A synergy of activity,stability, and inhibitor‐interaction of HIV‐1 protease mutants evolved under drug‐pressure

A clinically‐relevant, drug‐resistant mutant of HIV‐1 protease (PR), termed Flap+_(I54V) and containing L10I, G48V, I54V and V82A mutations, is known to produce significant changes in the entropy and enthalpy balance of drug‐PR interactions, compared to wild‐type PR. A similar mutant, Flap+_(I54A), which evolves from Flap+_(I54V) and contains the single change at residue 54 relative to Flap+_(I54V), does not. Yet, how Flap+_(I54A) behaves in solution is not known. To understand the molecular basis of V54A evolution, we compared nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and enzymatic assay data from four PR proteins: PR (pWT), Flap+_(I54V), Flap+_(I54A), and Flap+_(I54), a control mutant that contains only L10I, G48V and V82A mutations. Our data consistently show that selection to the smaller side chain at residue 54, not only decreases inhibitor affinity, but also restores the catalytic activity.     

A Hereditary Spastic Paraplegia Mouse Model Supports a Role of ZFYVE26/SPASTIZIN for the Endolysosomal System

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.     

Therapy of Venezuelan patients with severe mucocutaneous or early lesions of diffuse cutaneous leishmaniasis with a vaccine containing pasteurized Leishmania promastigotes and bacillus Calmette-Guerin: preliminary report

Severe mucocutaneous (MCL) and diffuse (DCL) forms of American cutaneous leishmaniasis (ACL) are infrequent in Venezuela. Chemotherapy produces only transitory remission in DCL, and occasional treatment failures are observed in MCL. We have evaluated therapy with an experimental vaccine in patients with severe leishmaniasis. Four patients with MCL and 3 with early DCL were treated with monthly intradermal injections of a vaccine containing promastigotes of Leishmania (Viannia) braziliensis killed by pasteurization and viable Bacillus Calmette- Guerin. Clinical and immunological responses were evaluated. Integrity of protein constituents in extracts of pasteurized promastigotes was evaluated by gel electrophoresis. Complete remission of lesions occurred after 5-9 injections in patients with MCL or 7-10 injections in patients with early DCL. DCL patients developed positive skin reactions, average size 18.7 mm. All have been free of active lesions for at least 10 months. Adverse effects of the vaccine were limited to local reactivity to BCG at the injection sites and fever in 2 patients. Extracts of pasteurized and fresh promastigotes did not reveal differences in the integrity of protein components detectable by gel electrophoresis. Immunotherapy with this modified vaccine offers an effective, safe option for the treatment of patients who do not respond to immunotherapy with vaccine containing autoclaved parasites or to chemotherapy.     

Two-photon laser-generated microtracks in 3D collagen lattices: principles of MMP-dependent and -independent collective cancer cell invasion

Cancer invasion into an extracellular matrix (ECM) results from a biophysical reciprocal interplay between the expanding cancer lesion and tissue barriers imposed by the adjacent microenvironment. In vivo, connective tissue provides both densely packed ECM barriers adjacent to channel/track-like spaces and loosely organized zones, both of which may impact cancer invasion mode and efficiency; however little is known about how three-dimensional (3D) spaces and aligned tracks present in interstitial tissue guide cell invasion. We here describe a two-photon laser ablation procedure to generate 3D microtracks in dense 3D collagen matrices that support and guide collective cancer cell invasion. Whereas collective invasion of mammary tumor (MMT) breast cancer cells into randomly organized collagen networks required matrix metalloproteinase (MMP) activity for cell-derived collagen breakdown, re-alignment and track generation, preformed tracks supported MMP-independent collective invasion down to a track caliber of 3 μm. Besides contact guidance along the track of least resistance and initial cell deformation (squeezing), MMP-independent collective cell strands led to secondary track expansion by a pushing mechanism. Thus, two-photon laser ablation is useful to generate barrier-free microtracks in a 3D ECM which guide collective invasion independently of pericellular proteolysis.     

Triterpenoids in Gypsophila trichotoma Wend.

A new triterpeniod saponin 3-O-β-arabinopyranosyl-(1 → 3)-[β-galactopyranosyl-(1 → 2)]-β-glucuronopyranosyl gypsogenin (1), together with the known saponin 3-O-β-xylopyranosyl-(1 → 3)-[β-galactopyranosyl-(1 → 2)]-β-glucuronopyranosyl gypsogenin (2), and three known triterpenes gypsogenic acid (3), quillaic acid (4) and gypsogenin (5) were isolated from the roots of Gypsophila trichotoma Wend. (Caryophyllaceae). Their structures were elucidated by chemical and spectral methods. Cytotoxic activity of compounds 1 and 2 were tested against seven human cancer cell lines. Compound 1 showed cytotoxic activity against all of them, while compound 2 only against two cell lines.     

