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We have studied the origin of the excess specific heat (anomaly) above 10 mK in bcc solid3 He near melting pressure. We applied strong magnetic fields to the sample to see whether the anomaly arises from spin polarons due to vacancies. The specific heat is the same before and after applying magnetic fields of 10-12 T. This result possibly indicates that the anomaly arises from the origin different from vacancies. Next, in order to check whether the anomaly comes from the surface magnetism, we measured the specific heat by coating the surface of sintered silver with three layers and two layers of4 He. The results showed that unexpected large heat capacity due to phase separation of solid3 He-4 He surpassed and smeared the original specific heat anomaly. We are investigating the origin of the anomaly further. [ABSTRACT FROM AUTHOR]

We observed the excess specific heat (anomaly) other than nuclear origins above 10 mK in bcc solid3 He of 24.21 cm3/ mole. We checked whether it arises from spin polarons due to vacancies out of equilibrium by applying a strong magnetic field, in which vacancies should diffuse and vanish due to high polarization. The specific heat is the same before and after applying a magnetic field of 10 T. This fact indicates that vacancies did not vanish even in a strong field or the anomaly arises from the origin different from vacancies. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy (DR) is the leading cause of visual impairment, particularly in the proliferative form (proliferative DR [PDR]). The impact of the PDR microenvironment on microglia, which are the resident immune cells in the central nervous system, and the specific pathological changes it may induce remain unclear. This study aimed to investigate the role of microglia in the progression of PDR under hypoxic and inflammatory conditions. We performed a comprehensive gene expression analysis using human-induced pluripotent stem cell-derived microglia under different stimuli (dimethyloxalylglycine (DMOG), lipopolysaccharide (LPS), and DMOG + LPS) to mimic the hypoxic inflammatory environment characteristic of PDR. Principal component analysis revealed distinct gene expression profiles, with 76 genes synergistically upregulated under combined stimulation. Notably, prostaglandin-endoperoxide synthase 2 (encoding cyclooxygenase (COX)-2) exhibited the most pronounced increase, leading to elevated prostaglandin E2 (PGE2) levels and driving pathological angiogenesis and inflammation via the COX-2/PGE2/PGE receptor 2 signaling axis. Additionally, the upregulation of the fibrogenic genes snail family transcriptional repressor 1 and collagen type I alpha 1 chain suggested a role for microglia in fibrosis. These findings underscore the critical involvement of microglia in PDR and suggest that targeting both the angiogenic and fibrotic pathways may present new therapeutic strategies for managing this condition.

Background: Macular edema is found in more than half of branch retinal vein occlusion (BRVO) cases, leading to visual loss in most of these cases. Intravitreal injection of anti-vascular endothelial growth factor is currently the standard treatment for macular edema due to BRVO (BRVO-ME). The difference in the effects of aflibercept and ranibizumab on the choroid in BRVO-ME is unknown. Therefore, we analyzed the effects of intravitreal injection of ranibizumab and aflibercept on BRVO-ME., Methods: We retrospectively observed changes in choroidal thickness in the subfoveal region in 36 patients with BRVO-ME who visited the Department of Ophthalmology at the Juntendo University Urayasu Hospital. The patients were treated with intravitreal injection of aflibercept or ranibizumab and followed up for 12 months or more., Results: The observed point bifurcated into the affected and non-affected sides 500 μm from the fovea. The central macular thickness (CMT) and subfoveal choroidal thickness (SFCT) were 564.2 ± 268.5 μm and 228.8 ± 50.1 μm, respectively, in the ranibizumab group (16 patients, 16 eyes) and 542.4 ± 172.5 μm and 246.1 ± 59.1 μm, respectively, in the aflibercept group (20 patients, 20 eyes). The changes in CMT at 12 months were 324.0 ± 262.6 μm and 326.55 ± 187.2 μm in the ranibizumab and aflibercept groups, respectively, with no significant difference (p = 0.97). Similarly, the changes in SFCT over 12 months were not significant between the groups (ranibizumab, 41.9 ± 33.0 μm; aflibercept, 43.8 ± 43.8 μm, p = 0.89)., Conclusion: The effects of ranibizumab and aflibercept on choroidal thickness in BRVO-ME were the same regardless of the site. Although BRVO is a retinal disease, we hope that we can further explore the mechanism of BRVO-ME by observing changes in the choroid in the future., (© 2022. The Author(s).)

