Site-directed mutagenesis shows that tyrosine 248 of carboxypeptidase A does not play a crucial role in catalysis

The residue Tyr 248 of carboxypeptidase A (CPA) is thought to play a role in catalysis by contributing a proton to the incipient amine anion generated during cleavage of peptide substrates. To test this hypothesis we have modified the rat CPA cDNA by site-directed mutagenesis so that the codon for Tyr 248 is replaced by that for Phe. Here, we report the expression of the cDNAs for proCPA and its Tyr-to-Phe variant in yeast via the alpha-factor system. Following zymogen activation by trypsin, wild-type CPA (CPA-WT) and variant CPA (CPA-Phe 248) were purified to homogeneity and characterized enzymatically. CPA-Phe 248 displays essentially undiminished values for the catalytic constant (kcat) towards various peptide and ester substrates. However, the Michaelis constants (Km values) of peptide substrates and the inhibition constant (Ki) of the potato carboxypeptidase inhibitor are increased 6-fold and 70-fold, respectively. These data suggest that the phenolic hydroxyl of Tyr 248 does not act as the requisite general acid catalyst but participates in ligand binding.     

Site-directed mutagenesis reveals new and essential elements for iron-coordination of the sulfur oxygenase reductase from the acidothermophilic Acidianus tengchongensis

Previous study on refolding of sulfur oxygenase reductase (SOR) inclusion bodies from recombinant Escherichia coli showed that iron was critical to the activity of the SOR from Acidianus ambivalens. In this study, enzymatic assays showed that 2,2′-Dipyridyl, Tiron and 8-hydroxyquinoline, which are specific for chelating ferrous or ferric ions, strongly inhibited the activity of SOR from A. tengchongensis, suggesting that iron atom is essential for SOR activity. Alignment of several functionally identified S...     

Angiotensin-converting enzyme 2 is an essential regulator of heart function

Cardiovascular diseases are predicted to be the most common cause of death worldwide by 2020. Here we show that angiotensin-converting enzyme 2 (ace2) maps to a defined quantitative trait locus (QTL) on the X chromosome in three different rat models of hypertension. In all hypertensive rat strains, ACE2 messenger RNA and protein expression were markedly reduced, suggesting that ace2 is a candidate gene for this QTL. Targeted disruption of ACE2 in mice results in a severe cardiac contractility defect, increased angiotensin II levels, and upregulation of hypoxia-induced genes in the heart. Genetic ablation of ACE on an ACE2 mutant background completely rescues the cardiac phenotype. But disruption of ACER, a Drosophila ACE2 homologue, results in a severe defect of heart morphogenesis. These genetic data for ACE2 show that it is an essential regulator of heart function in vivo.     

ICOS co-stimulatory receptor is essential for T-cell activation and function

T-lymphocyte activation and immune function are regulated by co-stimulatory molecules. CD28, a receptor for B7 gene products, has a chief role in initiating T-cell immune responses. CTLA4, which binds B7 with a higher affinity, is induced after T-cell activation and is involved in downregulating T-cell responses. The inducible co-stimulatory molecule (ICOS), a third member of the CD28/CTLA4 family, is expressed on activated T cells. Its ligand B7H/B7RP-1 is expressed on B cells and in non-immune tissues after injection of lipopolysaccharide into animals. To understand the role of ICOS in T-cell activation and function, we generated and analysed ICOS-deficient mice. Here we show that T-cell activation and proliferation are defective in the absence of ICOS. In addition, ICOS -/- T cells fail to produce interleukin-4 when differentiated in vitro or when primed in vivo. ICOS is required for humoral immune responses after immunization with several antigens. ICOS-/- mice showed greatly enhanced susceptibility to experimental autoimmune encephalomyelitis, indicating that ICOS has a protective role in inflammatory autoimmune diseases.     

Extra-embryonic function of Rb is essential for embryonic development and viability

The retinoblastoma (Rb) gene was the first tumour suppressor identified. Inactivation of Rb in mice results in unscheduled cell proliferation, apoptosis and widespread developmental defects, leading to embryonic death by day 14.5 (refs 2-4). However, the actual cause of the embryonic lethality has not been fully investigated. Here we show that loss of Rb leads to excessive proliferation of trophoblast cells and a severe disruption of the normal labyrinth architecture in the placenta. This is accompanied by a decrease in vascularization and a reduction in placental transport function. We used two complementary techniques-tetraploid aggregation and conditional knockout strategies-to demonstrate that Rb-deficient embryos supplied with a wild-type placenta can be carried to term, but die soon after birth. Most of the neurological and erythroid abnormalities thought to be responsible for the embryonic lethality of Rb-null animals were virtually absent in rescued Rb-null pups. These findings identify and define a key function of Rb in extra-embryonic cell lineages that is required for embryonic development and viability, and provide a mechanism for the cell autonomous versus non-cell autonomous roles of Rb in development.     

