Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium.

Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.

The crystal structure of the active domain of Anopheles anti-platelet protein, a powerful anti-coagulant, in complex with an antibody.

Blood clotting is a vitally important process that must be carefully regulated to prevent blood loss on one hand and thrombosis on the other. Severe injury and hemophilia may be treated with pro-coagulants, whereas risk of obstructive clotting or embolism may be reduced with anti-coagulants. Anti-coagulants are an extremely important class of drug, one of the most widely used types of medication, but there remains a pressing need for novel treatments, however, as present drugs such as warfarin have significant drawbacks. Nature provides a number of examples of anti-coagulant proteins produced by blood-sucking animals, which may provide templates for the development of new small molecules with similar physiological effects. We have, therefore, studied an Anopheles anti-platelet protein from a malaria vector mosquito and report its crystal structure in complex with an antibody. Overall the protein is extremely sensitive to proteolysis, but the crystal structure reveals a stable domain built from two helices and a turn, which corresponds to the functional region. The antibody raised against Anopheles anti-platelet protein prevents it from binding collagen. Our work, therefore, opens new avenues to the development of both novel small molecule anti-clotting agents and anti-malarials.

Capturing the hemoglobin allosteric transition in a single crystal form.

Allostery in many oligomeric proteins has been postulated to occur via a ligand-binding-driven conformational transition from the tense (T) to relaxed (R) state, largely on the basis of the knowledge of the structure and function of hemoglobin, the most thoroughly studied of all allosteric proteins. However, a growing body of evidence suggests that hemoglobin possesses a variety of intermediates between the two end states. As such intermediate forms coexist with the end states in dynamic equilibrium and cannot be individually characterized by conventional techniques, very little is known about their properties and functions. Here we present complete structural and functional snapshots of nine equilibrium conformers of human hemoglobin in the half-liganded and fully liganded states by using a novel combination of X-ray diffraction analysis and microspectrophotometric O2 equilibrium measurements on three isomorphous crystals, each capturing three distinct equilibrium conformers. Notably, the conformational set of this crystal form varies according to shifts in the allosteric equilibrium, reflecting the differences in hemoglobin ligation state and crystallization solution conditions. We find that nine snapshot structures cover the complete conformational space of hemoglobin, ranging from T to R2 (the second relaxed quaternary structure) through R, with various relaxed intermediate forms between R and R2. Moreover, we find a previously unidentified intermediate conformer, between T and R, with an intermediate O2 affinity, sought by many research groups over a period of decades. These findings reveal a comprehensive picture of the equilibrium conformers and transition pathway for human hemoglobin.

Structural insight into the essential PB1-PB2 subunit contact of the influenza virus RNA polymerase.

Influenza virus RNA-dependent RNA polymerase is a multi-functional heterotrimer, which uses a 'cap-snatching' mechanism to produce viral mRNA. Host cell mRNA is cleaved to yield a cap-bearing oligonucleotide, which can be extended using viral genomic RNA as a template. The cap-binding and endonuclease activities are only activated once viral genomic RNA is bound. This requires signalling from the RNA-binding PB1 subunit to the cap-binding PB2 subunit, and the interface between these two subunits is essential for the polymerase activity. We have defined this interaction surface by protein crystallography and tested the effects of mutating contact residues on the function of the holo-enzyme. This novel interface is surprisingly small, yet, it has a crucial function in regulating the 250 kDa polymerase complex and is completely conserved among avian and human influenza viruses.

Structures and oxygen affinities of crystalline human hemoglobin C (ß6 Glu->Lys) in the R and R2 quaternary structures.