Currently, ERY974, a humanized IgG4 bispecific T cell-redirecting antibody recognizing glypican-3 and CD3, is in phase I clinical trials. After a first-in-human clinical trial of an anti-CD28 agonist monoclonal antibody resulting in severe life-threatening adverse events, the minimal anticipated biological effect level approach has been considered for determining the first-in-human dose of high-risk drugs. Accordingly, we aimed to determine the first-in-human dose of ERY974 using both the minimal anticipated biological effect level and no observed adverse effect level approaches. In the former, we used the 10% effective concentration value from a cytotoxicity assay using the huH-1 cell line with the highest sensitivity to ERY974 to calculate the first-in-human dose of 4.9 ng/kg, at which maximum drug concentration after 4 h of intravenous ERY974 infusion was equal to the 10% effective concentration value. To determine the no observed adverse effect level, we conducted a single-dose study in cynomolgus monkeys that were intravenously infused with ERY974 (0.1, 1, and 10 μg/kg). The lowest dose of 0.1 μg/kg was determined as the no observed adverse effect level, and the first-in-human dose of 3.2 ng/kg was calculated, considering body surface area and species difference. For the phase I clinical trial, we selected 3.0 ng/kg as a starting dose, which was lower than the first-in-human dose calculated from both the no observed adverse effect level and minimal anticipated biological effect level. Combining these two methods to determine the first-in-human dose of strong immune modulators such as T cell-redirecting antibodies would be a suitable approach from safety and efficacy perspectives.Clinical trial registration: JapicCTI-194805/NCT05022927., (© 2022. The Author(s).)

We prepared a high-molecular-weight modified dextrin (MWS-1000) from a partial hydrolysate of waxy corn starch with a weight average molecular weight of 1 × 106 (WS-1000) using Paenibacillus alginolyticus PP710 α-glucosyltransferase. The gel permeation chromatography showed that the weight average molecular weight of MWS-1000 was almost the same as that of WS-1000. The side chain lengths of WS-1000 and MWS-1000 after isomaltodextranase digestion were also shown to be similar to each other by high-performance anion exchange chromatography with pulsed amperometric detection. Since MWS-1000 confirmed the presence of α-1,6 bonds by enzyme digestibility, methylation, and 1H-NMR analyses, it was presumed that the structure of MWS-1000 was based on the introduction of α-1,6 glucosyl residues at the nonreducing ends of the partial hydrolysate of waxy corn starch. Furthermore, the MWS-1000 solution was not retrograded even during refrigerated storage or after repeated freeze-thaw cycles., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

High-molecular-weight dextrin (WS-1000) was produced from waxy corn starch and enzymatically modified to link glucose by α-1,6 glycosidic bond at the terminal point of the glucose chain, forming MWS-1000. In this study, the physical properties of MWS-1000 were characterized, and the advantages of its use as a food modifier were described. From rheological and calorimetric studies, it was found that MWS-1000 does not undergo retrogradation, but it does not prevent the retrogradation of WS-1000, suggesting that they have no intermolecular interaction in solution. Investigation of the effect of MWS-1000 on the viscoelasticity of gelatinized wheat starch showed that in the linear viscoelastic region, storage modulus decreased and tan δ increased with increase in replaced MWS-1000 content. In addition, it was confirmed that gelatinized starch containing MWS-1000 showed viscoelastic behavior similar to that of commercially available custard cream., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Fungus-derived GH-7 family cellobiohydrolase I (CBHI, EC 3.2.1.91) is one of the most important industrial enzymes for cellulosic biomass saccharification. Talaromyces cellulolyticus is well known as a mesophilic fungus producing a high amount of CBHI. Thermostability enhances the economic value of enzymes by making them more robust. However, CBHI has proven difficult to engineer, a fact that stems in part from its low expression in heterozygous hosts and its complex structure. Here, we report the successful improvement of the thermostability of CBHI from T. cellulolyticus using our homologous expression system and protein engineering method. We examined the key structures that seem to contribute to its thermostability using the 3D structural information of CBHI. Some parts of the structure of the Talaromyces emersonii CBHI were grafted into T. cellulolyticus CBHI and thermostable mutant CBHIs were constructed. The thermostability was primarily because of the improvement in the loop structures, and the positive effects of the mutations for thermostability were additive. By combing the mutations, the constructed thermophilic CBHI exhibits high hydrolytic activity toward crystalline cellulose with an optimum temperature at over 70°C. In addition, the strategy can be applied to the construction of the other thermostable CBHIs., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Oncological and functional results of open conservation surgery for hypopharyngeal cancer have been desired., Methods: We performed a chart review of 33 patients with hypopharyngeal cancer who underwent open conservation surgery. Oncological and functional results were evaluated in surgery with primary closure (Group A) and surgery with reconstruction (Group B). Postoperative functions were evaluated by interval to resumption of oral intake, Functional Outcome Swallowing Scale (FOSS) and Communication Scale (CS)., Results: Five-year disease-specific and overall cumulative survival rates by Kaplan-Meier method for all cases were 95.7% and 82.3%, respectively. Duration from surgery to full oral intake was 12 days in Group A and 14 days in Group B. FOSS rates were 83.3 in Group A and 95.5 in Group B. CS was 0 in both groups., Conclusion: Oncological and functional results of open conservation surgery were comparable to those with transoral surgery and chemo/radiotherapy. Our technique represents a reliable treatment for hypopharyngeal cancer.

