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Conserved Oligomeric Golgi (COG) complex controls Golgi trafficking and glycosylation, but the precise COG mechanism is unknown. The auxin-inducible acute degradation system was employed to investigate initial defects resulting from COG dysfunction. We found that acute COG inactivation caused a massive accumulation of COG-dependent (CCD) vesicles that carry the bulk of Golgi enzymes and resident proteins. v-SNAREs (GS15, GS28) and v-tethers (giantin, golgin84, and TMF1) were relocalized into CCD vesicles, while t-SNAREs (STX5, YKT6), t-tethers (GM130, p115), and most of Rab proteins remained Golgi-associated. Airyscan microscopy and velocity gradient analysis revealed that different Golgi residents are segregated into different populations of CCD vesicles. Acute COG depletion significantly affected three Golgi-based vesicular coats-COPI, AP1, and GGA, suggesting that COG uniquely orchestrates tethering of multiple types of intra-Golgi CCD vesicles produced by different coat machineries. This study provided the first detailed view of primary cellular defects associated with COG dysfunction in human cells.

Rhodospirillum rubrum L‐asparaginase mutant E149R, V150P, F151T (RrA) down‐regulates telomerase activity due to its ability to inhibit the expression of telomerase catalytic subunit hTERT. The aim of this study was to define the effect of short‐term and long‐term RrA exposure on proliferation of cancer Jurkat cell line and normal human CD4+ T lymphocytes. RrA could inhibit telomerase activity in dose‐ and time‐dependent manner in both Jurkat and normal CD4+ T cells. Continuous RrA exposure of these cells resulted in shortening of telomeres followed by cell cycle inhibition, replicative senescence, and development of apoptosis. Complete death of Jurkat cells was observed at the day 25 of RrA exposure while normal CD4+ T cells died at the day 50 due to the initial longer length of telomeres. Removal of RrA from senescent cells led to a reactivation of hTERT expression, restoration telomerase activity, re‐elongation of telomeres after 48 h of cultivation, and survival of cells. These findings demonstrate that proliferation of cancer and normal telomerase‐positive cells can be limited by continuous telomerase inhibition with RrA. Longer telomeres of normal CD4+ T lymphocytes make such cells more sustainable to RrA exposure that could give them an advantage during anti‐telomerase therapy. These results should facilitate further investigations of RrA as a potent anti‐telomerase therapeutic protein.

De-N-acetylases of β-(1→6)-D-N-acetylglucosamine polymers (PNAG) and β-(1→4)-D-N-acetylglucosamine residues in peptidoglycan are attractive targets for antimicrobial agents. PNAG de-N-acetylases are necessary for biofilm formation in numerous pathogenic bacteria. Peptidoglycan de-N-acetylation facilitates bacterial evasion of innate immune defenses. To target these enzymes, transition-state analogue inhibitors containing a methylphosphonamidate have been synthesized through a direct Staudinger-phosphonite reaction. The inhibitors were tested on purified PgaB, a PNAG de-N-acetylase from Escherichia coli, and PgdA, a peptidoglycan de-N-acetylase from Streptococcus pneumonia. Herein, we describe the most potent inhibitor of peptidoglycan de-N-acetylases reported to date (Ki =80 μM). The minimal inhibition of PgaB observed provides insight into key structural and functional differences in these enzymes that will need to be considered during the development of future inhibitors.

Vesicular tethers and SNAREs are two key protein components that govern docking and fusion of intracellular membrane carriers in eukaryotic cells. The conserved oligomeric Golgi (COG) complex has been specifically implicated in the tethering of retrograde intra-Golgi vesicles. Using yeast two-hybrid and co-immunoprecipitation approaches, we show that the COG6 subunit of the COG complex is capable of interacting with a subset of Golgi SNAREs, namely STX5, STX6, GS27 and SNAP29. Interaction with SNAREs is accomplished via the universal SNARE-binding motif of COG6. Overexpression of COG6, or its depletion from cells, disrupts the integrity of the Golgi complex. Importantly, COG6 protein lacking the SNARE-binding domain is deficient in Golgi binding, and is not capable of inducing Golgi complex fragmentation when overexpressed. These results indicate that COG6-SNARE interactions are important for both COG6 localization and Golgi integrity.

Avian breeding colonies are generally in a continuous state of flux, some parts growing whilst others shrink as individuals move within the colony on the search for better nest-sites. We examined the role of experience in breeding patch choice by individually marked Barnacle Geese Branta leucopsis in a recently established colony in sub-arctic Russia. Individuals failing to reproduce successfully tended to shift nest location further the following season than did successful pairs, and they did so towards the most dense nest aggregations within the colony, where reproductive success was higher. We suggest that individual decisions on nest-site choice shape the spatial dynamics of this colony.

