Comparison of polyvinyl alcohol coated nasal packing with non-absorbable nasal packing.

BACKGROUND: A number of different nasal packing materials are available for prevention of nasal bleeding after endoscopic sinus surgery. Polyvinyl alcohol (PVA) coated nasal packing is an expandable packing for use in rhinological surgery. This innovative surf- ace treatment helps to reduce the possibility of adherence to tissue and of blood clotting within the sponge. The present study investigated the effects of PVA coated packing and non-absorbable packing with respect to pain, healing site and postoperative bleeding following endoscopic sinus surgery. METHODOLOGY: Patients between 18-80 years of age undergoing sinus surgery were enrolled. Each patient`s ethmoid cavities were randomised to receive PVA coated packing material or the standard non-absorbable sinus packs. The remaining nasal packing material was removed on the 2nd day in the clinic. We determined visual analog scale score, bleeding time and wound healing status. A single rhinologist graded postoperative endoscopic appearance. Length of follow-up was 3 months. RESULTS: Thirty three patients were recruited. There was a significant difference in the bleeding time between the two groups, but pain and wound healing were not significantly different between the two groups. CONCLUSION: PVA-coated nasal packing presents comparable characteristics with traditional nasal packing.

Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase assembly.

SCF ubiquitin ligases are composed of Skp1, Cdc53, Hrt1 and one member of a large family of substrate receptors known as F-box proteins (FBPs). Here we report the identification, using sequential rounds of epitope tagging, affinity purification and mass spectrometry, of 16 Skp1 and Cdc53-associated proteins in budding yeast, including all components of SCF, 9 FBPs, Yjr033 (Rav1) and Ydr202 (Rav2). Rav1, Rav2 and Skp1 form a complex that we have named 'regulator of the (H+)-ATPase of the vacuolar and endosomal membranes' (RAVE), which associates with the V1 domain of the vacuolar membrane (H+)-ATPase (V-ATPase). V-ATPases are conserved throughout eukaryotes, and have been implicated in tumour metastasis and multidrug resistance, and here we show that RAVE promotes glucose-triggered assembly of the V-ATPase holoenzyme. Previous systematic genome-wide two-hybrid screens yielded 17 proteins that interact with Skp1 and Cdc53, only 3 of which overlap with those reported here. Thus, our results provide a distinct view of the interactions that link proteins into a comprehensive cellular network.

Skp1p and the F-box protein Rcy1p form a non-SCF complex involved in recycling of the SNARE Snc1p in yeast.

Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin-ligases composed of a core complex including Skp1p, Cdc53p, Hrt1p, the E2 enzyme Cdc34p, and one of multiple F-box proteins which are thought to provide substrate specificity to the complex. Here we show that the F-box protein Rcy1p is required for recycling of the v-SNARE Snc1p in Saccharomyces cerevisiae. Rcy1p localized to areas of polarized growth, and this polarized localization required its CAAX box and an intact actin cytoskeleton. Rcy1p interacted with Skp1p in vivo in an F-box-dependent manner, and both deletion of its F box and loss of Skp1p function impaired recycling. In contrast, cells deficient in Cdc53p, Hrt1p, or Cdc34p did not exhibit recycling defects. Unlike the case for F-box proteins that are known to participate in SCF complexes, degradation of Rcy1p required neither its F box nor functional 26S proteasomes or other SCF core subunits. Importantly, Skp1p was the only major partner that copurified with Rcy1p. Our results thus suggest that a complex composed of Rcy1p and Skp1p but not other SCF components may play a direct role in recycling of internalized proteins.

Na+, K+-specific inhibition of protein and peptide hydrolyses by proteasomes from human hepatoma tissues.