Chinese hamster ovary (CHO) cells are the most commonly used mammalian host for large-scale commercial production of therapeutic monoclonal antibodies (mAbs). Chemically defined media are currently used for CHO cell-based mAb production. An adequate supply of nutrients, especially specific amino acids, is required for cell growth and mAb production, and chemically defined fed-batch processes that support rapid cell growth, high cell density, and high levels of mAb production is still challenging. Many studies have highlighted the benefits of various media designs, supplements, and feed addition strategies in cell cultures. In the present study, we used a strategy involving optimization of a chemically defined feed medium to improve mAb production. Amino acids that were consumed in substantial amounts during a control culture were added to the feed medium as supplements. Supplementation was controlled to minimize accumulation of waste products such as lactate and ammonia. In addition, we evaluated supplementation with tyrosine, which has poor solubility, in the form of a dipeptide or tripeptide to improve its solubility. Supplementation with serine, cysteine, and tyrosine enhanced mAb production, cell viability, and metabolic profiles. A cysteine-tyrosine-serine tripeptide showed high solubility and produced beneficial effects similar to those observed with the free amino acids and with a dipeptide in improving mAb titers and metabolic profiles., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

During the production of therapeutic monoclonal antibodies (mAbs), not only enhancement of mAb productivity but also control of quality attributes is critical. Charge variants, which are among the most important quality attributes, can substantially affect the in vitro and in vivo properties of mAbs. During process development for the production of mAbs in a Chinese hamster ovary cell line, we have observed that an improvement in mAb titer is accompanied by an increase in the content of acidic charge variants. Here, to help maintain comparability among mAbs, we aimed to identify the process parameters that controlled the content of acidic charge variants. First, we used a Plackett-Burman design to identify the effect of selected process parameters on the acidic charge variant content. Eight process parameters were selected by using a failure modes and effects analysis. Among these, temperature shift was identified from the Plackett-Burman design as the factor most influencing the acidic charge variant content. We then investigated in more detail the effects of shift temperature and temperature shift timing on this content. The content decreased with a shift to a lower temperature and with earlier timing of this temperature shift. Our observations suggest that Plackett-Burman designs are advantageous for preliminary screening of bioprocess parameters. We report here for the first time that temperature downshift is beneficial for effective control of the acidic peak variant content., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Background: Enzymatic removal of hemicellulose components such as xylan is an important factor for maintaining high glucose conversion from lignocelluloses subjected to low-severity pretreatment. Supplementation of xylanase in the cellulase mixture enhances glucose release from pretreated lignocellulose. Filamentous fungi produce multiple xylanases in their cellulase system, and some of them have modular structures consisting of a catalytic domain and a family 1 carbohydrate-binding module (CBM1). However, the role of CBM1 in xylanase in the synergistic hydrolysis of lignocellulose has not been investigated in depth., Results: Thermostable endo-β-1,4-xylanase (Xyl10A) from Talaromyces cellulolyticus, which is recognized as one of the core enzymes in the fungal cellulase system, has a modular structure consisting of a glycoside hydrolase family 10 catalytic domain and CBM1 at the C-terminus separated by a linker region. Three recombinant Xyl10A variants, that is, intact Xyl10A (Xyl10Awt), CBM1-deleted Xyl10A (Xyl10AdC), and CBM1 and linker region-deleted Xyl10A (Xyl10AdLC), were constructed and overexpressed in T. cellulolyticus. Cellulose-binding ability of Xyl10A CBM1 was demonstrated using quartz crystal microbalance with dissipation monitoring. Xyl10AdC and Xyl10AdLC showed relatively high catalytic activities for soluble and insoluble xylan substrates, whereas Xyl10Awt was more effective in xylan hydrolysis of wet disc-mill treated rice straw (WDM-RS). The enzyme mixture of cellulase monocomponents and intact or mutant Xyl10A enhanced the hydrolysis of WDM-RS glucan, with the most efficient synergism found in the interactions with Xyl10Awt. The increased glucan hydrolysis yield exhibited a linear relationship with the xylan hydrolysis yield by each enzyme. This relationship revealed significant hydrolysis of WDM-RS glucan with lower supplementation of Xyl10Awt., Conclusions: Our results suggest that Xyl10A CBM1 has the following two roles in synergistic hydrolysis of lignocellulose by Xyl10A and cellulases: enhancement of lignocellulosic xylan hydrolysis by binding to cellulose, and the efficient removal of xylan obstacles that interrupt the cellulase activity (because of similar binding target of CBM1). The combination of CBM-containing cellulases and xylanases in a fugal cellulase system could contribute to reduction of the enzyme loading in the hydrolysis of pretreated lignocelluloses.

Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) is one of the high cellulolytic enzyme-producing fungi. T. cellulolyticus exhibits the potential ability for high amount production of enzyme proteins. Using the homologous expression system under the control of a glucoamylase promoter, some kinds of cellulases of T. cellulolyticus can be expressed by T. cellulolyticus. On the other hand, hyperthermophilic cellulase has been expected to be useful in the industrial applications to biomass. The hyperthermophilic archaea Pyrococcus horikoshii and P. furiosus have GH family 5 and 12 hyperthermophilic endocellulase, respectively. The two kinds of hyperthermophilic endocellulases were successfully produced by T. cellulolyticus using the above expression system under the control of a glucoamylase promoter of T. cellulolyticus. These recombinant cellulases exhibited the same characteristics as those of the recombinant cellulases prepared in E. coli. The productions of the recombinant enzymes were estimated to be over 100 mg/L. In this study, we first report the overexpression of the hyperthermophilic enzymes of archaea using the fungal expression system.

Cellulose-inducible endo-β-1,4-xylanase (Xyl10A) from the mesophilic fungus Acremonium cellulolyticus was purified, characterized, and expressed by a homologous expression system. A. cellulolyticus CF-2612 produces a high level of xylanase upon induction by Solka-Floc cellulose. To identify this xylanase, the major fraction showing xylanase activity was purified from the CF-2612 culture supernatant, and its gene was identified from the genome sequence. Amino acid sequence homology of Xyl10A revealed that the purified xylanase, designated Xyl10A, exhibited significant homology to family 10 of the glycoside hydrolases (GH10), possessing a cellulose-binding module 1 in the C-terminal region. The xyl10A gene was cloned and expressed in A. cellulolyticus under the control of a glucoamylase promoter. Two recombinant Xyl10As (rXyl10A-I, 53kDa, and rXyl10A-II, 51kDa) were purified that have slightly different molecular weights based on SDS-PAGE. The rXyl10As had the same physicochemical and enzymatic properties as wtXyl10A: high thermostability (Tm 80.5°C), optimum pH 5.0 and specific activity 232-251U/mg for birchwood xylan. The molecular weights of N-deglycosylated rXyl10As were consistent with that of wild-type Xyl10A (wtXyl10A, 51kDa)., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A starch-inducible homologous expression system in Acremonium cellulolyticus was constructed to successfully produce recombinant cellulolytic enzymes. A. cellulolyticus Y-94 produced amylolytic enzymes and cellulolytic enzymes as major proteins in the culture supernatant when grown with soluble starch (SS) and Solka-Flock cellulose (SF), respectively. To isolate a strong starch-inducible promoter, glucoamylase (GlaA), which belongs to glycoside hydrolase family 15, was purified from the SS culture of Y-94, and its gene was identified in the genome sequence. The 1.4-kb promoter and 0.4-kb terminator regions of glaA were amplified by polymerase chain reaction (PCR) and used in the construction of a plasmid that drives the expression of the cellobiohydrolase I (Cel7A) gene from A. cellulolyticus. The resultant expression plasmid, containing pyrF as a selection marker, was randomly integrated into the genome of the A. cellulolyticus Y-94 uracil auxotroph. The prototrophic transformant, Y203, produced recombinant Cel7A as an extracellular protein under control of the glaA promoter in the SS culture. Recombinant and wild-type Cel7A were purified from the SS culture of Y203 and the SF culture of A. cellulolyticus CF-2612, respectively. Both enzymes were found to have the same apparent molecular weight (60 kDa), thermostability (T m 67.0 °C), and optimum pH (pH 4.5), and showed similar catalytic properties for soluble and insoluble substrates. These results suggest that the A. cellulolyticus starch-inducible expression system will be helpful for characterization and improvement of fungal cellulolytic enzymes.

