Isolation and properties of crystalline ferredoxin from lactuca sativa

Lettuce ferredoxin has been purified to homogeneity, with a yield of 18 mg/kg of denerved leaves. It crystallizes in magnificent needles, often clustered in broom-like sheaves. The absorption spectrum showed maxima at 460, 422, 330 and 274 nm,with a ratio A₄₂₂/A₂₇₄, of 0.46. The mM absorption coefficient was 9.74 at 422 nm, and 21.62 at 274 nm. This ferredoxin showed a pI = 4.7 and an E′₀ = ?425 mV (at pH = 7.7). MWs of 12 400, 11480 and 13000 were obtained by sucrose gradient centrifugation, and on the basis of the amino acid composition and the iron content, respectively, with an average of 12 300. The amino acid analysis showed the existence of one methionine residue per mole, with 105 amino acid residues. There are two iron atoms and two labile sulfide groups per mole; 4 half-cystine residues were found by performic acid oxidation, and 5 cysteine groups when determined by titration with pHMB. The native protein is not fixed on thiol-Sepharose 4B, but it is quantitatively retained after incubation with 8 M urea. Lettuce ferredoxin showed a 62, 58 and 78% effectiveness with the spinach ferredoxin-NADP reductase, nitrite reductase and fructose-1,6-diphosphatase (FDPase), respectively, when compared with the spinach ferredoxin. This different behaviour of both ferredoxins is joined to genetic-structural relationships, and suggests that the role of ferredoxin in FDPase activation is more sophisticated than that of a mere nonspecific reductant.     

Effect of papulacandin B on β-glucan synthesis in Schizosaccharomyces pombe

Abstract The antifungal antibiotic papulacandin β inhibited B(1,3)glucan-synthase activity, in vitro, from Schizosaccharomyces pombe . Levels of β(1,3)glucan-synthase from antibiotic-treated cultures were lower than the control cultures whereas mannan-synthase and β(1,3)glucanase activities were almost unaffected. The presence of an osmotic stabilizer reduced the inhibition of growth caused by the antibiotic. Addition of papulacandin β to a culture of S. pombe specifically inhibited incorporation of glucose into the β-glucan cell wall fraction. The fatty acids as well as the hydroxyl groups on the phenol residue of the papulacandin β molecule were essential for the inhibitory activity.     

Dipole moments of random coil polypeptides

The Rotational Isomeric States model is applied to calculate dipole moments of polypeptides of the twenty natural α-amino acids in the random coil state. Dipole moments of each repeat unit (μ_i), are evaluated using a quantum mechanics procedure. Dipole moment ratios ($\text{D}{}_{\mathrm{x}}\text{=}\text{〈μ}{}^2\text{〉}\text{x}\text{μ}{}_{\mathrm{i}}{}^2\text{,}\text{x = number of repeat units}$) of homopolypeptides are calculated and extrapolated to x →?. With a few exceptions, D_? = 0.36 ± 0.1. Ten actual proteins and three enzymes are also studied; their dipole ratios (D_x′ =〈μ〉/x) range from 7.34 to 10.57 in 10^?59 C² m² (6.6–9.5 D²). Diffferences in the values of D_x′ are due mainly to the different contributions, μ_i, of the amino acid residues contained in each polymer, whereas the sequence of amino acids has a very minor effect.     

Mammalian wildlife diseases as hazards to man and livestock in an area of the Llanos Orientales of Colombia

Development of the LLanos Orientales of Colombia, and access to underdeveloped areas in the Llanos, may create disease hazards to man and domestic animals or introduce exotic pathogens, creating reservoirs of infection for domestic animals and acting as limiting factors on the native wild species. A survey of wild animals common to the Llanos revealed a number of parasites indigenous to the area. A total total of 59 mammalian species, representing eight orders were examined. Haematozoa were represented by Trypanosoma cruzi, T. evansi and T. rangeli. Eight species of ticks were found: Amblyomma cajennense, A. auricularium, A. rotundatum, A. maculatum, A. longirostre, A. pacae, Ixodes luciae and Boophilus microplus. Four species of fleas were found: Rhopalopsyllus lugubris lugubris, R. australis tupinus, R. cacicus saevus and Polygenis klagesi samuelis. A species of Echinococcus was commonly found in Cuniculus paca. Serologic titers and/or isolations of pathogenic viral and bacterial agents generally indicated that the wildlife population had not been exposed to the diseases common to the domestic population. A low prevalence of titers to Venezuelan equine encephalomyelitis was found in Cebus apella and Proechimys sp. Neutralizing antibodies to Group B viruses were found in Proechimys sp., Coendor sp. and Nectomys squamipes. Antibodies to Group C viruses were found in Proechimys sp. Serologic titers to Leptospira sejroe and L. tarassovi were found in Proechimys sp. and Didelphis marsupialis. L. tarassovi was isolated from Proechimys sp. Titers to Brucella were not found in 1964 animals. The significance of these findings are discussed.     

