol saturase, Retsat. It also expressed all Rar and Rxr isotypes, Crabp1&2, the three Cyp26 genes, and both β-carotene-cleaving genes, Bcmo1 and Bco2. The basal expression levels and retinol responsiveness of 25 pathway-related genes were quantitated by RT-qPCR. A test of the Aldh1a2 inhibitor, citral, showed that the disruption of the pathway was easily detected and quantitated showing that the P19 cell provides an in vitro model system for identifying and exploring the mechanism of action of chemicals that interfere with this critical cellular pathway.     

番石榴实蝇性别决定基因 Bcotra 和 Bcotra-2 的克隆、序列特征及表达分析

【目的】对番石榴实蝇 Bactrocera correcta (Bezzi)性别决定基因 transformer 和 transformer 2 的cDNA和基因组DNA序列进行克隆和分析,明确这2个基因的结构特征及其在不同发育阶段和雌、雄成虫不同组织中的表达模式,为进一步的功能研究和番石榴实蝇遗传性别品系(genetic sexing strain, GSS)的建立奠定基础。【方法】利用PCR结合RACE技术克隆番石榴实蝇2个性别决定基因的cDNA全长和内含子序列,利用不同的生物信息学软件对序列进行结构预测、序列比对和进化树分析;利用半定量RT-PCR检测这2个基因在番石榴实蝇的不同发育阶段及雌、雄成虫不同组织(精巢、卵巢、中肠和脂肪体)中的表达分布。【结果】克隆得到番石榴实蝇 transformer 和 transformer 2 的cDNA全长序列,分别命名为 Bcotra 和 Bcotra-2 。 Bcotra 存在性别特异剪接,雌虫 Bcotra 的cDNA全长1 673 bp,其开放读码框(ORF)为1 242 bp,编码413个氨基酸(GenBank登录号为KP712876);雄虫Bcotra cDNA全长2 025 bp,比雌虫多2个外显子,但由于外显子上有多个终止密码子,因此,不能编码完整的有功能的Tra蛋白(GenBank登录号为KP712877)。 Bcotra-2 不存在性别特异剪接,cDNA全长1 458 bp,其开放读码框(ORF)为756 bp,编码251个氨基酸,具有RNA结合蛋白的典型特征(GenBank登录号为KM658207)。Bcotra-2有8个外显子,7个内含子。氨基酸序列比对和系统进化关系表明,两个基因的系统发育关系一致,Tra-2与目前已报道的双翅目Tra-2具有很高的同源性,而Tra的保守性较Tra-2要低。半定量RT-PCR结果显示, Bcotra 和 Bcotra-2 在番石榴实蝇的不同发育阶段及雌、雄成虫不同组织中都有表达。【结论】本研究明确了Bcotra 和 Bcotra-2 的基因组DNA和cDNA结构特征,Bcotra 和 Bcotra-2 在番石榴实蝇的不同发育阶段和成虫不同组织中均有表达,序列分析发现这两个性别决定基因均具有Tra/Tra-2结合位点、内含子剪接抑制序列位点,其中 Bcotra 具有RNA结合蛋白的结合位点,暗示了这2个基因可能通过翻译后相互作用调控雌、雄体性发育。Bcotra 存在性别特异剪接,雌虫特有的一段963 bp的内含子序列可以用于番石榴实蝇遗传定性品系的载体构建。     