Proteasomes were purified from human hepatoma tissues, and their sensitivities to Na+ and K+ were examined. At concentrations of 10 mM or more, these cations were found to inhibit completely polylysine-activated casein degradation by the purified proteasomes. They also strongly inhibited the hydrolyses of peptides, although to a lesser extent. On the other hand, they reversed the inhibitory and stimulatory effects of polylysine on the hydrolyses of Suc-Leu-Tyr-AMC and Cbz-Ala-Arg-Arg-MNA, respectively. These results suggest that Na+ and/or K+ may be involved in the regulation of intracellular protein breakdown by controlling the multicatalytic activity of proteasomes.

The 65-kDa protein derived from the internal translational start site of the clpA gene blocks autodegradation of ClpA by the ATP-dependent protease Ti in Escherichia coli.

The ATP-dependent protease Ti consists of two different components: ClpA containing ATP-cleaving sites and ClpP having serine active sites for proteolysis. The clpA gene has dual translational start sites and therefore encodes two polypeptides with sizes of 84 and 65 kDa (referred to as ClpA84 and ClpA65, respectively). Here we show that ClpA84, but not ClpA65, is degraded in vitro by ClpP in the presence of ATP. The ClpP-mediated hydrolysis of ClpA84 could be prevented by casein, which is an excellent substrate of protease Ti (i.e. ClpA84/ClpP complex). Thus, it appears that free form of ClpA84 competes with casein for the degradation by ClpA/ClpP complex. Furthermore, ClpA65 inhibited the auto-degradation of ClpA84 by the complex. These results suggest that ClpA65 may play an important role in the control of the ClpA84 level and in turn in the regulation of ATP-dependent protein breakdown in E. coli.

ATP-dependent degradation of SulA, a cell division inhibitor, by the HslVU protease in Escherichia coli.

HslVU is an ATP-dependent protease consisting of two multimeric components, the HslU ATPase and the HslV peptidase. To gain an insight into the role of HslVU in regulation of cell division, the reconstituted enzyme was incubated with SulA, an inhibitor of cell division in Escherichia coli, or its fusion protein with maltose binding protein (MBP). HslVU degraded both proteins upon incubation with ATP but not with its nonhydrolyzable analog, ATPgammaS, indicating that the degradation of SulA requires ATP hydrolysis. The pulse-chase experiment using an antibody raised against MBP-SulA revealed that the stability of SulA increased in hsl mutants and further increased in lon/hsl double mutants, indicating that SulA is an in vivo substrate of HslVU as well as of protease La (Lon). These results suggest that HslVU in addition to Lon plays an important role in regulation of cell division through degradation of SulA.

Mutational analysis of the ATP-binding site in HslU, the ATPase component of HslVU protease in Escherichia coli.

HslU is the ATPase component of the ATP-dependent HslVU protease in Escherichia coli. To gain an insight into the structure and function of HslU, site-directed mutagenesis was performed to generate a mutation in the ATP-binding site of the ATPase (i.e., to replace the Lys63 with Thr). Unlike the wild-type HslU, the mutant form (referred to as HslU/K63T) could not hydrolyze ATP or support the ATP-dependent hydrolysis of N-carbobenzoxy-Gly-Gly-Leu-7-amido-4-methyl coumarin by HslV. The wild-type HslU (a mixture of monomer and dimer) formed a multimer containing 6-8 subunits in the presence of either ATP or ADP, indicating that ATP-binding, but not its hydrolysis, is required for oligomerization of HslU. However, HslU/K63T remained as a monomer whether or not the adenine nucleotides were present. Furthermore, ATP or ADP could protect HslU, but not HslU/K63T, from degradation by trypsin. These results suggest that the mutation in the ATP-binding site results in prevention of the binding of the adenine nucleotides to HslU and hence in impairment of both oligomerization and ATPase function of HslU.

Mutagenesis of two N-terminal Thr and five Ser residues in HslV, the proteolytic component of the ATP-dependent HslVU protease.