Objective: Vascular malformations may appear anywhere in the body; 14-65% are in the head and neck. There are several treatments (sclerotherapy, surgery, laser treatment, and embolization, etc.), but standardized guidelines for these treatments are lacking. We conducted a retrospective review of venous or capillary malformations of the head and neck, and analyzed the epidemiology, pathology and treatment., Methods: We retrospectively reviewed 67 patients with pathologically diagnosed venous or capillary malformations of the head and neck; we analyzed the location, pathology and treatment, as well as recurrent/residual cases., Results: The oral cavity (59%) and nasal cavity (35%) were the most common locations. The frequency of each pathological type depended upon location. Surgery was undertaken in 65 cases, and sclerotherapy done in one patient. Sixty-one cases (92%) had resectable lesions. However polycystic masses (≥3 cysts) and large masses (diameter, ≥5cm) were significantly difficult to cure by single treatment., Conclusions: Surgery is indicated for localized small vascular malformations. However if the lesions ≥5cm or polycystic lesions were more likely to recur after surgery alone in our study population., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background: Primary intraosseous squamous cell carcinoma is very rare. We report two cases of primary intraosseous squamous cell carcinoma of the jaw, one arising from an odontogenic cyst and the other arising de novo., Methods and Results: The first case was a 76-year-old man with right mandible pain. A panoramic radiography and computed tomography revealed a large mandibular radiolucency. A biopsy revealed squamous cell carcinoma, and radiotherapy and hemimandibulectomy were performed. The second case was a 50-year-old man with lymph node swelling on the left neck. Squamous cell carcinoma of the lymph node was suspected after fine needle biopsy. After left neck dissection, histological testing of the odontogenic cyst revealed squamous cell carcinoma, of which the mandible was thought to be the primary site., Conclusion: Our two cases have no recurrence, and panoramic radiography was a useful tool in the detection of mandible disease.

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that functions as a sensor to maintain energy balance at both the cellular and the whole-body levels and is therefore a potential target for drug design against metabolic syndrome, obesity and type 2 diabetes. Here, the crystal structure of the phosphorylated-state mimic T172D mutant kinase domain from the human AMPK α2 subunit is reported in the apo form and in complex with a selective inhibitor, compound C. The AMPK α2 kinase domain exhibits a typical bilobal kinase fold and exists as a monomer in the crystal. Like the wild-type apo form, the T172D mutant apo form adopts the autoinhibited structure of the `DFG-out' conformation, with the Phe residue of the DFG motif anchored within the putative ATP-binding pocket. Compound C binding dramatically alters the conformation of the activation loop, which adopts an intermediate conformation between DFG-out and DFG-in. This induced fit forms a compound-C binding pocket composed of the N-lobe, the C-lobe and the hinge of the kinase domain. The pocket partially overlaps with the putative ATP-binding pocket. These three-dimensional structures will be useful to guide drug discovery.

The 70 kDa heat-shock proteins (Hsp70s) are highly conserved chaperones that are involved in several cellular processes, such as protein folding, disaggregation and translocation. In this study, the crystal structures of the human Hsp70 nucleotide-binding domain (NBD) fragment were determined in the nucleotide-free state and in complex with adenosine 5'-(beta,gamma-imido)triphosphate (AMPPNP). The structure of the nucleotide-free NBD fragment is similar to that of the AMPPNP-bound NBD fragment and is designated as the ;closed form'. In the nucleotide-free NBD fragment the closed form is intrinsically supported through interactions between Tyr15, Lys56 and Glu268 which connect subdomains IA, IB and IIB at the centre of the protein. Interaction with the substrate-binding domain (SBD) of Hsp70 or the BAG domain of BAG1 impairs this subdomain connection and triggers the rotation of subdomain IIA around a hydrophobic helix from subdomain IA. The subdomain rotation is limited by Asp199 and Asp206 from subdomain IIA and clearly defines the open form of the NBD. The open form is further stabilized by a new interaction between Gly230 from subdomain IIB and Ser340 from subdomain IIA. The structure of the NBD in the nucleotide-free state is determined by switching of the inter-subdomain interactions.

Interactions of Bcl-2 family proteins play a regulatory role in mitochondrial apoptosis. The pro-apoptotic protein Bak resides in the outer mitochondrial membrane, and the formation of Bak homo- or heterodimers is involved in the regulation of apoptosis. The previously reported structure of the human Bak protein (residues Glu16-Gly186) revealed that a zinc ion was coordinated with two pairs of Asp160 and His164 residues from the symmetry-related molecules. This zinc-dependent homodimer was regarded as an anti-apoptotic dimer. In the present study, we determined the crystal structure of the human Bak residues Ser23-Asn185 at 2.5A, and found a distinct type of homodimerization through Cys166 disulfide bridging between the symmetry-related molecules. In the two modes of homodimerization, the molecular interfaces are completely different. In the membrane-targeted model of the S-S bridged dimer, the BH3 motifs are too close to the membrane to interact directly with the anti-apoptotic relatives, such as Bcl-x(L). Therefore, the Bak dimer structure reported here may represent a pro-apoptotic mode under oxidized conditions.