Recent crystallographic studies suggested that fully liganded human hemoglobin can adopt multiple quaternary conformations that include the two previously solved relaxed conformations, R and R2, whereas fully unliganded deoxyhemoglobin may adopt only one T (tense) quaternary conformation. An important unanswered question is whether R, R2, and other relaxed quaternary conformations represent different physiological states with different oxygen affinities. Here, we answer this question by showing the oxygen equilibrium curves of single crystals of human hemoglobin in the R and R2 state. In this study, we have used a naturally occurring mutant hemoglobin C (ß6 Glu→Lys) to stabilize the R and R2 crystals. Additionally, we have refined the x-ray crystal structure of carbonmonoxyhemoglobin C, in the R and R2 state, to 1.4 and 1.8 Å resolution, respectively, to compare precisely the structures of both types of relaxed states. Despite the large quaternary structural difference between the R and R2 state, both crystals exhibit similar noncooperative oxygen equilibrium curves with a very high affinity for oxygen, comparable with the fourth oxygen equilibrium constant (K(4)) of human hemoglobin in solution. One small difference is that the R2 crystals have an oxygen affinity that is 2-3 times higher than that of the R crystals. These results demonstrate that the functional difference between the two typical relaxed quaternary conformations is small and physiologically less important, indicating that these relaxed conformations simply reflect a structural polymorphism of a high affinity relaxed state.

Crystallization and preliminary crystallographic studies of the butyrolactone autoregulator receptor protein (BarA) from Streptomyces virginiae.

The Streptomyces butyrolactone autoregulator receptor protein (BarA) is a DNA-binding protein that regulates the biosynthesis of the antibiotic virginiamycin. In this study, BarA from S. virginiae was overexpressed in Escherichia coli, purified and crystallized. Crystals of purified protein have been grown that diffracted to beyond 3.0 A resolution at 100 K using synchrotron radiation. The protein crystals belonged to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 128.0, c = 286.2 A. With four molecules per asymmetric unit, the crystal volume per unit protein mass (V(M)) was 3.2 A(3) Da(-1) and the solvent content was 62%.

Successful Bridge-to-Recovery Treatment in a Young Patient with Fulminant Eosinophilic Myocarditis: Roles of a Percutaneous Ventricular Assist Device and Endomyocardial Biopsy.

Eosinophilic myocarditis (EM) is a rare condition characterized by myocardial eosinophilic infiltration due to various underlying etiologies. The patient with EM may benefit from appropriate use of mechanical circulatory support (MCS) that acts as a bridge to myocardial recovery in response to effective immunosuppressive therapy. A 16-year-old boy presented with cardiogenic shock due to fulminant myocarditis, for which a percutaneous ventricular assist device (PVAD) was immediately inserted. Based on the histological diagnosis of EM, immunosuppressive therapy was immediately commenced, leading to improvement of left-ventricular ejection fraction (27% to 47%). The PVAD was successfully removed on day 7. Cardiac magnetic resonance imaging and dual-tracer myocardial scintigraphy suggested limited extent of irreversible myocardial damage. For fulminant EM, the short-term use of PVAD, together with immunosuppressive therapy guided by an immediate histological investigation, may be an effective bridging strategy to myocardial recovery.

[A case of sarcoidosis with diabetes insipidus].

A 15-year-old girl was admitted to our hospital because of polydipsia, polyuria, bilateral hilar lymphadenopathy and uveitis. A diagnosis of sarcoidosis with central diabetes insipidus was made by radiological, serological, bronchoalveolar lavage examinations, fluid restriction test and vasopression test. Prednisolone therapy improved all of her clinical findings except diabetes insipidus. So she had to continue intranasal 1-desamino-8-arginine vasopressin (DDAVP) therapy. In addition, we reviewed the clinical features of 27 patients of sarcoidosis with diabetes insipidus reported in Japan. They included 12 patients in young men and 21 patients having uveitis. These patients showed low frequency of lung complications in comparison with sarcoidosis without diabetes insipidus. Steroid therapy improved the symptoms of diabetes insipidus in only 3 patients, and all these 3 patients started steroid therapy within 1 month after the onset. Therefore we think that early diagnosis and treatment are important. Though central neurosarcoidosis was generally considered to have poor prognosis, there were only 3 patients who had recurrence by steroid tapering.

G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity.

