Features of interactions responsible for antifungal activity against resistant type cytochrome bc1: A data-driven analysis based on the binding free energy at the atomic level.

Quinone outside inhibitors (QoIs), which inhibit the mitochondrial respiratory system by binding to the Qo site of Complex III in fungi, are widely used as pesticides with broad spectrum antifungal activity. However, excessive use of QoIs leads to pesticide resistance through mutation of amino acid residues in the Qo site. Recently, metyltetraprole, a novel QoI that is effective against wild-type and resistant mutant fungi, was developed. Interestingly, metyltetraprole has a very similar structure to other QoIs, azoxystrobin and pyraclostrobin, which do not act on resistant mutants. However, it is unknown how slight structural differences in these inhibitors alter their effectiveness towards fungi with amino acid mutations in the Qo site of Complex III. Therefore, we studied the features of interactions of inhibitors effective towards resistant mutants by quantitatively comparing the interaction profiles of three QoIs at the atomic level. First, we reproduced the binding affinity by the thermodynamic integration (TI) method, which treated explicitly environmental molecules and considered the pseudo-binding pathway. As such, a good correlation (R2 = 0.74) was observed between the binding free energy calculated using the TI method and experimentally observed pIC50 value in 12 inhibitor-target pairs, including wild-type and mutant Complex III in two fungal species, Zymoseptoria tritici and Pyrenophora teres. Trajectory analysis of this TI calculation revealed that the effectiveness against resistant mutant fungi strongly depended on the interaction of constituent parts of the inhibitor disposed near the active center of the target protein. Specifically, the key in the effectiveness against resistant mutant fungi is that the corresponding component part, tetrazolinone moiety of metyltetraprole, traded off Coulomb and van der Waals interactions in response to subtle changes in the binding pose.

Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase.

The Hsp90 molecular chaperone and its Cdc37 cochaperone help stabilize and activate more than half of the human kinome. However, both the mechanism by which these chaperones assist their "client" kinases and the reason why some kinases are addicted to Hsp90 while closely related family members are independent are unknown. Our structural understanding of these interactions is lacking, as no full-length structures of human Hsp90, Cdc37, or either of these proteins with a kinase have been elucidated. Here we report a 3.9 angstrom cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex. Surprisingly, the two lobes of Cdk4 are completely separated with the ß4-ß5 sheet unfolded. Cdc37 mimics part of the kinase N lobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase ß5 strand and interacts with exposed N- and C-lobe interfaces, protecting the kinase in a trapped unfolded state. On the basis of this structure and an extensive amount of previously collected data, we propose unifying conceptual and mechanistic models of chaperone-kinase interactions.

Colonic distention at screening CT colonography: role of spasmolytic agents and body habitus.

Sufficient colonic dilation is important when using CT colonography (CTC) for colorectal cancer screening. We investigated the effect of antispasmodic agents and the patient body habitus on the degree of colonic dilation in screening CTC.We assessed the effect of clinical characteristics [age, gender, body mass index (BMI), and the presence of diverticula] and the use of antispasmodics on colonic distention in 140 patients who underwent CTC for colorectal cancer screening. The CTC was performed in both the supine- and prone positions. Seventy patients received antispasmodics prior to CT examination and the other 70 did not. Colonic distention was scored using a 5-point scale: 1=collapsed, 2=poorly visualized, 3=visualized but underdistended, 4=acceptable, and 5=excellent. Images scored as 4 or 5 were considered to be of diagnostic quality. The mean visual evaluation score was significantly higher in the supine- than the prone position (4.2±0.5 vs. 4.0±0.5, p<0.01). For the supine position, only the use of antispasmodic was statistically associated with sufficient colonic dilation by univariate logistic analysis (odds ratio=2.365, p=0.03). For the prone position, age, BMI, and the use of antispasmodic were statistically associated with sufficient colonic dilation by multivariate analysis. The odds ratio of these parameters was 0.955 (p=0.02), 0.874 (p=0.03), and 2.391 (p=0.02), respectively.We obtained sufficient colonic dilation with an antispasmodic for CTC in both positions. Younger age and a lower BMI were also associated with better colonic dilation in the prone position.

