Successful therapy with argatroban for superior mesenteric vein thrombosis in a patient with congenital antithrombin deficiency

A 38-year-old woman was admitted with superior mesenteric vein (SMV) thrombosis, which was refractory to anticoagulation therapy. The plasma antithrombin activity was decreased and hardly compensated by concentrated antithrombin preparation due to high consumption rate. However, successful anticoagulation was achieved by administration of direct thrombin inhibitor, argatroban. Family studies of antithrombin activity revealed that she had type I congenital antithrombin deficiency. A novel heterozygous mutation in the gene for antithrombin (single nucleotide T insertion at 7916 and 7917, Glu 272 to stop in exon 4) was identified. Argatroban administration would be effective in the treatment of congenital antithrombin deficiency with SMV thrombosis.     

Complete cure of metastatic gastric cancer treated with combination chemotherapy using S-1

A 65-year-old man had gastric cancer with liver and lung metastases, and received three cycles of FLEP (5-fluorouracil, leucovorin, etoposide, cisplatin) chemotherapy on June, 2001. The primary lesion became scar ulceration and adenocarcinoma tissue was recognized by biopsy pathologically. The lung metastases and lymph node metastases disappeared. The metastatic liver lesion decreased more than 95% in size and was judged partial response. Thereafter he received only oral administration of 100mg S-1 (70mg/m2). The metastatic liver lesion was shown to have disappeared on CT of the abdomen and was judged complete response, on June, 2002. Since then, he was receiving S-1 and continued to be complete response for two and half years until June, 2004. The clinical trial of S-1 as an adjuvant therapy for patients with advanced gastric cancer, who underwent curative resection, is ongoing in a multicenter study. Although the outcome is still unknown, the effect of S-1 for adjuvant chemotherapy may be also promising. We think that S-1 is useful as an adjuvant therapy and maintenance therapy of prior treatment including surgical resection.     

Hepatic portal venous gas disappearing within 24 hours

Hepatic portal venous gas (HPVG) was detected by CT in a 64-year-old woman who suddenly complained of lower abdominal pain. However, the abdominal symptoms disappeared rapidly, and lower gastrointestinal endoscopy indicated only terminal ileitis. Conservative treatment alone was performed, and HPVG completely disappeared approximately 18 hours later. The use of CT proved to be useful for following the course of HPVG.     

Prognostic significance of extracellular volume fraction with equilibrium contrast-enhanced computed tomography for pancreatic neuroendocrine neoplasms

Background/Objectives: Identifying reliable pretreatment imaging biomarkers for pancreatic neuroendocrine neoplasm (PanNEN) is a key imperative. Extracellular volume (ECV) fraction quantified with equilibrium contrast-enhanced CT can be easily integrated into routine examinations. This study aimed to determine whether ECV fraction with equilibrium contrast-enhanced computed tomography (CECT) could predict long-term outcomes in patients with PanNEN.MethodsThis study was a retrospective observational study of 80 patients pathologically diagnosed with PanNEN at a single institution. ECV fraction of the primary lesion was calculated using region-of-interest measurement within PanNEN and the aorta on unenhanced and equilibrium CECT. The impact of clinical factors and tumor ECV fraction on progression-free survival (PFS) and overall survival (OS) was assessed with univariate and multivariate analyses using Cox proportional hazards models. The correlation between WHO classification and tumor ECV fraction was evaluated using Kendall rank correlation coefficients.ResultsPFS and OS rates were estimated as 93.4% and 94.6%, 78.7% and 86.2%, 78.7% and 77.0%, and 78.7% and 66.6% at 1, 3, 5, and 10 years, respectively. Multivariate analysis revealed that Union for International Cancer Control (UICC) stage (hazard ratio [HR] = 3.95, P = 0.003), WHO classification (HR = 12.27, P = 0.003), and tumor ECV fraction (HR = 11.93, P = 0.039) were independent predictors of PFS. Patient age (HR = 1.11, P < 0.001), UICC stage (HR = 3.14, P = 0.001), and tumor ECV fraction (HR = 5.27, P = 0.024) were independent significant variables for predicting OS. Tumor ECV fraction had a weak inverse relationship with WHO classification (P = 0.045, τ = ?0.178).ConclusionsECV fraction determined by equilibrium CECT and UICC stage may predict survival in patients with PanNEN.     

MuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition

MuB is an ATP-dependent nonspecific DNA-binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. Mechanistic understanding of MuB function has previously been hindered by MuB''s poor solubility. Here we combine bioinformatic, mutagenic, biochemical, and electron microscopic analyses to unmask the structure and function of MuB. We demonstrate that MuB is an ATPase associated with diverse cellular activities (AAA+ ATPase) and forms ATP-dependent filaments with or without DNA. We also identify critical residues for MuB’s ATPase, DNA binding, protein polymerization, and MuA interaction activities. Using single-particle electron microscopy, we show that MuB assembles into a helical filament, which binds the DNA in the axial channel. The helical parameters of the MuB filament do not match those of the coated DNA. Despite this protein–DNA symmetry mismatch, MuB does not deform the DNA duplex. These findings, together with the influence of MuB filament size on strand-transfer efficiency, lead to a model in which MuB-imposed symmetry transiently deforms the DNA at the boundary of the MuB filament and results in a bent DNA favored by MuA for transposition.DNA transposons are ubiquitous in the genomes of all forms of life and play important evolutionary roles in generating gene diversity and in shaping genomic landscapes (1). Although typically transposons exhibit no strong sequence selectivity for the target DNA site, certain transposons avoid self-destructive insertion (reviewed in ref. 2), a phenomenon called “target immunity” because the presence of a copy of the transposon renders nearby DNA sites “immune” to additional insertion by the same transposon (3–8). MuB plays critical roles in this self-immunity in the bacteriophage Mu transposition process.Phage Mu is one of the most complex and efficient transposable elements (reviewed in refs. 3 and 4). Two phage-coded proteins, MuA and MuB, are essential for efficient Mu transposition. MuA is the transposase responsible for synapsing the two Mu end sequences and for all of the DNA cutting and joining steps in the initial stages of transposition. However, transposition is inefficient in the absence of MuB, and the residual Mu insertion that takes place uses only DNA target sites near or within the transposing element, often leading to self-destruction (9–11). MuB is a small (35-kDa) ATP-dependent nonspecific DNA-binding protein with relatively low ATPase activity (10, 12, 13). Upon ATP binding, MuB polymerizes preferentially on DNA, but in the absence of DNA it still can form polymers of variable sizes (14, 15). When observed by total internal reflection fluorescence microscopy, GFP-MuB-ATP binds along the DNA molecule forming many short separate segments of polymers, and, as more GFP-MuB is added, the protein-covered segments elongate to form an apparently continuous polymer that fully coats the DNA. Hydrolysis of ATP reverses this process, triggering disassembly of the MuB polymer (16–18).The MuB–DNA complex controls MuA activity at several steps, including stimulation of the strand-transfer reaction, which joins Mu DNA ends to the target DNA site (19, 20), leading to utilization of MuB-bound DNA as the preferred target for transposition (11). MuB-stimulated transposition displays target immunity, avoiding target sites near preexisting Mu-end sequences where MuA binds. This is because MuA stimulates the ATPase activity of MuB, and because this interaction takes place more frequently when the two proteins are bound on the same DNA molecule relatively close to each other, MuA triggers the dissociation of MuB from DNA sites near the Mu ends more efficiently than from those sites at a distance (21). Thus, by the time MuA assembles the active complex with synapsed Mu DNA ends ready to capture target DNA, MuB has been depleted from the vicinity of the active complex and accumulates on DNA sites away from the Mu-end sequence. This explains the strong preference of the transposing Mu to target DNA sites at substantial distances away (5–25 kb) from its original site (22).Beyond the mechanistic framework outlined above, study of MuB function has been hampered by the lack of structural information. The 312-amino-acid MuB polypeptide can be divided into two fragments by limited proteolysis (23). The N-terminal 25-kDa fragment contains Walker A and Walker B motifs (24) and also a putative helix-turn-helix motif (12), but it lacks ATPase activity and is prone to aggregate. The C-terminal 10-kDa fragment consists of a four-helix bundle, whose structure was determined by NMR spectroscopy and reported to resemble the N-terminal domain of Escherichia coli replicative helicase DnaB (25) (Fig. S1). Beyond this, no sequence similarity between MuB and other proteins has been reported. Studies of mutant proteins have been limited mostly to those involving the Walker motifs (26). MuB’s tendency to aggregate in the presence of ATP hampered efforts at structural characterization. Without such information, it has not been clear how ATP controls MuB polymerization, what kind of polymer MuB forms, how MuB polymer interacts with DNA, or how MuA interacts with MuB polymer and triggers ATP hydrolysis and DNA dissociation.In this study we combine bioinformatic, mutagenic, biochemical, and electron microscopy (EM) techniques to characterize the structure of MuB and its role in DNA binding and transposition. First, we present evidence that MuB is a member of the AAA+ (ATPases Associated with diverse cellular Activities) ATPase superfamily. A large number of these proteins have been studied structurally and functionally (27, 28). Guided by this finding, we identified the critical residues that control MuB ATPase, DNA substrate binding, and protein polymerization, yielding a clear picture of the communication between MuB and its interaction partners, MuA and DNA. Furthermore, we studied the structure of MuB polymers by cryo-EM and image reconstruction. We demonstrate that MuB-ATP forms a solenoid-like filament (150 Å in diameter with a pitch of 48 Å) and that DNA is bound in the axial channel. The helical parameters of the MuB filament (5.4 subunits and 48-Å per turn) do not match those of the B-form DNA (10 bp and 36 Å per turn). However, despite this mismatched symmetry between the protein and DNA, MuB does not significantly distort the bound DNA duplex. Therefore, individual MuB subunits in the filament must interact with DNA nonuniformly.     