HslVU in E. coli is a new type of ATP-dependent protease consisting of two heat shock proteins: the HslU ATPase and the HslV peptidase that has two repeated Thr residues at its N terminus, like certain beta-type subunit of the 20S proteasomes. To gain an insight into the catalytic mechanism of HslV, site-directed mutagenesis was performed to replace each of the Thr residues with Ser or Val and to delete the first or both Thr. Also each of the five internal Ser residues in HslV were replaced with Ala. The results obtained by the mutational analysis revealed that the N-terminal Thr acts as the active site nucleophile and that certain Ser residues, particularly Ser124 and Ser172, also contribute to the peptide hydrolysis by the HslVU protease. The mutational studies also revealed that both Thr, Ser103, and Ser172, but not Ser124, are involved in the interaction of HslV with HslU and hence in the activation of HslU ATPase as well as in the HslVU complex formation.

Hydrolysis of the IciA protein, an inhibitor of DNA replication initiation, by protease Do in Escherichia coli.

The 33 kDa IciA protein, an inhibitor of replication initiation of the Escherichia coli chromosome, was found to be specifically cleaved to 27 kDa fragment by protease Do, the htrA gene product. The 27 kDa polypeptide could no longer interact with the oriC region, and therefore the cleavage-site is likely to reside within the N-terminal DNA-binding domain of the IciA protein. In addition, protease Do was found to localize primarily to the cytoplasm although it also could bind to membranes through an ionic interaction. These results suggest that intracellular breakdown of the IciA protein by protease Do may provide a potential mechanism involving the regulation of initiation of DNA replication in Escherichia coli.

Robotic sialoadenectomy of the submandibular gland via a modified face-lift approach.

The purpose of this study was to describe and analyse the advantages and disadvantages of submandibular gland (SMG) resection using a robotic surgical system through a modified face-lift approach. The authors performed robotic sialoadenectomy of the SMG on 5 patients using the daVinci robot system through a modified face-lift approach. Three robotic arms were inserted through a modified face-lift incision; a face-down 30-degree endoscopic arm and two operative arms. The right arm was equipped with a harmonic scalpel and the left arm with a Maryland forceps. In all patients, robotic sialoadenectomy of the SMG was completed successfully. Diagnoses were sialolithiasis in two patients, pleomophic adenoma in two patients, and ranula in one patient. The mean robotic operative time was 90.2 min (range 62-185 min) and that for setting the robotic system was 8.2 min (range 5-15 min). No significant intra-operative or postoperative complications were observed. All patients were satisfied with the outcome and especially the cosmetic results at their last follow-up visit. In the authors opinion robotic sialoadenectomy of the SMG is technically feasible and secures a better cosmetic outcome than endoscopic submandibular resection.

Control of AIF-mediated cell death by antagonistic functions of CHIP ubiquitin E3 ligase and USP2 deubiquitinating enzyme.

Apoptosis inducing factor (AIF) is a mitochondrial oxidoreductase that scavenges reactive oxygen species under normal conditions. Under certain stresses, such as exposure to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG), AIF is truncated and released from the mitochondria and translocated into the nucleus, where the truncated AIF (tAIF) induces caspase-independent cell death. However, it is unknown how cells decide to kill themselves or operate ways to survive when they encounter stresses that induce the release of tAIF. Here, we demonstrated that USP2 and CHIP contribute to the control of tAIF stability. USP2 deubiquitinated and stabilized tAIF, thus promoting AIF-mediated cell death. In contrast, CHIP ubiquitinated and destabilized tAIF, thus preventing the cell death. Consistently, CHIP-deficient cells showed an increased sensitivity to MNNG. On the other hand, knockdown of USP2 attenuated MNNG-induced cell death. Moreover, exposure to MNNG caused a dramatic decrease in CHIP level, but not that of USP2, concurrent with cell shrinkage and chromatin condensation. These findings indicate that CHIP and USP2 show antagonistic functions in the control of AIF-mediated cell death, and implicate the role of the enzymes as a switch for cells to live or die under stresses that cause tAIF release.

Protease Ti (Clp), a multi-component ATP-dependent protease in Escherichia coli.