Kranz anatomy is not essential for terrestrial C4 plant photosynthesis.

An important adaptation to CO2-limited photosynthesis in cyanobacteria, algae and some plants was development of CO2-concentrating mechanisms (CCM). Evolution of a CCM occurred many times in flowering plants, beginning at least 15-20 million years ago, in response to atmospheric CO2 reduction, climate change, geological trends, and evolutionary diversification of species. In plants, this is achieved through a biochemical inorganic carbon pump called C4 photosynthesis, discovered 35 years ago. C4 photosynthesis is advantageous when limitations on carbon acquisition are imposed by high temperature, drought and saline conditions. It has been thought that a specialized leaf anatomy, composed of two, distinctive photosynthetic cell types (Kranz anatomy), is required for C4 photosynthesis. We provide evidence that C4 photosynthesis can function within a single photosynthetic cell in terrestrial plants. Borszczowia aralocaspica (Chenopodiaceae) has the photosynthetic features of C4 plants, yet lacks Kranz anatomy. This species accomplishes C4 photosynthesis through spatial compartmentation of photosynthetic enzymes, and by separation of two types of chloroplasts and other organelles in distinct positions within the chlorenchyma cell cytoplasm.     

Genetic ablation reveals that the roof plate is essential for dorsal interneuron specification

During neural development in vertebrates, a spatially ordered array of neurons is generated in response to inductive signals derived from localized organizing centres. One organizing centre that has been proposed to have a role in the control of neural patterning is the roof plate. To define the contribution of signals derived from the roof plate to the specification of neuronal cell types in the dorsal neural tube, we devised a genetic strategy to ablate the roof plate selectively in mouse embryos. Embryos without a roof plate lack all the interneuron subtypes that are normally generated in the dorsal third of the neural tube. Using a genetically based lineage analysis and in vitro assays, we show that the loss of these neurons results from the elimination of non-autonomous signals provided by the roof plate. These results reveal that the roof plate is essential for specifying multiple classes of neurons in the mammalian central nervous system.     

The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore

A sudden increase in permeability of the inner mitochondrial membrane, the so-called mitochondrial permeability transition, is a common feature of apoptosis and is mediated by the mitochondrial permeability transition pore (mtPTP). It is thought that the mtPTP is a protein complex formed by the voltage-dependent anion channel, members of the pro- and anti-apoptotic BAX-BCL2 protein family, cyclophilin D, and the adenine nucleotide (ADP/ATP) translocators (ANTs). The latter exchange mitochondrial ATP for cytosolic ADP and have been implicated in cell death. To investigate the role of the ANTs in the mtPTP, we genetically inactivated the two isoforms of ANT in mouse liver and analysed mtPTP activation in isolated mitochondria and the induction of cell death in hepatocytes. Mitochondria lacking ANT could still be induced to undergo permeability transition, resulting in release of cytochrome c. However, more Ca2+ than usual was required to activate the mtPTP, and the pore could no longer be regulated by ANT ligands. Moreover, hepatocytes without ANT remained competent to respond to various initiators of cell death. Therefore, ANTs are non-essential structural components of the mtPTP, although they do contribute to its regulation.     

仿射变换中的等积问题

探讨了空间仿射变换中平面图形经变换后面积不变的问题,并得到了求全部此类平面的方程式,进一步充实了空间仿射变换的理论。     

Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules

The four-way junction between DNA helices is the central intermediate in recombination, and the manner of its interaction with resolvase enzymes can determine the genetic outcome of the process. A knowledge of its structure is a prerequisite to understanding the interaction with proteins, and there has been recent progress. Here we use fluorescence energy transfer to determine the relative distances between the ends of a small DNA junction, and hence the path of the strands. Our results are consistent with the geometry of an 'X'. The interconnected helices are juxtaposed so that the continuous strands of each helix generate an antiparallel alignment, and the two interchanged strands do not cross at the centre. The acute angle of the X structure is defined by a right-handed rotation of the helical axes about the axis perpendicular to the X plane, as viewed from the centre of the X.     

