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Abstract 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 α-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 α-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 α-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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (j36 GIu-~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 A 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 (K4) 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Background: Epithelial neutrophil-activating peptide 78 (ENA-78) and interferon -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 (), idiopathic NSIP () and healthy subjects () 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. [Copyright &y& Elsevier]

Defensins are cysteine-rich cationic antimicrobial peptides that play an important role in innate immunity and are known to contribute to the regulation of host adaptive immunity. In addition to direct antimicrobial activities, it has been recently reported that α-defensins, mainly present in neutrophils in the lung, have a cytotoxic effect and induce IL-8 production from airway epithelial cells. Although β-defensins are expressed in epithelial cells in various tissues, including lung, there are no reports of their effects on cytokine synthesis in airway epithelial cells. The aim of the present study was to determine the effects of both α- and β-defensins on the cytokine production, transcription factor binding activity, and cytotoxicity in primary cultured human bronchial epithelial cells (HBECs). We used human neutrophil peptide-1 (HNP-1; α-defensin) and human β-defensin-2 (HBD-2) to stimulate HBECs. The results showed that treatment of HBECs with HNP-1, but not HBD-2, increased IL-8 and IL-1β mRNA expression in a dose-dependent manner and also enhanced IL-8 protein secretion and NF-κB DNA binding activity. The 24-h treatments with >20 μg/ml of HNP-1 or >50 μg/ml of HBD-2 were cytotoxic to HBECs. These results suggest that α- and β-defensins have different effects on cytokine synthesis by airway epithelial cells, and we speculate that they play different roles in inflammatory lung diseases. [ABSTRACT FROM AUTHOR]

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 α-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 α-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 a-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. [ABSTRACT FROM AUTHOR]

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 Å resolution at 100 K using synchrotron radiation. The protein crystals belonged to the hexagonal space group P6522, with unit-cell parameters a = b = 128.0, c = 286.2 Å. With four molecules per asymmetric unit, the crystal volume per unit protein mass ( VM) was 3.2 Å3 Da−1 and the solvent content was 62%. [ABSTRACT FROM AUTHOR]

Abstract: The photoswitching behavior of green fluorescent proteins (GFPs) or GFP-like proteins is increasingly recognized as a new technique for optical marking. Recently, Ando and his colleagues developed a new green fluorescent protein Dronpa, which possesses the unique photochromic property of being photoswitchable in a non-destructive manner. To better understand this mechanism, we determined the crystal structures of a new GFP Dronpa and its mutant C62S, at 1.9Å and 1.8Å, respectively. Determination of the structures demonstrates that a unique hydrogen-bonding network and the sulfur atom of the chromophore are critical to the photoswitching property of Dronpa. Reversible photoswitching was lost in cells expressing the Dronpa-C62S upon repetitive irradiation compared to the native protein. Structural and mutational analyses reveal the chemical basis for the functional properties of photoswitchable fluorescent proteins and provide the basis for subsequent coherent engineering of this subfamily of Dronpa homolog’s. [Copyright &y& Elsevier]