Assigning a function to a conserved archaeal metallo-β-lactamase from Haloferax volcanii

The metallo-β-lactamase family of enzymes comprises a large group of proteins with diverse functions in the metabolism of the cell. Among others, this superfamily contains proteins which are involved in DNA and RNA metabolism, acting as nucleases in e.g. repair and maturation. Many proteins have been annotated in prokaryotic genomes as being potential metallo-β-lactamases, but very often the function has not been proven. The protein HVO_2763 from Haloferax volcanii is such a potential metallo-β-lactamase. HVO_2763 has sequence similarity to the metallo-β-lactamase tRNase Z, a tRNA 3′ processing endonuclease. Here, we report the characterisation of this metallo-β-lactamase HVO_2763 in the halophilic archaeon Haloferax volcanii. Using different in vitro assays with the recombinant HVO_2763, we could show that the protein does not have tRNA 3′ processing or exonuclease activity. According to transcriptome analyses of the HVO_2763 deletion strain, expression of proteins involved in membrane transport is downregulated in the mutant. Therefore, HVO_2763 might be involved directly or indirectly in membrane transport

Cyclin-Dependent Kinase Activity Controls the Onset of the HCMV Lytic Cycle

The onset of human cytomegalovirus (HCMV) lytic infection is strictly synchronized with the host cell cycle. Infected G0/G1 cells support viral immediate early (IE) gene expression and proceed to the G1/S boundary where they finally arrest. In contrast, S/G2 cells can be infected but effectively block IE gene expression and this inhibition is not relieved until host cells have divided and reentered G1. During latent infection IE gene expression is also inhibited, and for reactivation to occur this block to IE gene expression must be overcome. It is only poorly understood which viral and/or cellular activities maintain the block to cell cycle or latency-associated viral IE gene repression and whether the two mechanisms may be linked. Here, we show that the block to IE gene expression during S and G2 phase can be overcome by both genotoxic stress and chemical inhibitors of cellular DNA replication, pointing to the involvement of checkpoint-dependent signaling pathways in controlling IE gene repression. Checkpoint-dependent rescue of IE expression strictly requires p53 and in the absence of checkpoint activation is mimicked by proteasomal inhibition in a p53 dependent manner. Requirement for the cyclin dependent kinase (CDK) inhibitor p21 downstream of p53 suggests a pivotal role for CDKs in controlling IE gene repression in S/G2 and treatment of S/G2 cells with the CDK inhibitor roscovitine alleviates IE repression independently of p53. Importantly, CDK inhibiton also overcomes the block to IE expression during quiescent infection of NTera2 (NT2) cells. Thus, a timely block to CDK activity not only secures phase specificity of the cell cycle dependent HCMV IE gene expression program, but in addition plays a hitherto unrecognized role in preventing the establishment of a latent-like state

Genome-Wide Screen of Three Herpesviruses for Protein Subcellular Localization and Alteration of PML Nuclear Bodies

Herpesviruses are large, ubiquitous DNA viruses with complex host interactions, yet many of the proteins encoded by these viruses have not been functionally characterized. As a first step in functional characterization, we determined the subcellular localization of 234 epitope-tagged proteins from herpes simplex virus, cytomegalovirus, and Epstein–Barr virus. Twenty-four of the 93 proteins with nuclear localization formed subnuclear structures. Twelve of these localized to the nucleolus, and five at least partially localized with promyelocytic leukemia (PML) bodies, which are known to suppress viral lytic infection. In addition, two proteins disrupted Cajal bodies, and 19 of the nuclear proteins significantly decreased the number of PML bodies per cell, including six that were shown to be SUMO-modified. These results have provided the first functional insights into over 120 previously unstudied proteins and suggest that herpesviruses employ multiple strategies for manipulating nuclear bodies that control key cellular processes

Exercise linked to transient increase in expression and activity of cation channels in newly formed hind-limb collaterals