Rapid and efficient detection method of Norwalk virus

A rapid and efficient RT-PCR with fluorogenic probe(TaqMan-PCR) was developed for detection of Norwalk virus(NV) genomes in clinical specimens. We designed NV genogroup specific primers and fluorogenic probes in the junction of open reading frame (ORF)1 and ORF2. Eighty specimens from patients of gastroenteritis, in which NV-like particles were detected by electron microscopy, were examined by TaqMan-PCR and RT-PCR using primer sets previously reported; two sets in RdRp region of NV and one set in capsid region. TaqMan-PCR detected NV genome from 79 of 80 specimens(98.8%); this method showed the highest sensitivity of all other RT-PCR tested. Moreover, TaqMan-PCR was considered to be useful to recognize genogroup of NV correctly.     

Case of intracranial hypotension responsive to oral prednisolone]

A 31-year-old female patient with headache and nausea was admitted to our hospital, although there were no apparent neurological abnormalities except headache. Cerebrospinal fluid (CSF) pressure was 40 mmH2O on the first lumbar puncture and CT showed some fluid in the left maxillary sinus. She gradually developed orthostatic headache despite antibiotics, hydration and analgesics. MRI showed diffuse meningeal thickening and enhancement. CSF pressure was 0 mmH2O on the second lumbar puncture and RI cisternography demonstrated early excretion to the kidneys. She was diagnosed with intracranial hypotension due to CSF leakage. An autologous 10ml blood patch on the lumbar epidural space did not relieve the orthostatic headache. However, headache disappeared one day after oral intake of 40mg prednisolone. During the next three months, oral prednisolone was tapered off. Three months after the onset of the illness, MRI did not show either meningeal thickening or enhancement. We concluded that oral prednisolone was effective in a case of intracranial hypotension.