Toxin trafficking studies provide valuable information about endogenous pathways of intracellular transport. Subtilase cytotoxin (SubAB) is transported in a retrograde manner through the endosome to the Golgi and then to the endoplasmic reticulum (ER), where it specifically cleaves the ER chaperone BiP/GRP78 (Binding immunoglobin protein/Glucose-Regulated Protein of 78 kDa). To identify the SubAB Golgi trafficking route, we have used siRNA-mediated silencing and immunofluorescence microscopy in HeLa and Vero cells. Knockdown (KD) of subunits of the conserved oligomeric Golgi (COG) complex significantly delays SubAB cytotoxicity and blocks SubAB trafficking to the cis Golgi. Depletion of Rab6 and beta-COP proteins causes a similar delay in SubAB-mediated GRP78 cleavage and did not augment the trafficking block observed in COG KD cells, indicating that all three Golgi factors operate on the same 'fast' retrograde trafficking pathway. SubAB trafficking is completely blocked in cells deficient in the Golgi SNARE Syntaxin 5 and does not require the activity of endosomal sorting nexins SNX1 and SNX2. Surprisingly, depletion of Golgi tethers p115 and golgin-84 that regulates two previously described coat protein I (COPI) vesicle-mediated pathways did not interfere with SubAB trafficking, indicating that SubAB is exploiting a novel COG/Rab6/COPI-dependent retrograde trafficking pathway.

Reverse transcriptase of human immunodeficiency virus type 1 is a vitalenzyme in the HIV-1 replication cycle and an attractive target of attempts to arrest a primary viral infection. We designed a vector for eukaryotic expression of the 66 kDa subunit of reverse transcriptase under the control of the immediate early cytomegalovirus promoter. Efficient transient expression of the 66 kDa subunit of reverse transcriptase was achieved in a variety of cells. Immunostaining of the transfected cells revealed the cytoplasmatic localization of reverse transcriptase. Reverse transcriptase activity was detected in all transfected cell lines. Injection of this plasmid encoding the 66 kDa subunit of reverse transcriptase into mice resulted in strong reverse transcriptase-specific immune responses indicating that the 66 kDa subunit of reverse transcriptase is expressed in vivo. Sera from DNA-immunized mice inhibited reverse transcription in vitro.

A study was carried out of alkylation, acylation and aminomethylation reactions were studied in the 5-alkyl-4,5-dihydro-1,2,4-triazin-4-one series. Alkylation by alkyl halides proceeds in the presence of sodium methylate at the N(1)and N(4)atoms with the formation of a mixture of alkylated compounds of three types: derivatives of 4,5-dihydrotriazine, unsaturated triazine, and their dimers. Acylation with isocyanates leads to the formation of heterocyclic ureas — derivatives at the N(4)atom. Mono- and bisaminals were obtained by the reaction of triazines with ethoxymethyldiethylamine.

6(E)- and 6(Z)-(3-Ethoxycarbonylpropyl)- and -(3-carboxypropyl)oximes of 16α,17α-cyclohexanopregn-4-ene-3,6,20-trione were synthesized. The reactions of these ester ligands with pentarane-binding proteins of the uterine cytosol and blood serum were studied; the latter exhibits a higher affinity. The preferred binding of the oxime (E)-isomer relative to the (Z)-isomer was noted.

New 16α,17α-cyclohexanopregnanes (pregna-D′6-pentaranes) containing alkyl or heteroatomic substituents in position 19, namely, 19-methylidene-, 19-methyl-, 19-methoxyimino-, and 19-(3-methoxycarbonylpropoxyimino)-16α,17α-cyclohexanopregn-4-ene-3,20-diones were synthesized, and their interactions with the progesterone receptor and pentarane-binding proteins of rat uterus cytosol and rat blood serum was studied. The same substituent in position 19 of the steroid molecule can affect in opposite direction its affinity to these proteins. These results suggest differences in the structures of the ligand-binding pockets of the three proteins in the region surrounding the C(10) atom of the steroid nucleus.