Mechanism of action of inter-alpha-trypsin inhibitor

Inter-alpha-trypsin inhibitor (I alpha I) is a unique proteinase inhibitor that can be proteolyzed by the same enzymes that are inhibited, to generate smaller inhibitors. This study examines the reactions of I alpha I with trypsin, chymotrypsin, plasmin, and leukocyte elastase. Complexes of I alpha I and proteinase were demonstrated by gel filtration chromatography. Complete digestion of I alpha I by each proteinase was not accompanied by a comparable loss of inhibition of that enzyme or a different enzyme. Following proteolysis, inhibitory activity was identified in I alpha I fragments of molecular weight 50,000-100,000 and less than 40,000. Addition of a second proteinase inhibitor prevented proteolysis. Both I alpha I and its complex with proteinase were susceptible to degradation. Kinetic parameters for both the inhibition and proteolysis reactions of I alpha I with four proteinases were measured under physiological conditions. On the basis of these results, a model for the mechanism of action of I alpha I is proposed: Proteinase can react with either of two independent sites on I alpha I to form an inhibitory complex or a complex that leads to proteolysis. Both reactions occur simultaneously, but the inhibitory capacity of I alpha I is not significantly affected by proteolysis since the product of proteolysis is also an inhibitor. For a given proteinase, the inhibition equilibrium constant and the Michaelis constant for proteolysis describe the relative stability of the inhibition and proteolysis complexes; the second-order rate constants for inhibition and proteolysis indicate the likelihood of either reaction. The incidence of inhibition or proteolysis reactions involving I alpha I in vivo cannot be assessed without knowledge of the exact concentrations of inhibitor and proteinases; however, analysis of inhibition rate constants suggests that I alpha I might be involved in plasmin inhibition.     

A revision of Ifloga in southern Africa, with comments on the northern hemisphere species

Ifloga in southern Africa is revised and comments are offered on the northern hemisphere species, I. spicata (together with I. rueppellii ) and I. obovata. Twelve species are recognized in southern Africa, two being described as new. Two natural groups, based on floral characters, are recognized and the rank of subgenus is proposed for these. Ifloga spicata, I. obovata, I. glomerata, I. anomala, I. thellungiana and I. molluginoides constitute subgenus Ifloga; I. verticillata, I. polycnemoides, I. paronychioides, I. Candida, I. ambigua, I. repens, I. pilulifera, I. decumbens , subgenus Trichogyne. A key to the species is provided. The morphology and phytogeography of the genus are discussed.     

进口美国松木中截获齿小蠹的鉴定(鞘翅目:小蠹科)

本文对2010—2015年全国口岸从美国进境松木中截获的齿小蠹进行了分析,分别对美云杉齿小蠹Ips borealis、美雕齿小蠹I.calligraphus、混点齿小蠹I.confusus、重齿小蠹I.duplicatus、南部松齿小蠹I.grandicollis、蒙大拿齿小蠹I.montanus、似混齿小蠹I.paraconfusus、白云杉齿小蠹I.perturbatus、美松齿小蠹I.pini、平额重齿小蠹I.plastographus、十二齿小蠹I.sexdentatus、云杉八齿小蠹I.typographus 12种昆虫的进境寄主进行统计,对截获频次进行分析对其成虫的主要形态特征进行了描述,编制了分种检索表,对检疫鉴定工作具有指导意义。     

国产香茶菜属十三个分类群的细胞学研究

对国产11种2变种共16个居群的香茶菜属植物的染色体数目进行了研究。除线纹香茶菜细花变种以外,其它种类的染色体数目均为首次报道。研究结果表明,有12个物种为二倍体,其染色体数目均为2n=24,推测该属植物的染色体基数为x=12。而细锥香茶菜既有染色体数目为2n=24的居群,也存在2n=48的居群,表明该种为二倍体或四倍体,同时2n=48的染色体数目也是香茶菜属内的首次报道。     

Effect of salt and pH on the activation of photoactive yellow protein and gateway mutants Y98Q and Y98F