Coenzyme Q is an obligatory cofactor for uncoupling protein function

Uncoupling proteins (UCPs) are thought to be intricately controlled uncouplers that are responsible for the futile dissipation of mitochondrial chemiosmotic gradients, producing heat rather than ATP. They occur in many animal and plant cells and form a subfamily of the mitochondrial carrier family. Physiological uncoupling of oxidative phosphorylation must be strongly regulated to avoid deterioration of the energy supply and cell death, which is caused by toxic uncouplers. However, an H+ transporting uncoupling function is well established only for UCP1 from brown adipose tissue, and the regulation of UCP1 by fatty acids, nucleotides and pH remains controversial. The failure of UCP1 expressed in Escherichia coli inclusion bodies to carry out fatty-acid-dependent H+ transport activity inclusion bodies made us seek a native UCP cofactor. Here we report the identification of coenzyme Q (ubiquinone) as such a cofactor. On addition of CoQ10 to reconstituted UCP1 from inclusion bodies, fatty-acid-dependent H+ transport reached the same rate as with native UCP1. The H+ transport was highly sensitive to purine nucleotides, and activated only by oxidized but not reduced CoQ. H+ transport of native UCP1 correlated with the endogenous CoQ content.     

带有随机干扰的经典风险过程下的破产时罚金折现期望

当风险模型为带有随机干扰的经典风险过程时,破产时罚金折现期望函数Φ(u,ω)及其分解表达式Φd(u)和Φs(u,ω)的积分表达被得到,并且它们的二次连续可微性也得到证明．所有这些都为Gerber and Landry(1998)和Tsai and Willmot(2002)中结论的前提假定提供了可靠的保证,同时,关于破产时赤字的分布及破产概率的一些结果也被得到．     

Orai1 is an essential pore subunit of the CRAC channel

Stimulation of immune cells causes depletion of Ca2+ from endoplasmic reticulum (ER) stores, thereby triggering sustained Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels, an essential signal for lymphocyte activation and proliferation. Recent evidence indicates that activation of CRAC current is initiated by STIM proteins, which sense ER Ca2+ levels through an EF-hand located in the ER lumen and relocalize upon store depletion into puncta closely associated with the plasma membrane. We and others recently identified Drosophila Orai and human Orai1 (also called TMEM142A) as critical components of store-operated Ca2+ entry downstream of STIM. Combined overexpression of Orai and Stim in Drosophila cells, or Orai1 and STIM1 in mammalian cells, leads to a marked increase in CRAC current. However, these experiments did not establish whether Orai is an essential intracellular link between STIM and the CRAC channel, an accessory protein in the plasma membrane, or an actual pore subunit. Here we show that Orai1 is a plasma membrane protein, and that CRAC channel function is sensitive to mutation of two conserved acidic residues in the transmembrane segments. E106D and E190Q substitutions in transmembrane helices 1 and 3, respectively, diminish Ca2+ influx, increase current carried by monovalent cations, and render the channel permeable to Cs+. These changes in ion selectivity provide strong evidence that Orai1 is a pore subunit of the CRAC channel.     

Fibulin-5 is an elastin-binding protein essential for elastic fibre development in vivo.

Extracellular elastic fibres provide mechanical elasticity to tissues and contribute towards the processes of organ remodelling by affecting cell-cell signalling. The formation of elastic fibres requires the assembly and crosslinking of tropoelastin monomers, and organization of the resulting insoluble elastin matrix into functional fibres. The molecules and mechanisms involved in this process are unknown. Fibulin-5 (also known as EVEC/DANCE) is an extracellular matrix protein abundantly expressed in great vessels and cardiac valves during embryogenesis, and in many adult tissues including the aorta, lung, uterus and skin, all of which contain abundant elastic fibres. Here we show that fibulin-5 is a calcium-dependent, elastin-binding protein that localizes to the surface of elastic fibres in vivo. fibulin-5-/- mice develop marked elastinopathy owing to the disorganization of elastic fibres, with resulting loose skin, vascular abnormalities and emphysematous lung. This phenotype, which resembles the cutis laxa syndrome in humans, reveals a critical function for fibulin-5 as a scaffold protein that organizes and links elastic fibres to cells. This function may be mediated by the RGD motif in fibulin-5, which binds to cell surface integrins, and the Ca2+-binding epidermal growth factor (EGF) repeats, which bind elastin.