The ATP-dependent protease Ti(Clp) consists of two different multimeric components: ClpA containing ATP-cleaving sites and ClpP, with serine active sites for proteolysis. Here we summarize the most recent results on the structure and function of protease Ti. (1) The clpA gene has dual translational initiation sites and therefore encodes two polypeptides with sizes of 84 and 65 kDa. The abbreviated form of ClpA may play an important role in regulation of the ATP-dependent proteolysis, since it inhibits the ability of the 84-kDa ClpA in supporting the ClpP-mediated protein breakdown and the autodegradation of the 84-kDa ClpA. (2) ClpA contains two highly conserved sequences for ATP-binding: the first site is essential for oligomerization and the second site is responsible for ATP hydrolysis. (3) ATP hydrolysis by ClpA is required not only for assembly of the ClpA/ClpP complex but also for its rapid dissociation.

Poly-L-lysine activates both peptide and ATP hydrolysis by the ATP-dependent HslVU protease in Escherichia coli.

Hs1VU in E. coli is a new type of ATP-dependent protease composed of two heat shock proteins, the HslU ATPase and the HslV peptidase related to certain beta-type subunits of the 20S proteasome. Here we show that the ATP-dependent hydrolysis of N-carbobenzoxy-Gly-Gly-Leu-7-amido-4-methylcoumarin by the HslVU protease can be markedly stimulated by poly-L-lysine, that is known to activate the casein-degrading activity of the 20S proteasome. However, poly-L-lysine showed little or no effect on the peptidase activity of HslV itself. Instead, it stimulated the hydrolysis of ATP by HslU several-fold. Histone that could stimulate the ATPase activity of HslU also increased the rate of the ATP-dependent peptide hydrolysis by HslV, although to a much lesser extent than by poly-L-lysine. Thus, the poly-L-lysine-mediated increase in the ATPase activity of HslU appears to be responsible for the dramatic activation of the ATP-dependent peptide hydrolysis by HslV. These results suggest that, in the reconstituted HslVU complex, the peptide hydrolysis by HslV occurs in a tightly coupled process with the cleavage of ATP by HslU.

Requirement of ATP hydrolysis for assembly of ClpA/ClpP complex, the ATP-dependent protease Ti in Escherichia coli.

The ATP-dependent protease Ti (Clp) consists of two distinct components, ClpP containing the serine active sites for proteolysis and ClpA having two ATP-binding sites. A ClpA variant (ClpAT) carrying Thr in place of Met169 is highly soluble but indistinguishable from the wild-type ClpA in its ability to hydrolyze ATP and to support the ClpP-mediated proteolysis. Here we show that ATP hydrolysis is essential for assembly of ClpAT/ClpP complex upon analysis of the mixture of its components by gel filtration followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Either ADP or adenosine 5'-(beta,gamma-imido)-triphosphate could not support the complex formation. Furthermore, ClpAT/K501T which carries a mutation in the second ATP-binding site and therefore is unable to cleave ATP could not interact with ClpP. On the other hand, ClpAT/K220T carrying a mutation in the first site and ClpP could be assembled into a complex at 2 mM ATP but not at 0.5 mM, at which concentration the trimeric mutant protein can not form a hexamer. These results indicate that assembly of protease Ti requires hydrolysis of ATP by ClpA in addition to its binding for hexamer formation.

Distinctive roles of the two ATP-binding sites in ClpA, the ATPase component of protease Ti in Escherichia coli.