We investigated the photocycle of mutants Y98Q and Y98F of the photoactive yellow protein (PYP) from Halorhodospira halophila. Y98 is located in the beta4-beta5 loop and is thought to interact with R52 in the alpha3-alpha4 loop thereby stabilizing this region. Y98 is conserved in all known PYP species, except in Ppr and Ppd where it is replaced by F. We find that replacement of Y98 by F has no significant effect on the photocycle kinetics. However, major changes were observed with the Y98Q mutant. Our results indicate a requirement for an aromatic ring at position 98, especially for recovery and a normal I1/I2 equilibrium. The ring of Y98 could stabilize the beta4-beta5 loop. Alternatively, the Y98 ring could transiently interact with the isomerized chromophore ring, thereby stabilizing the I2 intermediate in the I1/I2 equilibrium. For Y98Q, the decay of the signaling state I2' was slowed by a factor of approximately 40, and the rise of the I2 and I2' intermediates was slowed by a factor of 2-3. Moreover, the I1 intermediate is in a pH-dependent equilibrium with I2/I2' with the ratio of the I1 and I2 populations close to one at pH 7 and 50 mM KCl. From pH 5.5 to 8, the equilibrium shifts toward I1, with a pKa of approximately 6.3. Above pH 8, the populations of I1 and I2/I2' decrease due to an equilibrium between I1 and an additional species I1' which absorbs at approximately 425 nm (pKa approximately 9.8) and which we believe to be an I2-like form with a surface-exposed deprotonated chromophore. The I1/I2/I2' equilibrium was found to be strongly dependent on the KCl concentration, with salt stabilizing the signaling state I2' up to 600 mM KCl. This salt-induced transition to I2' was analyzed and interpreted as ion binding to a specific site. Moreover, from analysis of the amplitude spectra, we conclude that KCl exerts its major effect on the I2 to I2' transition, i.e., the global conformational change leading to the signaling state I2' and the exposure of a hydrophobic surface patch. In wild type and Y98F, the I1/I2 equilibrium is more on the side of I2/I2' as compared to Y98Q but is also salt-dependent at pH 7. The I2 to I2' transition appears to be controlled by an ionic lock, possibly involving the salt bridge between K110 on the beta-scaffold and E12 on the N-terminal cap. Salt binding would break the salt bridge and weaken the interaction between the two domains, facilitating the release of the N-terminal domain from the beta-scaffold in the formation of I2'.     

Contributions of illegitimate and premaritally conceived legitimate births to total fertility

As causes of death, influenza and pneumonia are typically analyzed together. We quantify influenza's contribution to the combined pneumonia and influenza mortality time series for the United States, 1959–2009. A key statistic is I/(P + I), the proportion of pneumonia and influenza mortality accounted for by influenza. Year-to-year, I/(P + I) is highly variable and shows long-term decline. Extreme values of I/(P + I) are associated with extreme P + I death rates and vice versa, but I/(P + I) is a weak predictor of P + I mortality overall. Prominence of influenza in the medical news is associated with high I/(P + I). Influenza and pneumonia should be analyzed as a combined cause.     

Polymorphism of cellulose I family: reinvestigation of cellulose IVI

Polymorphs of cellulose I, III(I), and IV(I) have been investigated by X-ray diffraction, FT-IR, and solid-state (13)C NMR spectroscopy. Highly crystalline cellulose III(I) samples were prepared by treating cellulose samples in supercritical ammonia at 140 degrees C for 1 h, and conventional cellulose III(I) samples were prepared by liquid ammonia treatment. The cellulose IV(I) sample of highest crystallinity was that prepared from Cladophora cellulose III(I) in supercritical ammonia, followed by the sample treated in glycerol at 260 degrees C for 0.5 h, whereas the lowest crystallinity was observed in ramie cellulose prepared by conventional liquid ammonia treatment followed by glycerol annealing. In general, the perfection of cellulose IV(I) depends on the crystallinity of the original material: either of the starting cellulose I or of the cellulose III(I) after ammonia treatment. The product thus obtained was analogous to cellulose I(beta), which is what it should be called rather than cellulose IV(I). If the existence of the polymorph cellulose IV(I) is not accepted, the observations on which it has been based may be explained by the fact that the structure termed cellulose IV(I) is cellulose I(beta) which contains lateral disorder.     

Nucleolar delocalization of human topoisomerase I in response to topotecan correlates with sumoylation of the protein.

DNA topoisomerase (topo) I is an essential nuclear protein and a target for anticancer drug camptothecin derivatives. As a nuclear protein, topo I is concentrated in the nucleolus. However, this nucleolar distribution of topo I is dynamic. It has been shown recently that topo I rapidly moves out of the nucleolus (nucleolar delocalization) in response to topo I inhibitors. In the present study, we demonstrated that nucleolar delocalization of topo I is associated with its conjugation by SUMOs (small ubiquitin-like modifiers) in response to the topo I inhibitor topotecan. Time-course experiments revealed that SUMO-topo I conjugation occurred at as early as 5 min after drug treatment, which was earlier than its observed nucleolar delocalization. Furthermore, heat shock blocked sumoylation of topo I; it also blocked the nucleolar delocalization of topo I fusion proteins. UBC9 is an E2 (ubiquitin carrier protein)-conjugating enzyme essential for sumoylation. Although overexpression of wild-type UBC9 enhanced both sumoylation and nuclear delocalization of topo I, overexpression of a UBC9 dominant negative mutant attenuated topo I sumoylation and its nucleolar delocalization. Taken together, our results suggest that sumoylation of topo I might serve as an addressing tag for its nucleolar delocalization in response to topo I inhibitors.     