Introduction : The UGT1A1*28 (rs8175347) polymorphism is associated with hyperbilirubinemia. The presence of 6 TA-repeats in the UGT1A1 gene promoter region corresponds to normal UGT1TA1 activity. A detection of 7 TA-repeats in hetero- or homozygous individuals [(TA)6/(TA)7 and (TA)7/(TA)7] is associated with lower UGT1TA1 activity, which may eventually result in the development of Gilbert syndrome and/or modified individual response to drugs metabolized by this enzyme. ATV contributes to the decreased levels of UGT1A1, which may lead to elevations of indirect bilirubin, jaundice and even to therapy discontinuation. We evaluated the prevalence of the UGT1A1*28 among HIV-infected patients and the dependence of the frequency and severity of AE during ATV treatment on individual genetic characteristics. Materials and Methods : 47 HIV-infected patients was screen for UGT1A1 genotype and the presence of UGT1A1*28. All patients received ATV in the HAART regimen for 48 weeks. Changes in the total, direct and indirect bilirubin, ALT, AST, GGT and jaundice were evaluated. Statistical analysis was performed using Microsoft Office Excel for Windows XP Professional 2007 and Biostat. Results : All patients were followed up in the AIDS Center (males 72.3%, median age 33 years, median CD4+ count-282 cells/µl (19.5%)). HBV/HCV was in 36.2% patients. Ten patients had risk factors that could affect bilirubin turnover (chronic cholecystitis, biliary dyskinesia, etc.). Genotype (TA)6/(TA)6 was found in 42.6% patients, (TA)6/(TA)7-42.6% and (TA)7/(TA)7-14.9%. Overall prevalence of UGT1A1*28 was 57.4%, and homozygous allele frequency was 14.9%. G3/4 of indirect bilirubin were detected in 36.2% patients [(TA)6/(TA)6 in 10–20%, (TA)6/(TA)7-25-40%, (TA)7/(TA)7-72-86%], and significant jaundice in 10.6% [80% with (TA)7/(TA)7]. The OR for hyperbilirubinemia>40 µmol/L in patients with heterozygous UGT1A1*28 was increased 3 times over patients without this allele (OR 3.07, 95% CI 1.54–4.6) and 34 times as compared with homozygotes (OR 33.9, 95% CI 31.45–36.35). The presence of additional risk factors increased the probability of G3/4 hyperbilirubinemia. No significant changes in the ALT, AST, and GGT levels were observed. Conclusions : The risk of severe hyperbilirubinemia during ATV treatment is minimal for patients without UGT1A1*28 and no more than one additional risk factor and for patients with UGT1A1*28 and no additional risk factors; patients with homozygous genotype UGT1A1*28 are at the highest risk. (Published: 2 November 2014) Citation : Abstracts of the HIV Drug Therapy Glasgow Congress 2014 Kanestri V et al. Journal of the International AIDS Society 2014, 17(Suppl 3) :19579 http://www.jiasociety.org/index.php/jias/article/view/19579 | http://dx.doi.org/10.7448/IAS.17.4.19579

Germanium MIS-structures with a double-layer dielectric (germanium dioxide, silicon nitride) are developed. Conditions of crack appearance in the dielectric layer are investigated. In the structures under study two kinds of cracks are observed: oriented ones in crystalline directions 〈112〉 of the substrate and non-oriented ones. It is shown that the character of film cracking depends on the hardness of the mechanical connection of Si3N4 with the substrate conditioned by viscoelastic properties of the germanium dioxide film. The GeO2 layer viscosity is found to increase during the dilution of SiO2 and Ge3N4 resulting from the reaction between GeO2 and Si3N4 at their interfaces at 700 to 750 °C. This is the cause of changing the cracking type in Si3N4 from the non-oriented to the oriented one on dccreasing GeO2 thickness in Ge-GeO2-Si3N4 structures or during their annealing as well as of the fall of the critical film thickness for crack formation with the thickness increase of GeO2. The general electrical properties of Ge–GeO2–Si3N4–Al MIS- structures are investigated. Germanium-MIS-Strukturen mit einem Doppelschicht-Dielektrikum (Germaniumdioxid, Silizium-nitrid) werden entwickelt. Die Bedingungen fur das Auftreten von Rissen in der dielektrischen Schirht wcrden untersucht. In der untersuchten Struktur werden zwei Arten von Rissen beobachtet, in 〈112〉-Kristallrichtungen des Substrats orientierte und nichtorientierte. Es wird gezeigt, das der Charakter der Schichtrisse von der Harte der mechanischen Verbindung des Si3N4 mit dem Substrat abhangt, bedingt durch die viskoelastischen Eigenschaften der Germaniumdioxidschicht. Es wird gefunden, das die GeO2-Schicht wahrend der Verdunnung von SiO2 und Ge3N4 die aus der Reaktion zwischen GeO2, und Si3N4 an ihren Grenzschichten bei 700 bis 750 °C herruhrt, an-wachst. Dies ist der Grund fur die Anderung des Ristyps im Si3N4 vom nichtorientierten zum orientierten nach Abnahme der GeO2-Dicke in der Ge-GeO2-Si3N4-Struktur oder wahrend ihrer Ausheilung sowie auch fur den Abfall der kritischen Schichtdicke fur die Risbildung mit wachsender Schichtdicke von GeO2. Die allgemeinen elektrischcn Eigenschaften der Ge–GeO2–Si3N4–Al–MIS-Strukturen werden ebenfalls untersuoht.

SiO2 pyrolitic films are studied by ellipsometry, transmission electron microscopy (TEM), and electron diffraction techniques. During the condensation the films are shown densified starting from interface by a heterogeneous mechanism which results in the formation of more porous layer near the film surface. Between the SiO2 film and Ge substrate a region with higher concentration of GeO2 appears. Synthesized in oxygen flow this region is hundreds Angstroms thick and a phase separation occurs. All the films have dome-like defects as potential centres of spherulite crystallization. On the interface polycrystalline diamond-like germanium and in some cases the germanium high pressure phase, Ge III, is found. [Russian text Ignored.]

Das Reaktionsverhalten des Pho spholans (I) bei der Umsetzung mit Alkoholen (II) wird in Abhangigkeit von Temperatur, Reaktionsbedingungen und Kettenlange der einge- Vsetzten Alkohole untersucht.