The recognition specificity of monoclonal antibodies (mAbs) has made mAbs among the most frequently used tools in both basic science research and in clinical diagnosis and therapies. Precise determination of the epitope allows the development of epitope tag systems to be used with recombinant proteins for various purposes. Here we describe a new family of tag derived from the epitope recognized by a highly specific mAb G196. The minimal epitope was identified as the five amino acid sequence Asp-Leu-Val-Pro-Arg. Permutation analysis was used to characterize the binding requirements of mAb G196, and the variable regions of the mAb G196 were identified and structurally analyzed by X-ray crystallography. Isothermal titration calorimetry revealed the high affinity (Kd = 1.25 nM) of the mAb G196/G196-epitope peptide interaction, and G196-tag was used to detect several recombinant cytosolic and nuclear proteins in human and yeast cells. mAb G196 is valuable for developing a new peptide tagging system for cell biology and biochemistry research.

[A case of rheumatoid lung complicated by SELAPINA-induced pneumonia].

SELAPINA is generic product of PL granule which is one of the most common forms of combination remedies for the common cold in Japan, and includes acetaminophen. We report a case of SELAPINA-induced pneumonia successfully treated with glucocorticoid pulse therapy followed by orally administered prednisolone. A 68-year-old woman, who had been treated for rheumatoid arthiritis with pulmonary involvement, took SELAPINA with an antibiotic for 6 days because of her cold symptoms. She then suffered a high fever, cough, dyspnea, vomiting and diarrhea. Chest radiograph and high-resolution computed tomography (HRCT) scan revealed diffuse interstitial shadows. SELAPINA-induced pneumonia was diagnosed because the blastoid transformation test using her peripheral blood lymphocytes was positive on stimulation with SELAPINA, but negative for other medicines.

[Three cases of chronic bird fancier's lung associated with the use of feather bedclothes].

We report here 3 cases of chronic bird fancier's lung diagnosed immunologically using antibodies to pigeon dropping extract. None of the patients were bird fanciers but had indirect exposure to birds in their living environment, and had been using feather-filled duvets or pillows for a long time. Two of 3 cases were positive for environmental provocation tests and 2 cases had pathological findings of hypersensitivity pneumonitis such as multinucleated giant cells and non-necrotizing epithelioid cell granulomas. One case was resistant to steroid therapy alone and was successfully treated by coadministration of prednisolone and cyclosporin A. Another case was treated by steroid alone but died of acute exacerbation of unknown cause. These cases suggest that not only feathers but two or more kinds of bird-related antigens were involved in the sensitization immunology and development of bird fancier's disease, and that clinicians should perform thorough history taking with environmental surveillance relevant to birds.

Expression of HSP47 in usual interstitial pneumonia and nonspecific interstitial pneumonia.

BACKGROUND: Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagens, and its expression is increased in various fibrotic diseases. The aim of this study was to determine whether quantitative immunohistochemical evaluation of the expression levels of HSP47, type I procollagen and alpha-smooth muscle actin (SMA) allows the differentiation of idiopathic usual interstitial pneumonia (UIP) from UIP associated with collagen vascular disease (CVD) and idiopathic nonspecific interstitial pneumonia (NSIP). METHODS: We reviewed surgical lung biopsy specimens of 19 patients with idiopathic UIP, 7 with CVD-associated UIP and 16 with idiopathic NSIP and assigned a score for the expression of HSP47, type I procollagen and alpha-SMA in type II pneumocytes and/or lung fibroblasts (score 0 = no; 1 = weak; 2 = moderate; 3 = strong staining). RESULTS: The expression level of HSP47 in type II pneumocytes of idiopathic UIP was significantly higher than in CVD-associated UIP and idiopathic NSIP. The expression of HSP47 in fibroblasts was significantly higher in idiopathic UIP and idiopathic NSIP than in CVD-associated UIP. The expression of type I procollagen in type II pneumocytes was significantly higher in idiopathic UIP than in idiopathic NSIP. The expression of type I procollagen in fibroblasts was not different in the three groups, while the expression of alpha-SMA in fibroblasts was significantly higher in idiopathic UIP than in idiopathic NSIP. CONCLUSION: Our results suggest the existence of different fibrotic pathways among these groups involved in the expression of HSP47 and type I procollagen.