Ultrastructural localization of acid phosphatase and thiamine pyrophosphatase activities and of phosphotungstic staining at low pH in the placental labyrinth of the cat]

The localizations of acid phosphatase (ACPase) and thiamine pyrophosphatase (TPPase) activities were studied in the placental labyrinth of the cat during the last days of gestation. ACPase activities were observed essentially in the syncytiotrophoblast and in the "interstitial inert substance" (S.I.I.) that separates maternal from foetal tissues. Reaction product was localized in lysosomes, multivesicular bodies, and in the network of smooth-membraned tubules which open on the cell surface of the syncytiotrophoblast facing the S.I.I. The S.I.I. exhibit a strong activity, probably of syncytiotrophoblastic origin. TPPase activities were found in the syncytiotrophoblast, rarely in the Golgi apparatus but always in the lumen of the network of smooth-membrande tubules. The S.I.I. shows a moderate activity. These TPPase positive tubules being frequently observed very close to the Golgi cisternae, it is proposed that they arise from the Golgi complex and convey phosphatases to the S.I.I. After phosphotungstic acid staining at low pH, luminal surface coat of maternal endothelium is always strongly and continuously visualized, while the plasma membrane of the syncytiotrophoblast facing the S.I.I. is never stained. Staining is intense in the lumen of tubules, and continuous with the stain of the S.I.I. ACPase activity of the S.I.I. could be implicated in enzymatic degradation of maternal molecules during their transfer from maternal to foetal blood. The S.I.I. may correspond to the immunological buffer zone at the interface between maternal and foetal tissues.     

Molecular interactions between poly(ADP-ribose) polymerase (PARP I) and topoisomerase I (Topo I): identification of topology of binding

The molecular interactions of poly(ADP-ribose) polymerase I (PARP I) and topoisomerase I (Topo I) have been determined by the analysis of physical binding of the two proteins and some of their polypeptide components and by the effect of PARP I on the enzymatic catalysis of Topo I. Direct association of Topo I and PARP I as well as the binding of two Topo I polypeptides to PARP I are demonstrated. The effect of PARP I on the 'global' Topo I reaction (scission and religation), and the activation of Topo I by the 36 kDa polypeptide of PARP I and catalytic modifications by poly(ADP-ribosyl)ation are also shown. The covalent binding of Topo I to circular DNA is activated by PARP I similar to the degree of activation of the 'global' Topo I reaction, whereas the religation of DNA is unaffected by PARP I. The geometry of PARP I-Topo I interaction compared to automodified PARP I was reconstructed from direct binding assays between glutathione S-transferase fusion polypeptides of Topo I and PARP I demonstrating highly selective binding, which was correlated with amino acid sequences and with the 'C clamp' model derived from X-ray crystallography.     

Synthesis and intracellular transportation of type I procollagen during functional differentiation of odontoblasts

The expression of type I collagen, the most component of dentin extracellular matrix proteins (ECMs) in odontoblast is correlated with the activity of dentin formation. Since odontoblast possesses a distinct cellular process for protein transport into the dentinal tubule, it is important to examine the intracellular protein localization. However, a study focusing on odontoblast processes has not been performed. Type I collagen is synthesized as procollagen, which is immediately converted to collagen upon secretion. After characterization of antiserum to rat type I procollagen, we investigated the intracellular localization of type I procollagen in odontoblasts during and after dentinogenesis, using immunohistochemistry and in situ hybridization. The level of mRNA expression decreased during dentinogenesis, whereas the intracellular localization of type I procollagen in odontoblast processes become more distinct. The percentage of dentinal tubules with type I procollagen increased significantly with aging. Odontoblasts in pulp horn, in particular, showed moderate expression of type I procollagen after dentinogenesis. Since loss of occlusion also caused a significant decrease in type I procollagen, we concluded that occlusal stimulation activated type I procollagen synthesis in odontoblasts. We also suggest that analysis of intracellular transport of type I procollagen via odontoblast processes may be a new approach to evaluation of odontoblast function.     