[A case of a patient with advanced rectal cancer and urinary bladder fistula surviving for 6 years after chemoradiotherapy without surgery].

A 60-year-old man with advanced rectal cancer and urinary bladder fistula received preoperative chemoradiotherapy with S-1(120mg/m / 2day)on weekdays and concurrent radiotherapy(65 Gy). After chemoradiotherapy, the clinical symptoms resolved and the tumor shrunk, as observed on endoscopic and radiologic examinations. However, remnant cancer was suspected; therefore, modified oxaliplatin, 5-fluorouracil, and Leucovorin(mFOLFOX6)therapy was initiated, although it was stopped after 3 cycles because of numbness in the lower extremities. Finally, clinical and pathological complete response(CR) was achieved by administering additional doses of S-1 for approximately 1 year after treatment initiation; CR was confirmed by using endoscopy and computed tomography(CT), and there has been no recurrence for 6 years. This case suggests that treatment without surgery is a viable alternative for advanced rectal cancer with pathological CR after chemoradiotherapy.

Spinal imaging features in Japanese patients with Marfan syndrome: a case-control study.

PURPOSE: The purpose of our study was to evaluate the morphology of the lumbosacral spine, i.e. the dura and vertebral body shape, of Japanese patients with Marfan syndrome (MFS) by comparing it with sex- and age-matched controls. MATERIALS AND METHODS: Spinal MR or CT images of 32 MFS patients and 32 controls were retrospectively reviewed. The anteroposterior dural sac diameter (DSD), anteroposterior vertebral body diameter (VBD), and vertebral body height (VBH) were measured from L1 to S1 levels and the dural sac ratio [DSR = (DSD/VBD)] and vertebral body aspect ratio [VAR = (VBH/VBD)] were calculated. RESULTS: At each level, mean DSD and DSR were significantly higher in MFS patients; VBD was not. The cutoff values for DSR to differentiate between MFS patients and the controls were 0.59, 0.46, 0.42, 0.45, 0.47, and 0.47 from the level of L1 to S1. At a sensitivity of 93.8 % and a specificity of 84.4 % the cutoff value at S1 was most diagnostic. In MFS patients VAR was significantly higher at L3 and L4. CONCLUSION: Our cutoff value for DSR >0.47 at S1 may help to identify MFS in the Japanese population. A square-like appearance of the L3 and L4 vertebral bodies is a supplementary finding in MFS patients.

Crystal structure of the guanylate kinase domain from discs large homolog 1 (DLG1/SAP97).

Discs large homolog 1 (DLG1/SAP97) is involved in the development and regulation of neuronal and immunological synapses. DLG1 is a member of the membrane associated guanylate kinase (MAGUK) family of proteins, which function as molecular scaffolds. The C-terminal guanylate kinase (GK) domain of DLG1 binds peptides with a phosphorylated serine residue. In this study, we solved the crystal structure of the GK domain of human DLG1. The C-terminal tail of DLG1 is bound to the peptide-binding site of an adjacent symmetry-related DLG1 GK molecule. The binding direction of the C-terminal tail to the peptide-binding site is opposite to that of the phosphorylated LGN peptide in complex with the rat DLG1 GK domain. The C-terminal tail forms a 310 helix, which is also different from the conformation of the phosphorylated LGN peptide. Nevertheless, the side chain interactions of the C-terminal tail with the DLG1 GK domain are similar to those of the phosphorylated LGN peptide.

Biochemical and structural studies on the high affinity of Hsp70 for ADP.