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of the cyclic nucleotides cAMP and cGMP, which are important second messengers. Five of the 11 mammalian PDE families have tandem GAF domains at their N termini. PDE10A may be the only mammalian PDE for which cAMP is the GAF domain ligand, and it may be allosterically stimulated by cAMP. PDE10A is highly expressed in striatal medium spiny neurons. Here we report the crystal structure of the C-terminal GAF domain (GAF-B) of human PDE10A complexed with cAMP at 2.1-angstroms resolution. The conformation of the PDE10A GAF-B domain monomer closely resembles those of the GAF domains of PDE2A and the cyanobacterium Anabaena cyaB2 adenylyl cyclase, except for the helical bundle consisting of alpha1, alpha2, and alpha5. The PDE10A GAF-B domain forms a dimer in the crystal and in solution. The dimerization is mainly mediated by hydrophobic interactions between the helical bundles in a parallel arrangement, with a large buried surface area. In the PDE10A GAF-B domain, cAMP tightly binds to a cNMP-binding pocket. The residues in the alpha3 and alpha4 helices, the beta6 strand, the loop between 3(10) and alpha4, and the loop between alpha4 and beta5 are involved in the recognition of the phosphate and ribose moieties. This recognition mode is similar to those of the GAF domains of PDE2A and cyaB2. In contrast, the adenine base is specifically recognized by the PDE10A GAF-B domain in a unique manner, through residues in the beta1 and beta2 strands.

The target of diphtheria toxin is the diphthamide residue in translation elongation factor 2 (EF-2), which is generated by a three-step post-translational modification of a specific histidine residue in the EF-2 precursor. In the second modification step, an S-adenosylmethionine-dependent methyltransferase, diphthine synthase (DS), catalyzes the trimethylation of the EF-2 precursor. The homodimeric crystal structures of the archaeal diphthine synthases from Pyrococcus horikoshii OT3 and Aeropyrum pernix K1 have been determined. These structures share essentially the same overall fold as the cobalt-precorrin-4 methyltransferase CbiF, confirming that DS belongs to the dimeric class III family of methyltransferases. In the P. horikoshii DS dimer, only one of the two active sites binds the reaction product S-adenosyl-L-homocysteine (AdoHcy), while the other active site contains no ligand. This asymmetric AdoHcy binding may be a consequence of intra-domain and inter-domain movements upon binding of AdoHcy at one of the two sites. These movements disrupt the twofold dimeric symmetry of the DS dimer and probably cause lower AdoHcy affinity at the other binding site.

Interleukin (IL)-15 is a pleiotropic cytokine that plays a pivotal role in both innate and adaptive immunity. IL-15 is unique among cytokines due to its participation in a trans signaling mechanism in which IL-15 receptor alpha (IL-15Ralpha) from one subset of cells presents IL-15 to neighboring IL-2Rbeta/gammac-expressing cells. Here we present the crystal structure of IL-15 in complex with the sushi domain of IL-15Ralpha. The structure reveals that the alpha receptor-binding epitope of IL-15 adopts a unique conformation, which, together with amino acid substitutions, permits specific interactions with IL-15Ralpha that account for the exceptionally high affinity of the IL-15.IL-15Ralpha complex. Interestingly, analysis of the topology of IL-15 and IL-15Ralpha at the IL-15.IL-15Ralpha interface suggests that IL-15 should be capable of participating in a cis signaling mechanism similar to that of the related cytokine IL-2. Indeed, we present biochemical data demonstrating that IL-15 is capable of efficiently signaling in cis through IL-15Ralpha and IL-2Rbeta/gammac expressed on the surface of a single cell. Based on our data we propose that cis presentation of IL-15 may be important in certain biological contexts and that flexibility of IL-15Ralpha permits IL-15 and its three receptor components to be assembled identically at the ligand-receptor interface whether IL-15 is presented in cis or trans. Finally, we have gained insights into IL-15.IL-15Ralpha.IL-2Rbeta.gammac quaternary complex assembly through the use of molecular modeling.

The gene encoding TTHA1544 is a singleton found in the Thermus thermophilus HB8 genome and encodes a 131-amino-acid protein. The crystal structure of TTHA1544 has been determined at 2.0 A resolution by the single-wavelength anomalous dispersion method in order to elucidate its function. There are two molecules in the asymmetric unit. Each molecule consists of four alpha-helices and six beta-strands, with the beta-strands composing a central beta-sheet. A structural homology search revealed that the overall structure of TTHA1544 resembles the alpha/beta-hydrolase fold, although TTHA1544 lacks the catalytic residues of a hydrolase. These results suggest that TTHA1544 represents the minimized alpha/beta-hydrolase fold and that an additional component would be required for its activity.

Familial oncocytic thyroid carcinoma is associated with a missense mutation, P308Q, in the C-terminal domain of Tim44. Tim44 is the mitochondrial inner-membrane translocase subunit and it functions as a membrane anchor for the mitochondrial heat-shock protein 70 (mtHsp70). Here, the crystal structure of the human Tim44 C-terminal domain complexed with pentaethylene glycol has been determined at 1.9 A resolution. The overall structure resembles that of the nuclear transport factor 2-like domain. In the crystal structure, pentaethylene glycol molecules are associated at two potential membrane-binding sites: the large hydrophobic cavity and the highly conserved loop between the alpha1 and alpha2 helices near Pro308. A comparison with the yeast homolog revealed that lipid binding induces conformational changes around the alpha1-alpha2 loop, leading to slippage of the alpha1 helix along the large beta-sheet. These changes may play important roles in the translocation of polypeptides across the mitochondrial inner membrane.