The capybara (Hydrochoerus hydrochaeris) as a reservoir host for Trypanosoma evansi

Discovery of two ill horses and three dogs naturally infected with Trypanosoma evansi near an experimental station in the Eastern Plains of Colombia led to a search for reservoir hosts of the parasite. Infection was detected in 8/33 healthy capybaras (Hydrochoerus hydrochaeris), none of the remaining 14 horses, and none of 32 Zebu cattle (Bos indicus), 18 paca (Cuniculus paca) and 20 spiny rats (Proechimys sp.). Contrary to common opinion, the results indicated a carrier state in the capybara. Diagnosis was based on morphology, behaviour in albino rats, and pathogenicity and host range in domestic animals.     

Activity of prostaglandin E, F, A and B on sphincter, dilator and ciliary muscle preparations of the cat eye

Both sphincter and dilator muscle preparations of the cat iris contract to prostaglandins; F_2α and E₂ are the most potent and A₁ and B₁ the least. Ciliary muscle strips relax to PG's provided that the strips are precontracted. E₁, E₂ and often F_2α are more potent relaxants than the remaining PG's. The effects of PG's are not altered by α or β blockade nor by atropine; however, propranolol blocks the PG induced relaxation of the ciliary muscle. The effects of PG's on the sphincter are antagonized by catecholamines; but the latter act synergistically in contracting the dilator and in relaxing the ciliary muscle. Indomethacin markedly potentiates the effects of PG's on all three muscle preparations.     

Conjugation of chemical handles and functional moieties to DNA during solid phase synthesis with sulfonyl azides

Labelling of oligonucleotides with dyes, targeting ligands, and other moieties has become ever more essential in life-sciences. Conventionally, modifications are introduced to oligonucleotides during solid phase synthesis by special phosphoramidites functionalised with a chemical handle or the desired functional group. In this work, we present a facile and inexpensive method to introduce modifications to oligonucleotides without the need for special phosphoramidites. Sulfonyl azides are applied to react with one or more selected phosphite intermediates during solid phase synthesis. We have prepared 11 sulfonyl azides with different chemical handles such as amine, azide, alkyne, and thiol, and we have further introduced functionalities such as pyrene, other dyes, photo-switchable azobenzenes, and a steroid. The method is compatible with current phosphoramidite-based automated oligonucleotide synthesis and serves as a simple alternative to the unstable and expensive special phosphoramidites currently used for conjugation to oligonucleotides.     

A mouse model for Li-Fraumeni-Like Syndrome with cardiac angiosarcomas associated to POT1 mutations

The shelterin protein POT1 has been found mutated in many different familial and sporadic cancers, however, no mouse models to understand the pathobiology of these mutations have been developed so far. To address the molecular mechanisms underlying the tumorigenic effects of POT1 mutant proteins in humans, we have generated a mouse model for the human POT1^R117C mutation found in Li-Fraumeni-Like families with cases of cardiac angiosarcoma by introducing this mutation in the Pot1a endogenous locus, knock-in for Pot1a^R117C. We find here that both mouse embryonic fibroblasts (MEFs) and tissues from Pot1a^+/ki mice show longer telomeres than wild-type controls. Longer telomeres in Pot1a^+/ki MEFs are dependent on telomerase activity as they are not found in double mutant Pot1a^+/ki Tert^-/- telomerase-deficient MEFs. By using complementation assays we further show that POT1a pR117C exerts dominant-negative effects at telomeres. As in human Li-Fraumeni patients, heterozygous Pot1a^+/ki mice spontaneously develop a high incidence of angiosarcomas, including cardiac angiosarcomas, and this is associated to the presence of abnormally long telomeres in endothelial cells as well as in the tumors. The Pot1a^+/R117C mouse model constitutes a useful tool to understand human cancers initiated by POT1 mutations.     