DNase-I-dependent dissociation of erythrocyte cytoskeletons

The human erythrocyte contains a complex of peripheral membrane proteins which forms an extensive network or cytoskeleton on the cytoplasmic membrane surface. When I treat erythrocyte cytoskeletons with deoxyribonuclease I (DNase I), the cytoskeletons dissociate and erythrocyte actin is solubilized. The dissociation of the cytoskeletons by DNase I parallels the disruption of actin filaments in vitro by DNase I and is blocked by the addition of action to the DNase I. Large protein complexes remain after DNase I disrupts the cytoskeletons, but these complexes are no longer visible in the light microscope nor sedimentable and are selectively depleted with respect to actin. From these studies, I suggest that DNase I binds to and solubilizes actin, which serves as a structural link between protein complexes in the erythrocyte cytoskeleton.     

Binding and dissociation of human topoisomerase I with hairpin-loop RNAs: implications for the regulation of HIV-1 replication

Cellular topoisomerase I has been reported to be present in retroviral particles and to enhance viral cDNA synthesis; however, the mechanisms involved remain unknown. In the present study, it has been demonstrated that human topoisomerase I combines with a stem-loop RNA and that the bound topoisomerase I can be dissociated from RNA substrates in the presence of ATP. In addition, in vitro cleaved synthetic RNA bound by topoisomerase I is subsequently relegated when the topoisomerase I is dissociated by ATP. A mechanism is proposed in which human topoisomerase I is carried into virions and regulates the repair of genomic RNA by its ligation activity.     

Role of I(K) and I(f) in the pacemaker mechanisms of sino-atrial node myocytes

The role of I(K) (delayed rectifier current) and I(f) (hyperpolarization-activated current) in dominant and subsidiary pacemaker ranges was studied in single myocytes isolated from the guinea pig sino-atrial node by means of a perforated patch-clamp technique. In the dominant pacemaker range (approx. -55 to -40 mV), I(K) tails are present whereas I(f) is not activated. In the subsidiary pacemaker range (approx. -80 to -70 mV), I(f) is large whereas I(K) is minimal and reversing. The threshold for I(f) activation is more negative at short time intervals. Larger or longer depolarizations to -40 mV and +20 mV deactivate I(f) more and are followed by faster reactivation of I(f). Steps of 200-300 ms duration to +20 mV completely deactivate I(f). The slope conductance decreases during depolarizations at -40 and +20 mV and quickly re-increases after the steps. The I(f) deactivation range is between -70 and +10 mV, with a V(1/2) of -35 mV. Depolarizations from -80 to +20 mV at a rate of 120/min limit the subsequent I(f) reactivation owing to the short diastole. We conclude that I(K) plays a predominant role in the dominant pacemaker range and I(f) does so in the subsidiary pacemaker range. Either pacemaker mechanism is used by sino-atrial node cells depending on the diastolic potential range. A previous depolarization markedly increases the amplitude and rate of I(f) reactivation.     

Rapid measurement of deoxyribonuclease I activity with the use of microchip electrophoresis based on DNA degradation

Deoxyribonuclease I (DNase I) activity in serum has been shown to be a novel diagnostic marker for the early detection of acute myocardial infarction (AMI). However, the conventional method to measure DNase I activity is time-consuming. In the current study, to develop a rapid assay method for DNase I activity for clinical purposes, a microchip electrophoresis device was used to measure DNase I activity. Because DNase I is an endonuclease that degrades double-stranded DNA endo-nucleolytically to produce oligonucleotides, degradation of the DNA standard caused by DNase I action was detected using microchip electrophoresis. We detected DNase I activity within 10 min. This is the first study to apply microchip electrophoresis for the detection of DNase I activity; furthermore, it seems plausible that reduction of analysis time for DNase I activity could make this novel assay method using microchip electrophoresis applicable in clinical use.