ClpA is the ATPase component of the ATP-dependent protease Ti (Clp) in Escherichia coli and contains two ATP-binding sites. A ClpA variant (referred to as ClpAT) carrying threonine in place of the 169th methionine has recently been shown to be highly soluble but indistinguishable from the wild-type, 84-kDa ClpA in its ability to hydrolyze ATP and to support the casein-degrading activity of ClpP. Therefore, site-directed mutagenesis was performed to generate mutations in either of the two ATP-binding sites of ClpAT (i.e. to replace the Lys220 or Lys501 with Thr). ClpAT/K220T hydrolyzed ATP and supported the ClpP-mediated proteolysis 10-50% as well as ClpAT depending on ATP concentration, while ClpAT/K501T was unable to cleave ATP or to support the proteolysis. Without ATP, ClpAT and both of its mutant forms behaved as trimeric molecules as analyzed by gel filtration on a Sephacryl S-300 column. With 0.5 mM ATP, ClpAT and ClpAT/K501T became hexamers, but ClpAT/K220T remained trimeric. With 2 mM ATP, however, ClpAT/K220T also behaved as a hexamer. These results suggest that the first ATP-binding site of ClpA is responsible for hexamer formation, while the second is essential for ATP hydrolysis. When trimeric ClpAT/K220T was incubated with the same amount of hexameric ClpAT/K501T (i.e. at 0.5 mM ATP) and then subjected to gel filtration as above, a majority of ClpAT/K220T ran together with ClpAT/K501T as hexameric molecules. Furthermore, ClpAT/K501T in the mixture strongly inhibited the ability of ClpAT/K220T to cleave ATP and to support the ClpP-mediated proteolysis. Similar results were obtained in the presence of 2 mM ATP and also with the mixture with ClpAT. On the other hand, the ATPase activity of the mixture of ClpAT and ClpAT/K220T was significantly higher than the sum of that of each protein, particularly in the presence of 2 mM ATP, although its ability to support the proteolysis by ClpP remained unchanged. These results suggest that a rapid exchange of the subunits, possibly as a trimeric unit, occurs between the ClpAT proteins in the presence of ATP and leads to the formation of mixed hexameric molecules.

clpX encoding an alternative ATP-binding subunit of protease Ti (Clp) can be expressed independently from clpP in Escherichia coli.

ClpX, an alternative ATP-binding subunit for protease Ti (also called Clp), has been shown to support the ATP-dependent hydrolysis of lambda O-protein by ClpP. clpX has also been reported to be in an operon with clpP, and therefore both are co-transcribed in a single mRNA using the promoter proximal to clpP. Here, we show that clpX can be expressed independently from clpP using its own promoter. The cells carrying clpX alone on a multicopy plasmid successively produced the 46-kDa ClpX protein. Moreover, in vitro translation analysis revealed that the recombinant plasmid containing clpX generates the 46-kDa protein that can be immunoprecipitated with anti-ClpX antibody. In addition, it has recently been reported that CipX, but not ClpP, is required for normal replication of bacteriophage Mu. Thus, it appears that clpX can be expressed alone and/or co-expressed with clpP in cells depending on physiological conditions.

ATP binding, but not its hydrolysis, is required for assembly and proteolytic activity of the HslVU protease in Escherichia coli.

HslVU is an ATP-dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase. To gain an insight into the role of ATP hydrolysis in protein breakdown, we determined the insulin B-chain-degrading activity and assembly of HslVU in the presence of ATP and its nonhydrolyzable analogs. While beta,gamma-methylene-ATP could not support the proteolytic activity, beta,gamma-imido-ATP supported it to an extent less than 10% of that seen with ATP. Surprisingly, however, HslVU degraded insulin B-chain even more rapidly in the presence of ATPgammaS than with ATP. Furthermore, the ability of ATP and its analogs in supporting the proteolytic activity was closely correlated with their ability in supporting the oligomerization of HslU and the formation of the HslVU complex. However, ADP, which is capable of supporting the HslU oligomerization, could not support the HslVU complex formation or the proteolytic activity, suggesting that the conformation of the ADP-bound HslU oligomer is different from that of ATP-bound form. Thus, it appears that ATP-binding, but not its hydrolysis, is essential for assembly and proteolytic activity of HslVU.

Site-directed mutagenesis of the Cys residues in ClpA, the ATPase component of protease Ti (ClpAP) in Escherichia coli.

