Crystal structure of Legionella DotD: insights into the relationship between type IVB and type II/III secretion systems.

The Dot/Icm type IVB secretion system (T4BSS) is a pivotal determinant of Legionella pneumophila pathogenesis. L. pneumophila translocate more than 100 effector proteins into host cytoplasm using Dot/Icm T4BSS, modulating host cellular functions to establish a replicative niche within host cells. The T4BSS core complex spanning the inner and outer membranes is thought to be made up of at least five proteins: DotC, DotD, DotF, DotG and DotH. DotH is the outer membrane protein; its targeting depends on lipoproteins DotC and DotD. However, the core complex structure and assembly mechanism are still unknown. Here, we report the crystal structure of DotD at 2.0 Å resolution. The structure of DotD is distinct from that of VirB7, the outer membrane lipoprotein of the type IVA secretion system. In contrast, the C-terminal domain of DotD is remarkably similar to the N-terminal subdomain of secretins, the integral outer membrane proteins that form substrate conduits for the type II and the type III secretion systems (T2SS and T3SS). A short ß-segment in the otherwise disordered N-terminal region, located on the hydrophobic cleft of the C-terminal domain, is essential for outer membrane targeting of DotH and Dot/Icm T4BSS core complex formation. These findings uncover an intriguing link between T4BSS and T2SS/T3SS.

Crystal structure of glutamine receptor protein from Sulfolobus tokodaii strain 7 in complex with its effector L-glutamine: implications of effector binding in molecular association and DNA binding.

Genome analyses have revealed that members of the Lrp/AsnC family of transcriptional regulators are widely distributed among prokaryotes, including both bacteria and archaea. These regulatory proteins are involved in cellular metabolism in both global and specific manners, depending on the availability of the exogenous amino acid effectors. Here we report the first crystal structure of glutamine receptor protein (Grp) from Sulfolobus tokodaii strain 7, in the ligand-free and glutamine-bound (Grp-Gln) forms. Although the overall structures of both molecules are similar, a significant conformational change was observed at the ligand [L-glutamine (Gln)] binding site in the effector domain, which may be essential for further stabilization of the octameric structure, and in turn for facilitating DNA binding. In addition, we predicted promoter for the grp gene, and these analyses suggested the importance of cooperative binding to the protein. To gain insights into the ligand-induced conformational changes, we mutated all of the ligand-binding residues in Grp, and revealed the importance of Gln binding by biochemical and structural analyses. Further structural analyses showed that Y77 is crucial for ligand binding, and that the residues T132 and T134, which are highly conserved among the Lrp family of proteins, fluctuates between the active and inactive conformations, thus affecting protein oligomerization for DNA binding.

Purification, crystallization and preliminary X-ray crystallographic analysis of ST1022, a putative member of the Lrp/AsnC family of transcriptional regulators isolated from Sulfolobus tokodaii strain 7.

The Lrp/AsnC family of transcriptional regulators, also known as feast/famine transcriptional regulators, are widely distributed among bacteria and archaea. This family of proteins are likely to be involved in cellular metabolism, with exogenous amino acids functioning as effectors. Here, the crystallization and preliminary X-ray diffraction analysis of ST1022, a member of the Lrp/AsnC family of proteins, is reported with and without exogenous glutamine as the effector molecule. The crystals of native ST1022 and of the putative complex belong to the tetragonal space group I422, with unit-cell parameters a = b = 103.771, c = 73.297 A and a = b = 103.846, c = 73.992 A, respectively. Preliminary X-ray diffraction data analysis and molecular-replacement solution revealed the presence of one monomer per asymmetric unit.

Structure of the stand-alone RAM-domain protein from Thermus thermophilus HB8.

The stand-alone RAM (regulation of amino-acid metabolism) domain protein SraA from Thermus thermophilus HB8 (TTHA0845) was crystallized in the presence of zinc ions. The X-ray crystal structure was determined using a multiple-wavelength anomalous dispersion technique and was refined at 2.4 A resolution to a final R factor of 25.0%. The monomeric structure is a betaalphabetabetaalphabeta fold and it dimerizes mainly through interactions between the antiparallel beta-sheets. Furthermore, five SraA dimers form a ring with external and internal diameters of 70 and 20 A, respectively. This decameric structure is unique compared with the octameric and dodecameric structures found for other stand-alone RAM-domain proteins and the C-terminal RAM domains of Lrp/AsnC-family proteins.