Comparison of BALF concentrations of ENA-78 and IP10 in patients with idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia.

BACKGROUND: Epithelial neutrophil-activating peptide 78 (ENA-78) and interferon gamma-inducible protein 10 (IP10) belong to the CXC chemokine family and are considered to be important factors in idiopathic pulmonary fibrosis (IPF). Idiopathic nonspecific interstitial pneumonia (NSIP) and IPF are the two largest subsets of idiopathic interstitial pneumonias (IIP). In patients with NSIP, the prognosis is generally good compared with IPF. Therefore, the pathogenesis of NSIP seems to be different from that of IPF, but this remains unclear. The aim of the present study was to evaluate the contribution of ENA-78 and IP10 in the two diseases. METHODS: We measured the levels of ENA-78 and IP10 in serum and bronchoalveolar lavage fluid (BALF) of patients with IPF (n=17), idiopathic NSIP (n=10) and healthy subjects (n=12) by enzyme-linked immunosorbent assays. RESULTS: The level of ENA-78 in BALF was significantly higher in IPF patients than in NSIP patients and controls. Serum levels of ENA-78 and BALF levels of IP10 in NSIP patients were significantly higher than in patients with IPF and controls. In BALF of patients with NSIP, IP10 level significantly correlated with the absolute number of lymphocytes. In IPF patients, BALF IP10 levels also correlated with the proportion of lymphocytes in BALF. CONCLUSION: Our results show distinct profiles of CXC chemokines in IPF and NSIP, and suggest that these chemokines play an important role in inflammatory cell recruitment into the lung in patients with IIP.

[A case of drug-induced pneumonia possibly associated with simvastatin].

A 59-year-old woman was admitted because of general fatigue, cough and progressive dyspnea about 5 months after treatment with simvastatin for hyperlipidemia. A chest radiograph and computed tomography scans revealed ground glass and reticular opacities in the right middle and lower lung fields. The percentage of peripheral blood eosinophils was elevated. After simvastatin was discontinued and administration of prednisolone was started, eosinophilia and reticular shadows improved. Drug lymphocyte stimulation test (DLST) for simvastatin was positive, so we diagnosed drug induced eosinophilic pneumonia. Now hyperlipidemia is treated frequently with HMG-CoA reductase inhibitor, but there are few reports demonstrating lung injury by this drug. We should be aware of lung side effects of HMG-CoA reductase inhibitor.

[Familial summer-type hypersensitivity pneumonitis--case report and review of literature].

A 37-year-old-woman was admitted to our hospital because of chest bilateral reticular shadow with fever, cough, general malaise and exertional dyspnea in the summer. A diagnosis of summer-type hypersensitivity pneumonitis (SHP) was made by radiological, serological and histological examinations. Her 10-year-old daughter had chest reticular shadows and similar symptoms. These two patients were positive for serum anti-Trichosporon cutaneum (T. asahii, T. mucoides) antibodies and T. asahii was identified from cultured samples obtained from their house. They recovered spontaneously after hospitalization or isolation from the antigen. We reviewed the clinical features in sixteen families with familial SHP reported in Japan. Children aged under 15 years old accounted for 34% and there was no gender difference among patients. This finding differs from the conventionally defined features of patients with SHP. Measurements of serum KL-6, SP-D and SP-A seem to be useful for auxiliary diagnosis and monitoring the disease activity of SHP, especially in pediatric cases who cannot undergo invasive evaluation.

Structure-based analysis of domain function of chitin oligosaccharide deacetylase from Vibrio parahaemolyticus.