The molecular chaperone 70-kDa heat shock protein (Hsp70) is driven by ATP hydrolysis and ADP-ATP exchange. ADP dissociation from Hsp70 is reportedly slow in the presence of inorganic phosphate (P(i) ). In this study, we investigated the interaction of Hsp70 and its nucleotide-binding domain (NBD) with ADP in detail, by isothermal titration calorimetry measurements and found that Mg(2+) ion dramatically elevates the affinity of Hsp70 for ADP. On the other hand, P(i) increased the affinity in the presence of Mg(2+) ion, but not in its absence. Thus, P(i) enhances the effect of the Mg(2+) ion on the ADP binding. Next, we determined the crystal structures of the ADP-bound NBD with and without Mg(2+) ion. As compared with the Mg(2+) ion-free structure, the ADP- and Mg(2+) ion-bound NBD contains one Mg(2+) ion, which is coordinated with the ß-phosphate group of ADP and associates with Asp10, Glu175, and Asp199, through four water molecules. The Mg(2+) ion is also coordinated with one P(i) molecule, which interacts with Lys71, Glu175, and Thr204. In fact, the mutations of Asp10 and Asp199 reduced the affinity of the NBD for ADP, in both the presence and the absence of P(i) . Therefore, the Mg(2+) ion-mediated network, including the P(i) and water molecules, increases the affinity of Hsp70 for ADP, and thus the dissociation of ADP is slow. In ADP-ATP exchange, the slow ADP dissociation might be rate-limiting. However, the nucleotide-exchange factors actually enhance ADP release by disrupting the Mg(2+) ion-mediated network.

[A case of pancreatic cancer treated by gemcitabine with sequential radiotherapy].

A 65-year-old man suffering from acute pancreatitis underwent MRI scanning, which revealed a low signal on the T1 and T2 sequences, and hypovascularity in arterial phase in the head of the pancreas. This corresponded to the area showing the absence of the lower common bile duct. FDG-PET was highly suggestive of pancreatic cancer (T4N1M0, Stage IVa) with lymph node metastasis. He was treated with systemic chemotherapy using gemcitabine (GEM) followed by radiotherapy. His symptoms gradually improved with a reduction in size of the primary lesion. The patient has been receiving systemic chemotherapy using S-1 without recurrence.

The C-terminal BAG domain of BAG5 induces conformational changes of the Hsp70 nucleotide-binding domain for ADP-ATP exchange.

ADP-ATP exchange by the molecular chaperone Hsp70 is enhanced by several cochaperones. BAG5 consists of five BAG domains and associates with the nucleotide-binding domain (NBD) of Hsp70. The overexpression of BAG5 in the cytosol reportedly disturbs Hsp70-mediated protein refolding and induces Parkinson's disease. In the present study, we found that the fifth BAG domain (BD5) of BAG5 is responsible for the interaction between Hsp70 and BAG5. We also determined the crystal structures of the BD5*NBD complex. BD5 binding caused two different types of NBD conformational changes, which both disrupted the nucleotide-binding groove. In fact, BD5 reduced the affinity of the NBD for ADP. Moreover, BD5, as well as the full-length BAG5, accelerated Hsp70-mediated refolding in an in vitro assay. Therefore, BAG5 can function as the nucleotide exchange factor of Hsp70 for the enhancement of protein refolding.

Direct inter-subdomain interactions switch between the closed and open forms of the Hsp70 nucleotide-binding domain in the nucleotide-free state.

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.

Detection of hepatocellular carcinoma using double-echo FLASH sequence during the hepatic arterial phase.