Combined mediastinal node assessment by lymphadenectomy and intraoperative mediastinoscopy.

The purpose of this study was to determine the importance of combined mediastinal node assessment by lymphadenectomy and intraoperative mediastinoscopy to patients with left lung cancer. Forty-one patients with left lung cancer were divided into a group of 13 and a group of 28: in the first group, the aortic arch was mobilized, while in the second group the aortic arch was not mobilized. The mediastinal nodes of both groups were then dissected and mediastinoscopes were inserted into the operation field with an approach through thoracotomy to biopsy each mediastinal node. We compared nodal pathological assessment in these two groups. In the group with mobilized aortic arches, pathological assessment of lymphadenectomy at Naruke's station 3 was 11/13 (85%) and combined with intraoperative mediastinoscopy was 12/13 (92%). In the group without mobilized aortic arches, pathological assessment of lymphadenectomy of station 3 was 14/28 (50%) and combined use of intraoperative mediastinoscopy significantly improved the diagnoses to 24/28 (86%) (P=0.004). Combined use of lymphadenectomy and intraoperative mediastinoscopy could improve the assessment of station 3 in left lung cancer without mobilizing aortic arch.

Induction concurrent chemoradiation therapy for invading apical non-small cell lung cancer.

OBJECTIVE: Although non-small cell lung cancer (NSCLC) involving the superior sulcus has been generally treated with radiation therapy (RT) followed by surgery, local recurrence is still a big problem to be solved. We investigated a role of induction therapy, especially induction concurrent chemoradiation therapy (CRT), on the surgical results of this type of NSCLC. METHOD: We retrospectively reviewed 30 patients with NSCLC invading the apex of the chest wall who underwent surgery from 1987 to 1996. Ten patients (57 +/- 8 years) received surgery alone, 9 (55 +/- 13 years) received RT (42 +/- 7 Gy) followed by surgery and 11 (51 +/- 9 years) received cisplatin based chemotherapy and RT (47 +/- 5 Gy) as an induction therapy. RESULTS: Two and 4-year survival rates were 30% and 20% in patients with surgery alone, 22% and 11% in patients with induction RT, and 73% and 53% in patients with induction CRT, respectively. The survival was significantly better in patients with induction CRT than those with induction RT or surgery alone. Univariate analysis demonstrated that curability (yes versus no: p = 0.027) and induction therapy (surgery alone and RT versus CRT: p = 0.0173) were significant prognostic factors. Multivariate analysis revealed that only induction therapy (p = 0.0238) was a significant prognostic factor. CONCLUSIONS: Induction CRT seems to improve the survival in patients with NSCLC invading the apex of the chest wall compared with induction RT or surgery alone.

Immediate localization using ultrasound-guided hookwire marking of peripheral lung tumors in the operating room.

A new method of marking peripheral lung tumors using an ultrasound-guided hookwire has been developed. The procedure was done for nine tumors taking 15-20 min for each method in the operating room; all of them had no complications. In eight cases (89%), the wire tips were shown to be located within the tumor itself or within 5 mm from the targets, close enough to support appropriate surgery. Ultrasound-guided hookwire marking of peripheral tumors can provide appropriate guidance and prove effective in immediately facilitating subsequent thoracoscopic resection.

Duodenum-preserving resection of the pancreatic head with the ultrasonic coagulating shears.

Duodenum-preserving resection of the pancreatic head is a risk factor for pancreatic fistula because of the wide surface of the transected pancreas. We report on a 72-year-old woman undergoing this procedure for cystadenoma in the pancreatic head using the ultrasonic coagulating shears. The soft pancreatic parenchyma was extensively transected around the cyst with the coagulating shears. Suture of the cut stump was not necessary due to absence of bleeding. The distal pancreatic duct was well preserved for pancreatojejunostomy. We oversewed the possible opening of the main pancreatic duct on the remaining pancreas attached to the duodenum after confirming the location by intrabiliary dye injection. The postoperative course was uneventful. The draining fluid amylase level was low and there were no viscous materials from the drains. We compared histologic changes of a porcine pancreas transected with the coagulating shears or electrocautery to evaluate the sealing effect of the transected surface. The cut stump was covered by a continuing layer of thick protein coagulum in cases of coagulating shears, but by a disrupted layer of coagulating necrosis with charring in cases of electrocautery. The coagulating shears are useful for pancreatic transection in duodenum-preserving resection of the pancreatic head to prevent bleeding and pancreatic fistula from the cut surface.