Cysteine-to-Serine Mutants Dramatically Reorder the Active Site of Human ABO(H) Blood Group B Glycosyltransferase without Affecting Activity: Structural Insights into Cooperative Substrate Binding

A common feature in the structures of GT-A-fold-type glycosyltransferases is a mobile polypeptide loop that has been observed to participate in substrate recognition and enclose the active site upon substrate binding. This is the case for the human ABO(H) blood group B glycosyltransferase GTB, where amino acid residues 177-195 display significantly higher levels of disorder in the unliganded state than in the fully liganded state. Structural studies of mutant enzymes GTB/C80S/C196S and GTB/C80S/C196S/C209S at resolutions ranging from 1.93 to 1.40 Å display the opposite trend, where the unliganded structures show nearly complete ordering of the mobile loop residues that is lost upon substrate binding. In the liganded states of the mutant structures, while the UDP moiety of the donor molecule is observed to bind in the expected location, the galactose moiety is observed to bind in a conformation significantly different from that observed for the wild-type chimeric structures. Although this would be expected to impede catalytic turnover, the kinetics of the transfer reaction are largely unaffected. These structures demonstrate that the enzymes bind the donor in a conformation more similar to the dominant solution rotamer and facilitate its gyration into the catalytically competent form. Further, by preventing active-site closure, these structures provide a basis for recently observed cooperativity in substrate binding. Finally, the mutation of C80S introduces a fully occupied UDP binding site at the enzyme dimer interface that is observed to be dependent on the binding of H antigen acceptor analog.     

Dimebon attenuates methamphetamine, but not MPTP, striatal dopamine depletion

Dimebon is an anti-histamine with central nervous system activity. In this report the effects of dimebon as a neuroprotectant in animal models of Parkinson's disease were tested as assessed in methamphetamine- and MPTP-induced striatal dopaminergic toxicity. Dimebon (1mg/kg) administered at 30 min prior to methamphetamine (40mg/kg) significantly reduced the amount of striatal dopamine depletion in mice, without altering the initial methamphetamine-induced increase in body temperature. In contrast, dimebon at either 1 or 25mg/kg administered at 30 min prior to MPTP (35 mg/kg) was unable to prevent MPTP-induced striatal dopamine loss as determined at 7 days post-methamphetamine/MPTP. These data suggest that dimebon may be exerting a neurotoxin specific neuroprotective effect upon the striatal dopaminergic system and may serve as an important tool for discriminating the mechanistic basis of these two dopaminergic neurotoxins.     

Hyperglycemic responses to cold shock in the freshwater giant prawn, Macrobrachium rosenbergii

Crustacean hyperglycemic hormone (CHH), a neurohormone synthesized and released from the x-organ sinus gland complex, is primarily involved in carbohydrate metabolism; biogenic amines and peptidergic neuroregulators are known to modulate the release of CHH. Marked elevations of hemolymph glucose titers, which peaked within 2 h, were observed in both intact and bilaterally eyestalk-ablated prawns, Macrobrachium rosenbergii, when they were transferred directly from their optimal temperature of 28 °C to lower temperatures close to their lethal limit. Hyperglycemia can therefore be considered a characteristic response in this species under cold shock. Involvement of biogenic amines in the hyperglycemic response was also demonstrated. Hyperglycemic effects of epinephrine, dopamine and serotonin were mediated through CHH at the eyestalk level, but the response under cold shock was not exclusively mediated through CHH. It is suggested that factor(s) other than CHH are involved in the hyperglycemic response, possibly norepinephrine or/and octopamine. Accepted: 24 October 1998     