Ku antigen interacts with abasic sites

One of the most abundant lesions in DNA is the abasic (AP) sites arising spontaneously or as an intermediate in base excision repair. Certain proteins participating in the processing of these lesions form a Schiff base with the deoxyribose of the AP site. This intermediate can be stabilized by NaBH(4) treatment. By this method, DNA duplexes with AP sites were used to trap proteins in cell extracts. In HeLa cell extract, along with a prevalent trap product with an apparent molecular mass of 95 kDa, less intensive low-molecular-weight products were observed. The major one was identified as the p80-subunit of Ku antigen (Ku). Ku antigen, a DNA binding component of DNA-dependent protein kinase (DNA-PK), participates in double-stranded break repair and is responsible for the resistance of cells to ionizing radiation. The specificity of Ku interaction with AP sites was proven by more efficient competition of DNA duplexes with an analogue of abasic site than non-AP DNA. Ku80 was cross-linked to AP DNAs with different efficiencies depending on the size and position of strand interruptions opposite to AP sites. Ku antigen as a part of DNA-PK was shown to inhibit AP site cleavage by apurinic/apyrimidinic endonuclease 1.     

Solar UV‐B radiation and ethylene play a key role in modulating effective defenses against Anticarsia gemmatalis larvae in field‐grown soybean

Solar UV‐B radiation has been reported to enhance plant defenses against herbivore insects in many species. However, the mechanism and traits involved in the UV‐B mediated increment of plant resistance are unknown in crops species, such as soybean. Here, we studied defense‐related responses in undamaged and Anticarsia gemmatalis larvae‐damaged leaves of two soybean cultivars grown under attenuated or full solar UV‐B radiation. We determined changes in jasmonates, ethylene (ET), salicylic acid, trypsin protease inhibitor activity, flavonoids, and mRNA expression of genes related with defenses. ET emission induced by Anticarsia gemmatalis damage was synergistically increased in plants grown under solar UV‐B radiation and was positively correlated with malonyl genistin concentration, trypsin proteinase inhibitor activity and expression of IFS2, and the pathogenesis protein PR2, while was negatively correlated with leaf consumption. The precursor of ET, aminocyclopropane‐carboxylic acid, applied exogenously to soybean was sufficient to strongly induce leaf isoflavonoids. Our results showed that in field‐grown soybean isoflavonoids were regulated by both herbivory and solar UV‐B inducible ET, whereas flavonols were regulated by solar UV‐B radiation only and not by herbivory or ET. Our study suggests that, although ET can modulate UV‐B‐mediated priming of inducible plant defenses, some plant defenses, such as isoflavonoids, are regulated by ET alone.     

Interaction of poly(ADP-ribose) polymerase 1 with apurinic/apyrimidinic sites within clustered DNA damage

To study the interaction of poly(ADP-ribose) polymerase 1 (PARP1) with apurinic/apyrimidinic sites (AP sites) within clustered damages, DNA duplexes were created that contained an AP site in one strand and one of its analogs situated opposite the AP site in the complementary strand. Residues of 3-hydroxy-2-hydroxymethyltetrahydrofuran (THF), diethylene glycol (DEG), and decane-1,10-diol (DD) were used. It is shown for the first time that apurinic/apyrimidinic endonuclease 1 (APE1) cleaves the DNA strands at the positions of DEG and DD residues, and this suggests these groups as AP site analogs. Insertion of DEG and DD residues opposite an AP site decreased the rate of AP site hydrolysis by APE1 similarly to the effect of the THF residue, which is a well-known analog of the AP site, and this allowed us to use such AP DNAs to imitate DNA with particular types of clustered damages. PARP1, isolated and in cell extracts, efficiently interacted with AP DNA with analogs of AP sites producing a Schiff base. PARP1 competes with APE1 upon interaction with AP DNAs, decreasing the level of its cross-linking with AP DNA, and inhibits hydrolysis of AP sites within AP DNAs containing DEG and THF residues. Using glutaraldehyde as a linking agent, APE1 is shown to considerably decrease the amount of AP DNA-bound PARP1 dimer, which is the catalytically active form of this enzyme. Autopoly(ADP-ribosyl)ation of PARP1 decreased its inhibitory effect. The possible involvement of PARP1 and its automodification in the regulation of AP site processing within particular clustered damages is discussed.