TBX2 acts as a potent transcriptional silencer of tumour suppressor genes through interaction with the CoREST complex to sustain the proliferation of breast cancers

Chromosome 17q23 amplification occurs in 20% of primary breast tumours and is associated with poor outcome. The TBX2 gene is located on 17q23 and is often over-expressed in this breast tumour subset. TBX2 is an anti-senescence gene, promoting cell growth and survival through repression of Tumour Suppressor Genes (TSGs), such as NDRG1 and CST6. Previously we found that TBX2 cooperates with the PRC2 complex to repress several TSGs, and that PRC2 inhibition restored NDRG1 expression to impede cellular proliferation. Here, we now identify CoREST proteins, LSD1 and ZNF217, as novel interactors of TBX2. Genetic or pharmacological targeting of CoREST emulated TBX2 loss, inducing NDRG1 expression and abolishing breast cancer growth in vitro and in vivo. Furthermore, we uncover that TBX2/CoREST targeting of NDRG1 is achieved by recruitment of TBX2 to the NDRG1 promoter by Sp1, the abolishment of which resulted in NDRG1 upregulation and diminished cancer cell proliferation. Through ChIP-seq we reveal that 30% of TBX2-bound promoters are shared with ZNF217 and identify novel targets repressed by TBX2/CoREST; of these targets a lncRNA, LINC00111, behaves as a negative regulator of cell proliferation. Overall, these data indicate that inhibition of CoREST proteins represents a promising therapeutic intervention for TBX2-addicted breast tumours.     

Poliovirus induces autophagic signaling independent of the ULK1 complex

Poliovirus (PV), like many positive-strand RNA viruses, subverts the macroautophagy/autophagy pathway to promote its own replication. Here, we investigate whether the virus uses the canonical autophagic signaling complex, consisting of the ULK1/2 kinases, ATG13, RB1CC1, and ATG101, to activate autophagy. We find that the virus sends autophagic signals independent of the ULK1 complex, and that the members of the autophagic complex are not required for normal levels of viral replication. We also show that the SQSTM1/p62 receptor protein is not degraded in a conventional manner during infection, but is likely cleaved in a manner similar to that shown for coxsackievirus B3. This means that SQSTM1, normally used to monitor autophagic degradation, cannot be used to accurately monitor degradation during poliovirus infection. In fact, autophagic degradation may be affected by the loss of SQSTM1 at the same time as autophagic signals are being sent. Finally, we demonstrate that ULK1 and ULK2 protein levels are greatly reduced during PV infection, and ATG13, RB1CC1, and ATG101 protein levels are reduced as well. Surprisingly, autophagic signaling appears to increase as ULK1 levels decrease. Overexpression of wild-type or dominant-negative ULK1 constructs does not affect virus replication, indicating that ULK1 degradation may be a side effect of the ULK1-independent signaling mechanism used by PV, inducing complex instability. This demonstration of ULK1-independent autophagic signaling is novel and leads to a model by which the virus is signaling to generate autophagosomes downstream of ULK1, while at the same time, cleaving cargo receptors, which may affect cargo loading and autophagic degradative flux. Our data suggest that PV has a finely-tuned relationship with the autophagic machinery, generating autophagosomes without using the primary autophagy signaling pathway.

Abbreviations: ACTB - actin beta; ATG13 - autophagy related 13; ATG14 - autophagy related 14; ATG101 - autophagy related 101; BECN1 - beclin 1; CVB3 - coxsackievirus B3; DMV - double-membraned vesicles; EM - electron microscopy; EMCV - encephalomyocarditis virus; EV-71 - enterovirus 71; FMDV - foot and mouth disease virus; GFP - green fluorescent protein; MAP1LC3B/LC3B - microtubule associated protein 1 light chain 3 beta; MOI - multiplicity of infection; MTOR - mechanistic target of rapamycin kinase; PIK3C3 - phosphatidylinositol 3-kinase catalytic subunit type 3; PRKAA2 - protein kinase AMP-activated catalytic subunit alpha 2; PSMG1 - proteasome assembly chaperone 1; PSMG2 - proteasome assembly chaperone 2PV - poliovirus; RB1CC1 - RB1 inducible coiled-coil 1; SQSTM1 - sequestosome 1; ULK1 - unc-51 like autophagy activating kinase 1; ULK2 - unc-51 like autophagy activating kinase 2; WIPI1 - WD repeat domain, phosphoinositide interacting 1