多巴胺通过促进RNF20降解抑制神经细胞中组蛋白H2B泛素化

表观遗传修饰参与了药物成瘾的形成过程,而在药物成瘾过程中组蛋白泛素化水平的变化仍未可知。药物成瘾过程中常表现为多巴胺(dopamine, DA)表达量的升高,因此本研究欲探讨多巴胺升高对神经细胞组蛋白泛素化的影响及其机制。Western印迹结果显示,在终浓度0.8 mmol/L的多巴胺作用8 h后,人神经母细胞瘤细胞系SH-SY5Y细胞中环指蛋白20(ring finger protein 20, RNF20)表达量降低(0.29±0.032 vs. 1.0±0.025,P<0.0001),泛素化组蛋白H2B(H2Bub1)表达量下降(0.28±0.032 vs. 1.0±0.017,P<0.0001)。但是RT-PCR结果显示,多巴胺处理SH-SY5Y细胞后,RNF20在mRNA水平的表达无明显变化。在SH-SY5Y细胞中沉默RNF20的表达,H2Bub1在蛋白质水平的表达明显降低(0.20±0.069 vs. 1.0±0.060,P=0.001)。在加入多巴胺的基础上,分别加入蛋白酶体抑制剂MG132、自噬体形成抑制剂3-MA以及空泡型H^+-ATP酶特异性抑制剂Baf-A1等药物来检测RNF20的降解途径,结果发现,加入MG132、3-MA以及Baf-A1后,RNF20表达量均比DA处理组显著上升(1.51±0.095,P=0.0003; 0.89±0.075,P=0.0021; 2.74±0.099,P<0.0001;vs. 0.27±0.044)。上述结果表明,在SH-SY5Y细胞中,RNF20对H2Bub1具有调控作用,多巴胺可通过泛素化及自噬两种途径促进RNF20降解,从而抑制组蛋白H2B泛素化。     

Cyanobacterial bioactive metabolites—A review of their chemistry and biology

Cyanobacterial blooms occur when algal densities exceed baseline population concentrations. Cyanobacteria can produce a large number of secondary metabolites. Odorous metabolites affect the smell and flavor of aquatic animals, whereas bioactive metabolites cause a range of lethal and sub-lethal effects in plants, invertebrates, and vertebrates, including humans. Herein, the bioactivity, chemistry, origin, and biosynthesis of these cyanobacterial secondary metabolites were reviewed. With recent revision of cyanobacterial taxonomy by Anagnostidis and Komárek as part of the Süβwasserflora von Mitteleuropa volumes 19(1–3), names of many cyanobacteria that produce bioactive compounds have changed, thereby confusing readers. The original and new nomenclature are included in this review to clarify the origins of cyanobacterial bioactive compounds.Due to structural similarity, the 157 known bioactive classes produced by cyanobacteria have been condensed to 55 classes. This review will provide a basis for more formal procedures to adopt a logical naming system. This review is needed for efficient management of water resources to understand, identify, and manage cyanobacterial harmful algal bloom impacts.     

Induced plasmon mutations affecting the growth habit of peanuts, A. hypogaea L.

The effectiveness of the acridines ethidium bromide (EB) and acriflavine in inducing plasmon mutations was compared with the alkylating agents ethyl methanesulphonate (EMS) and diethyl sulphate and to γ-rays. The growth habit (trailing versus bunch) of peanuts (A. hypogaea), controlled by geniccytoplasmic interactions, was utilized. Breeding tests distinguishing nuclear from plasmon mutations were developed and are described in detail. Plasmon mutations were induced, but there were differences in mutation yields between the cultivars and the mutagens.In the trailing line, TBR[V4], 135 independent bunch mutations (in 1804 M₂ families) were recovered: 28 bred true while 97 continued to segregate into M₃ and M₄. Of the 28, 14 were nuclear from an Hb to an hb allele while 14 were in the plasmon. Of the latter, 6 were induced by EMS, 7 by γ-rays and 1 by acriflavine. Somatic segregation of heteroplasmons, i.e. more plasmon mutations, could be responsible for many of the mutations that continued to segregate, but in some cases chromosomal aberrations might be involved.In the bunch cultivars there were 32 independent trailing mutations (in 3895 M₂ families), one bred true for trailing, while the others continued to segregate into M₃ and M₄. Plasmon mutations could not be ascertained because of the continuing segregations, but these mutations manifested sorting out of heteroplasmons.     

Endogenous reductions in N-methyl-d-aspartate receptor activity inhibit nitric oxide production in the anoxic freshwater turtle cortex

Increased nitric oxide (NO) production from hypoxic mammalian neurons increases cerebral blood flow (CBF) but also glutamatergic excitotoxicity and DNA fragmentation. Anoxia-tolerant freshwater turtles have evolved NO-independent mechanisms to increase CBF; however, the mechanism(s) of NO regulation are not understood. In turtle cortex, anoxia or NMDAR blockade depressed NO production by 27+/-3% and 41+/-5%, respectively. NMDAR antagonists also reduced the subsequent anoxic decrease in NO by 74+/-6%, suggesting the majority of the anoxic decrease is due to endogenous suppression of NMDAR activity. Prevention of NO-mediated damage during the transition to and from anoxia may be incidental to natural reductions of NMDAR activity in the anoxic turtle cortex.     