OBJECTIVE: This study aimed to compare arteriogenesis after femoral artery occlusion as influenced by exercise or arteriovenous shunt and follow changes in collateral transient receptor potential cation channel, subfamily V, member 4 (Trpv4). DESIGN: A prospective, controlled study wherein rats were subjected to femoral artery ligation (FAL), or FAL+arteriovenous shunt. Collateral Trpv4 was determined 0.5 and 6h post exercise. METHODS: Rats were subjected to exercise for 15 min, twice daily. The number and diameter of collaterals were assessed after 7 days. Collateral Trpv4 expression was quantified by reverse transcription-polymerase chain reaction. RESULTS: Collateral number and diameter per limb were significantly higher in the shunt group (number: 16.0+/-2.4 and diameter: 216.0+/-34 microm) compared to the ligature (number: 9.4+/-2 and diameter: 144+/-21 microm) and exercise groups (number: 9.9+/-2.5 and diameter: 151+/-15 microm). Trpv4 expression in collaterals harvested 0.5h post exercise was not significantly different from expression in shunted rats. It was significantly lower in collaterals harvested 6h post exercise (comparable to that in ligated rats). CONCLUSION: Collateral formation was greater in the shunt group than in the exercise group. Exercise-induced Trpv4 up-regulation, not significantly different from that achieved with shunt, returned to control values when evaluated 6h post exercise. More frequent exercise to chronically increase fluid shear stress, as with a shunt model, may be required for sufficient arteriogenesis to compensate for peripheral occlusion

Određivanje krivulje magnetiziranja vektorski upravljanog asinkronog motora kod malih opterećenja

In many drives the induction motor torque is transferred to the load through complex mechanical connections which produce an initial load. In such cases, to identify the magnetization curve by the no-load test it is necessary to decouple the motor from the transfer gear what is expensively and time-consuming. In an attempt to avoid the motor mechanical decoupling in the case of hoist crane systems, in this paper a method is proposed for the magnetization curve identification of the indirect rotor flux oriented (IRFO) induction machine at low-load conditions. Two flux controllers with estimated feedback quantities are added to the basic structure of the IRFO control drive. One of these controllers is used to control desired operating point on the magnetization curve and the other for on-line adaption of the reference frame position. The proposed algorithm is implemented in a commercial drive as a part of the self-commissioning module. Afterward, the magnetization curve of the 22 kW induction motor was identified and compared with the one obtained by the no-load test. The method sensitivity to the load torque and the transient inductance has also been considered.Brojni su primjeri pogona u kojima se moment asinkronog stroja prenosi na radni mehanizam preko mehaničkih reduktora koji mogu predstavljati značajno opterećenje motoru i u režimu neopterećenog pogona. Da bi se u takvim slučajevima odredila krivulja magnetiziranja iz pokusa praznog hoda nužno je odspojiti motor od reduktora što je skupo i vremenski zahtjevno. U pokušaju da se izbjegne mehaničko odspajanje motora kod dizaličnih sustava, u ovom je radu predložena metoda za određivanje krivulje magnetizranja vektorski upravljanog asinkronog motora kod malih iznosa opterećenja. Dva regulatora ulančenog toka s estimiranim veličinama u granama povratne veze dodani su osnovnoj strukturi vektorski upravljanog pogona. Jedan od regulatora koristi se za upravljanje željenom radnom točkom na krivulji magnetiziranja dok se drugi koristi za podešavanje referentnog položaja vektora toka rotora. Predloženi algoritam implementiran je u komercijalnom uređaju kao jedan od samopodešavajućih modula koji se koriste prilikom puštanja u rad pogona. Nakon toga je opisanim postupkom određena krivulja magnetiziranja 22 kW asinkronog motora i uspoređena s krivuljom dobivenom iz klasičnog pokusa praznog hoda. Također je razmotrena i osjetljivost metode na iznos momenta opterećenja i tranzijentnog induktiviteta