The nuclear pore complex mediates the transport of macromolecules across the nuclear envelope (NE). The vertebrate nuclear pore protein Nup35, the ortholog of Saccharomyces cerevisiae Nup53p, is suggested to interact with the NE membrane and to be required for nuclear morphology. The highly conserved region between vertebrate Nup35 and yeast Nup53p is predicted to contain an RNA-recognition motif (RRM) domain. Due to its low level of sequence homology with other RRM domains, the RNP1 and RNP2 motifs have not been identified in its primary structure. In the present study, we solved the crystal structure of the RRM domain of mouse Nup35 at 2.7 A resolution. The Nup35 RRM domain monomer adopts the characteristic betaalphabetabetaalphabeta topology, as in other reported RRM domains. The structure allowed us to locate the atypical RNP1 and RNP2 motifs. Among the RNP motif residues, those on the beta-sheet surface are different from those of the canonical RRM domains, while those buried in the hydrophobic core are highly conserved. The RRM domain forms a homodimer in the crystal, in accordance with analytical ultracentrifugation experiments. The beta-sheet surface of the RRM domain, with its atypical RNP motifs, contributes to homodimerization mainly by hydrophobic interactions: the side-chain of Met236 in the beta4 strand of one Nup35 molecule is sandwiched by the aromatic side-chains of Phe178 in the beta1 strand and Trp209 in the beta3 strand of the other Nup35 molecule in the dimer. This structure reveals a new homodimerization mode of the RRM domain.

The topa quinone (TPQ) cofactor of copper amine oxidase is generated by copper-assisted self-processing of the precursor protein. Metal ion specificity for TPQ biogenesis has been reinvestigated with the recombinant phenylethylamine oxidase from Arthrobacter globiformis. Besides Cu2+ ion, some divalent metal ions such as Co2+, Ni2+, and Zn2+ were also bound to the metal site of the apoenzyme so tightly that they were not replaced by excess Cu2+ ions added subsequently. Although these noncupric metal ions could not initiate TPQ formation under the atmospheric conditions, we observed slow spectral changes in the enzyme bound with Co2+ or Ni2+ ion under the dioxygen-saturating conditions. Resonance Raman spectroscopy and titration with phenylhydrazine provided unambiguous evidence for TPQ formation by Co2+ and Ni2+ ions. Steady-state kinetic analysis showed that the enzymes activated by Co2+ and Ni2+ ions were indistinguishable from the corresponding metal-substituted enzymes prepared from the native copper enzyme (Kishishita, S., Okajima, T., Kim, M., Yamaguchi, H., Hirota, S., Suzuki, S., Kuroda, S., Tanizawa, K., and Mure, M. (2003) J. Am. Chem. Soc. 125, 1041-1055). X-ray crystallographic analysis has also revealed structural identity of the active sites of Co- and Ni-activated enzymes with Cu-enzyme. Thus Cu2+ ion is not the sole metal ion assisting TPQ formation. Co2+ and Ni2+ ions are also capable of forming TPQ, though much less efficiently than Cu2+.

The role of the active site Cu(2+) of phenylethylamine oxidase from Arthrobacter globiformis (AGAO) has been studied by substitution with other divalent cations, where we were able to remove >99.5% of Cu(2+) from the active site. The enzymes reconstituted with Co(2+) and Ni(2+) (Co- and Ni-AGAO) exhibited 2.2 and 0.9% activities, respectively, of the original Cu(2+)-enzyme (Cu-AGAO), but their K(m) values for amine substrate and dioxygen were comparable. X-ray crystal structures of the Co- and Ni-AGAO were solved at 2.0-1.8 A resolution. These structures revealed changes in the metal coordination environment when compared to that of Cu-AGAO. However, the hydrogen-bonding network around the active site involving metal-coordinating and noncoordinating water molecules was preserved. Upon anaerobic mixing of the Cu-, Co-, and Ni-AGAO with amine substrate, the 480 nm absorption band characteristic of the oxidized form of the topaquinone cofactor (TPQ(ox)) disappeared rapidly (< 6 ms), yielding the aminoresorcinol form of the reduced cofactor (TPQ(amr)). In contrast to the substrate-reduced Cu-AGAO, the semiquinone radical (TPQ(sq)) was not detected in Co- and Ni-AGAO. Further, in the latter, TPQ(amr) reacted reversibly with the product aldehyde to form a species with a lambda(max) at around 350 nm that was assigned as the neutral form of the product Schiff base (TPQ(pim)). Introduction of dioxygen to the substrate-reduced Co- and Ni-AGAO resulted in the formation of a TPQ-related intermediate absorbing at around 360 nm, which was assigned to the neutral iminoquinone form of the 2e(-)-oxidized cofactor (TPQ(imq)) and which decayed concomitantly with the generation of TPQ(ox). The rate of TPQ(imq) formation and its subsequent decay in Co- and Ni-AGAO was slow when compared to those of the corresponding reactions in Cu-AGAO. The low catalytic activities of the metal-substituted enzymes are due to the impaired efficiencies of the oxidative half-reaction in the catalytic cycle of amine oxidation. On the basis of these results, we propose that the native Cu(2+) ion has essential roles such as catalyzing the electron transfer between TPQ(amr) and dioxygen, in part by providing a binding site for 1e(-)- and 2e(-)-reduced dioxygen species to be efficiently protonated and released and also preventing the back reaction between the product aldehyde and TPQ(amr).