PURPOSE: The purpose of this study was to compare the performance of in-phase and opposed-phase gradient-recalled echo (GRE) pulse sequences in paramagnetic contrast-enhanced magnetic resonance (MR) imaging of hepatocellular carcinomas (HCCs) during the hepatic arterial phase. MATERIAL AND METHODS: Thirty-four patients with 84 lesions with known or suspected HCCs, nine of whom had a fatty liver, were examined with double-echo GRE techniques under 1.5T before and 30 s after injection of gadopentenate dimeglumine at a dose of 0.1 mmol/kg. Echo times were 2.4 ms (opposed phase) and 5.0 ms (in phase). Contrast enhancement of the HCC detected in both in-phase and opposed-phase images was evaluated. The liver signal-to-noise ratio (SNR), lesion-liver contrast-to-noise ratio (CNR), and enhancement ratio (ER) were calculated for the largest lesion of each patient. RESULTS: In dynamic gadolinium-enhanced images of the 84 HCCs, 81 (96.4%) were detected in both in-phase and opposed-phase images, two (2.4%) were detected in only in-phase images, and one (1.2%) was detected only in opposed-phase images. The liver SNR, CNR, and ER were 46.7+/-16.1, 15.2+/-10.3, and 0.637+/-0.268 for in-phase images, and 48.9+/-16.9, 16.3+/-11.8, and 0.647+/-0.309 for opposed-phase images, respectively. In patients with a fatty liver, the SNR, CNR, and ER were 46.0+/-18.1, 21.7+/-17.9, and 0.525+/-0.231 for in-phase images, and 44.3+/-18.7, 26.0+/-21.3, and 0.793+/-0.124 for opposed-phase images, respectively. No significant statistical differences were found between the in-phase and opposed-phase images. CONCLUSION: Opposed-phase GRE imaging is equivalent to in-phase GRE sequences in patients with or without fatty liver for detection of HCC in dynamic gadolinium-enhanced images.

[Clinical evaluation of gastric diverticulum on indirect radiograph for gastric mass survey].

The aim of this study was to evaluate the clinical conditions of gastric diverticulum. Fifty-four patients with gastric diverticulum (20 men and 34 women among 34,314 patients who underwent medical check-ups) were evaluated on indirect radiographs, for an incidence of 0.16% among the total number of examined cases, a rate lower than that of previous reports. Almost all cases were asymptomatic, had a single diverticulum, and showed a saccular shape. The age distribution indicated higher frequencies in the 5th and 6th decades, and the posterior wall of the fornix was the most common location. Size ranged from 0.6 cm to 12 cm, and 41 cases (75.9%) were between 1.0 cm and 4.0 cm in size. This entity should be kept in mind when reading radiographs of upper gastrointestinal series as well as recognition of pseudodiverticulum and aberrant pancreas as noted for the stomach in several past report. Diverticulum on the cardia, which was previously classified as gastric diverticulum, should be excluded because of the possibility of normal variation.

Evaluation of hypervascular hepatocellular carcinoma in cirrhotic liver by means of helical CT: comparison of different contrast medium concentrations within the same patient.

PURPOSE: To compare enhancement of the aorta, portal vein, liver, and tumor with contrast medium of a higher iodine concentration to that with one of a standard iodine concentration for liver dynamic CT within the same patient with known liver cirrhosis and hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Fifty-three patients with known cirrhosis and HCC underwent repeat computed tomographic (CT) examinations within three months, the first with either 300 (n=26) or 370 mgI/ml (n=27) of nonionic contrast material. In the second examination, only concentration was altered. In 28 patients, weight was equal to or more than 65 kg, and in 25 patients it was less than 65 kg. The CT numbers (HU) of the aorta, portal vein, liver, and tumor were obtained, and CT attenuation was compared between the two concentration studies. The degree of enhancement was scored qualitatively. RESULTS: Mean enhancement values of the aorta, liver, portal vein, and tumor were greater with the 370 mgI/ml injections than with the 300 mgI/ml injections throughout the study. In visual analysis, the difference in aortic, portal venous, liver, and tumor enhancement was not statistically significant between the two groups in patients weighing less than 65 kg. However, in patients weighing 65 kg or more, strong aortic and portal venous enhancement (rated as good or excellent) occurred more frequently with the 370 mgI/ml injections than with the 300 mgI/ml injections. CONCLUSION: Higher contrast enhancement was achieved in patients who received 370 mgI/ml of contrast material, resulting in better tissue contrast between liver parenchyma and HCCs. However, this difference was not visually significant in patients weighing less than 65 kg.