The X-ray crystal structure of chitin oligosaccharide deacetylase from Vibrio parahaemolyticus (Vp-COD) was determined at an 1.35 Å resolution. The amino acid sequence and structure of Vp-COD show that the enzyme comprises one polysaccharide deacetylase domain (PDD) and two carbohydrate-binding domains (CBDs). On the basis of a chitin-binding assay with Vp-COD and its CBDs-deleted mutant, it was confirmed that CBDs can adhere to chitin. The catalytic activity of the CBDs-deleted mutant was only mildly depressed compared with that of Vp-COD, indicating that CBDs are unlikely to affect the configuration of the active center residues in active site of PDD.

High-BAL fluid concentrations of RANTES in nonspecific interstitial pneumonia compared with usual interstitial pneumonia.

Chemokines such as regulated on activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP)-1, monocyte inflammatory protein (MIP)-lalpha have been reported to play an important role in the pathogenesis of interstitial lung diseases. Among idiopathic interstitial pneumonia (IIP), nonspecific interstitial pneumonia (NSIP) has elevated percentages of Lymphocytes in bronchoalveolar lavage (BAL) fluid compared with usual interstitial pneumonia (UIP). These chemokines are candidate mediators for lymphocyte attraction to the lung in NSIR Therefore, we measured the BAL fluid levels of RANTES, MCP-1 and MIP1-alpha in 15 patients with idiopathic NSIP, 20 with idiopathic UIP, 22 with sarcoidosis and 12 healthy volunteers to evaluate the contribution of these chemokines using enzyme-linked immunosorbent assays. The levels of RANTES in BAL fluid were significantly higher in patients with NSIP compared with healthy volunteers (P < 0.01), UIP and sarcoidosis (P < 0.05). In MCP-1, the levels in BAL fluid of NSIP and UIP patients were significantly elevated compared with healthy volunteers and sarcoidosis patients (P < 0.01). These results suggest that RANTES and MCP-1 in BAL fluid may play an important role in inflammatory cell recruitment to the lung in idiopathic NSIP as well as other interstitial lung diseases.

[A case of multiple pulmonary infarctions associated with Sjögren's syndrome and antipsychotic agent administration].

A 56-year-old woman presented with complaints of general malaise and left chest pain. Chest radiography and CT scanning revealed multiple nodules and infiltrations in both lung fields. Her symptoms diminished and the extent of some of the lung shadows decreased spontaneously. However, since new shadows appeared later in other parts of the lung, she was admitted to our hospital on September 3. A transbronchial lung biopsy was not adequate for diagnosing a particular disease. But thoracoscopic lung biopsy specimens revealed necrosis with localized pleural fibrosis, and so a diagnosis of pulmonary infarction was made. The patient did not have any underlying disease or coagulation abnormalities, but Sjögren's syndrome and an antipsychotic agent were suspected to be background factors.

Homopiperazine derivatives as a novel class of proteasome inhibitors with a unique mode of proteasome binding.

The proteasome is a proteolytic machinery that executes the degradation of polyubiquitinated proteins to maintain cellular homeostasis. Proteasome inhibition is a unique and effective way to kill cancer cells because they are sensitive to proteotoxic stress. Indeed, the proteasome inhibitor bortezomib is now indispensable for the treatment of multiple myeloma and other intractable malignancies, but is associated with patient inconvenience due to intravenous injection and emerging drug resistance. To resolve these problems, we attempted to develop orally bioavailable proteasome inhibitors with distinct mechanisms of action and identified homopiperazine derivatives (HPDs) as promising candidates. Biochemical and crystallographic studies revealed that some HPDs inhibit all three catalytic subunits (ß 1, ß 2 and ß 5) of the proteasome by direct binding, whereas bortezomib and other proteasome inhibitors mainly act on the ß5 subunit. Proteasome-inhibitory HPDs exhibited cytotoxic effects on cell lines from various hematological malignancies including myeloma. Furthermore, K-7174, one of the HPDs, was able to inhibit the growth of bortezomib-resistant myeloma cells carrying a ß5-subunit mutation. Finally, K-7174 had additive effects with bortezomib on proteasome inhibition and apoptosis induction in myeloma cells. Taken together, HPDs could be a new class of proteasome inhibitors, which compensate for the weak points of conventional ones and overcome the resistance to bortezomib.