The quinone cofactor TPQ in copper amine oxidase is generated by posttranslational modification of an active site tyrosine residue. Using X-ray crystallography, we have probed the copper-dependent autooxidation process of TPQ in the enzyme from Arthrobacter globiformis. Apo enzyme crystals were anaerobically soaked with copper; the structure determined from this crystal provides a view of the initial state: the unmodified tyrosine coordinated to the bound copper. Exposure of the copper-bound crystals to oxygen led to the formation of freeze-trapped intermediates; structural analyses indicate that these intermediates contain dihydroxyphenylalanine quinone and trihydroxyphenylalanine. These are the first visualized intermediates during TPQ biogenesis in copper amine oxidase.

p53 plays a key role in DNA damage-induced apoptosis. Recent studies have reported that the phosphatidylinositol 3-OH-kinase-Akt pathway inhibits p53-mediated transcription and apoptosis, although the underlying mechanisms have yet to be determined. Mdm2, a ubiquitin ligase for p53, plays a central role in regulation of the stability of p53 and serves as a good substrate for Akt. In this study, we find that expression of Akt reduces the protein levels of p53, at least in part by enhancing the degradation of p53. Both Akt expression and serum treatment induced phosphorylation of Mdm2 at Ser186. Akt-mediated phosphorylation of Mdm2 at Ser186 had little effect on the subcellular localization of Mdm2. However, both Akt expression and serum treatment increased Mdm2 ubiquitination of p53. The serum-induced increase in p53 ubiquitination was blocked by LY294002, a phosphatidylinositol 3-OH-kinase inhibitor. Moreover, when Ser186 was replaced by Ala, Mdm2 became resistant to Akt enhancement of p53 ubiquitination and degradation. Collectively, these results suggest that Akt enhances the ubiquitination-promoting function of Mdm2 by phosphorylation of Ser186, which results in reduction of p53 protein. This study may shed light on the mechanisms by which Akt promotes survival, proliferation, and tumorigenesis.

The catalytic reaction of copper/topa quinone (TPQ) containing amine oxidase consists of the initial, well-characterized, reductive half-reaction and the following, less studied, oxidative half-reaction. We have analyzed the oxidative half-reaction catalyzed by phenylethylamine oxidase from Arthrobacter globiformis (AGAO) by rapid-scan stopped-flow measurements. Upon addition of dioxygen to the substrate-reduced AGAO at pH 8.2, the absorption bands derived from the semiquinone (TPQ(sq)) and aminoresorcinol forms of the TPQ cofactor disappeared within the dead time (<1 ms) of the measurements, indicating that the reaction of the substrate-reduced enzyme with dioxygen is very rapid. Concomitantly, an early intermediate exhibiting an absorption band at about 410 nm was formed, which then decayed with a rate constant of 390 +/- 50 s(-1). This intermediate was detected more prominently in the reaction in D2O buffer (pD 8.1) and was assigned to a Cu(II)-peroxy species. The assignment was based on the observation that addition of H2O2 to the substrate-reduced AGAO under anaerobic conditions led to the formation of a new band at about 415 nm, accompanied by partial quenching of absorption bands derived from TPQ(sq). Other intermediates exhibiting absorption bands at about 310 and 340 nm were also observed in the oxidative half-reaction. Kinetics of the disappearance of these latter bands did not correspond with that of the Cu(II)-peroxy band at 410 nm but did well with that of the increase of the 480 nm absorption band due to the reoxidized TPQ. Rapid increase of the absorption in the 320-370 nm region was also observed for the reaction of the substrate-reduced, Ni-substituted enzyme with dioxygen. On the basis of these results, a possible mechanism is proposed for the oxidative half-reaction of the bacterial copper amine oxidase.