Effects of amphetamine on dopamine release in the rat nucleus accumbens shell region depend on cannabinoid CB1 receptor activation

The psychostimulant drug amphetamine is often prescribed to treat Attention-Deficit/Hyperactivity Disorder. The behavioral effects of the psychostimulant drug amphetamine depend on its ability to increase monoamine neurotransmission in brain regions such as the nucleus accumbens (NAC) and medial prefrontal cortex (mPFC). Recent behavioral data suggest that the endocannabinoid system also plays a role in this respect. Here we investigated the role of cannabinoid CB1 receptor activity in amphetamine-induced monoamine release in the NAC and/or mPFC of rats using in vivo microdialysis. Results show that systemic administration of a low, clinically relevant dose of amphetamine (0.5mg/kg) robustly increased dopamine and norepinephrine release (to ～175-350% of baseline values) in the NAC shell and core subregions as well as the ventral and dorsal parts of the mPFC, while moderately enhancing extracellular serotonin levels (to ～135% of baseline value) in the NAC core only. Although systemic administration of the CB1 receptor antagonist SR141716A (0-3mg/kg) alone did not affect monoamine release, it dose-dependently abolished amphetamine-induced dopamine release specifically in the NAC shell. SR141716A did not affect amphetamine-induced norepinephrine or serotonin release in any of the brain regions investigated. Thus, the effects of acute CB1 receptor blockade on amphetamine-induced monoamine transmission were restricted to dopamine, and more specifically to mesolimbic dopamine projections into the NAC shell. This brain region- and monoamine-selective role of CB1 receptors is suggested to subserve the behavioral effects of amphetamine.     

Quantification of the elevated rate of domain rearrangements in metazoa

Most eukaryotic proteins consist of multiple domains created through gene fusions or internal duplications. The most frequent change of a domain architecture (DA) is insertion or deletion of a domain at the N or C terminus. Still, the mechanisms underlying the evolution of multidomain proteins are not very well studied.Here, we have studied the evolution of multidomain architectures (MDA), guided by evolutionary information in the form of a phylogenetic tree. Our results show that Pfam domain families and MDAs have been created with comparable rates (0.1-1 per million years (My)). The major changes in DA evolution have occurred in the process of multicellularization and within the metazoan lineage. In contrast, creation of domains seems to have been frequent already in the early evolution. Furthermore, most of the architectures have been created from older domains or architectures, whereas novel domains are mainly found in single-domain proteins. However, a particular group of exon-bordering domains may have contributed to the rapid evolution of novel multidomain proteins in metazoan organisms. Finally, MDAs have evolved predominantly through insertions of domains, whereas domain deletions are less common.In conclusion, the rate of creation of multidomain proteins has accelerated in the metazoan lineage, which may partly be explained by the frequent insertion of exon-bordering domains into new architectures. However, our results indicate that other factors have contributed as well.     

c-kit delineates a distinct domain of progenitors in the developing kidney

Early inductive events in mammalian nephrogenesis depend on an interaction between the ureteric bud and the metanephric mesenchyme. However, mounting evidence points towards an involvement of additional cell types--such as stromal cells and angioblasts--in growth and patterning of the nephron. In this study, through analysis of the stem cell factor (SCF)/c-kit ligand receptor pair, we describe an additional distinct cell population in the early developing kidney. While SCF is restricted to the ureteric bud, c-kit-positive cells are located within the renal interstitium, but are negative for Foxd1, an established marker of stromal cells. In fact, the c-kit-positive domain is continuous with a central mesodermal cell mass ventral and lateral to the dorsal aorta, while Foxd1-expressing stromal cells are continuous with a dorsal perisomitic cell population suggesting distinct intraembryonic origins for these cell types. A subset of c-kit-positive cells expresses Flk-1 and podocalyxin, suggesting that this cell population includes angioblasts and their progenitors. c-kit activation is not required for the survival of these cells in vivo, because white spotting (c-kit(W/W)) mice, carrying a natural inactivating mutation of c-kit, display normal intrarenal distribution of the c-kit-positive cells at E13.5. In addition, early kidney development in these mutants is preserved up to the stage when anemia compromises global embryonic development. In contrast, under defined conditions in organ cultures of metanephric kidneys, c-kit-positive cells, including the Flk-1-positive subset, undergo apoptosis after treatment with STI-571, an inhibitor of c-kit tyrosine phosphorylation. This is associated with reductions in ureteric bud branching and nephron number. Conversely, exogenous SCF expands the c-kit-positive population, including Flk-1-positive angioblasts, and accelerates kidney development in vitro. These data suggest that ureteric bud-derived SCF elicits growth-promoting effects in the metanephric kidney by expanding one or more components of the interstitial c-kit-positive progenitor pool.