The ATP-dependent casein hydrolysis by protease Ti (ClpAP) has been shown to be inhibited by sulfhydryl blocking agents, such as N-ethylmaleimide (NEM), when preincubated with ClpA but not with ClpP. To define the role of three Cys residues in ClpA, site-directed mutagenesis was performed to substitute each of them with Ser or Ala. None of the mutations showed any effect on the ATPase activity of ClpA or its ability to support the casein degradation by ClpP. However, NEM could no longer block the ability of ClpA/C47S or ClpA/C47A in supporting the ClpP-mediated proteolysis, unlike that of ClpA, ClpA/C203S, or ClpA/C243S. Furthermore, in the presence of NEM, casein could stimulate the ATPase activities of ClpA/C47S and ClpA/C47A and protect from their degradation by ClpP, but not of the other ClpA proteins. These results suggest that the inhibitory effect of NEM is due to prevention of the interaction of ClpA with casein by introduction of a bulky alkyl group to Cys47, but not linked to the catalytic function of the ATPase.

The 65-kDa protein derived from the internal translational initiation site of the clpA gene inhibits the ATP-dependent protease Ti in Escherichia coli.

The clpA gene that encodes the ATPase subunit of the ATP-dependent protease Ti (Clp) in Escherichia coli contains a putative internal translational initiation site. Here we show that mutagenesis of its 5'-end AUG codon resulted in an exclusive synthesis of the 65-kDa protein (ClpA65), while mutation at the internal 169th AUG codon (Met) to ACG (Thr) produced only the 84-kDa protein (ClpA84T). On the other hand, the cells carrying the wild-type clpA gene produced both the 84- and 65-kDa proteins (ClpA84/65). While the purified ClpA84T and ClpA84/65 hydrolyzed ATP nearly as well as the 84-kDa ClpA alone (ClpA84), ClpA65 cleaved ATP at a rate less than 5% of that by ClpA84. Unlike ClpA84 and ClpA84T, ClpA65 could not support the casein-degrading activity of ClpP. Furthermore, ClpA65 inhibited the proteolysis by the mixture of ClpP with ClpA84 or ClpA84T but not that with ClpA84/65, which could support the proteolytic activity of ClpP only about 40% as well as ClpA84. Nevertheless, ClpA65 showed little or no effect on the basal or protein-activated ATPase activity of ClpA84, ClpA84T, or ClpA84/65 alone or in the presence of ClpP. These results suggest that ClpA65 may interfere the interaction of ClpA84 or ClpA84T with ClpP and, hence, impair their assembly into an active form of the ATP-dependent protease Ti.

The heat-shock protein HslVU from Escherichia coli is a protein-activated ATPase as well as an ATP-dependent proteinase.

HslVU in Escherichia coli a new two-component ATP-dependent protease composed of two heat-shock proteins, the HslU ATPase and the HslV peptidase which is related to proteasome beta-type subunits. Here we show that the reconstituted HslVU enzyme degrades not only certain hydrophobic peptides but also various polypeptides, including insulin B-chain, casein, and carboxymethylated lactalbumin. Maximal proteolytic activity was obtained with a 1:2 molar ratio of HslV (a 250-kDa complex) to HslU (a 450-kDa complex). By itself, HslV could slowly hydrolyze these polypeptides, but its activity was stimulated 20-fold by HslU in the presence of ATP. The ATPase activity of HslU was stimulated up to 50% by the protein substrates, but not by nonhydrolyzed proteins, and this stimulation further increased 2-3-fold in the presence of HslV. Concentrations of insulin B-chain that maximally stimulated the ATPase allowed maximal rates of the B-chain hydrolysis. Furthermore, addition of increasing amounts of ADP or N-ethylmaleimide reduced ATP and protein or peptide hydrolysis in parallel. Thus, HslVU is a protein-activated ATPase as well as an ATP-dependent proteinase, and these processes appear linked. Surprisingly, the protein and peptide substrates do not compete with each other for hydrolysis. Lactacystin strongly inhibits protein degradation, but has little effect on peptide hydrolysis, while the peptide aldehydes are potent inhibitors of hydrolysis of small peptides, but have little effect on proteins. Thus, the functional requirements for ATP-dependent hydrolysis of peptides and proteins appear different.