Problems in the current diagnostic standards of clinical N1 non-small cell lung cancer.

BACKGROUND: Although clinical N1 (cN1) non-small cell lung cancer (NSCLC) is considered to be locoregional, the postoperative outcome is disappointing, with a 5 year survival of less than 50%. One possible reason may be that cN1disease diagnosed by current standard imaging modalities often contains unexpected N2 disease. This study was conducted to evaluate the surgical and pathological results of patients with cN1 NSCLC. METHODS: Among 1782 patients with NSCLC who underwent intended curative resection from 1993 to 2003, 143 patients were identified as having cN1 disease and were enrolled in this study. The clinicopathological records and CT films of each patient were retrospectively reviewed to identify predictors for pN2-3 disease. RESULTS: The pathological nodal status was pN0 in 23% (n = 33), pN1 in 47% (n = 67) and pN2-3 in 30% (n = 43) of patients. Patients with pN2-3 showed a significantly worse 5 year survival rate of 38% compared with patients with pN0 (68%) and pN1 (60%) (p = 0.017 and 0.007, respectively). Multivariate analysis showed that adenocarcinoma histology was a significant predictor for pN2-3 disease (OR 3.312, 95% CI 1.439 to 7.784; p = 0.005). The presence of N1 node separate from the main tumour on CT scans tended to predict pN2-3 disease although this did not reach statistical significance (OR 2.103, 95% CI 0.955 to 4.693; p = 0.066). Pathological N2-3 disease was found in 53% of patients with adenocarcinoma with a separate N1 pattern and in only 12% of patients with non-adenocarcinoma with a continuous N1 pattern. CONCLUSIONS: The diagnosis of N1 status by contrast enhanced CT scans is unsatisfactory with a high rate of unexpected pN2 disease. To avoid infertile lung resection, patients with CT diagnosed N1 adenocarcinoma, especially with a separate N1 pattern on CT, should be considered for additional invasive node biopsy modalities, including mediastinoscopy.

A yeast gene, MGS1, encoding a DNA-dependent AAA(+) ATPase is required to maintain genome stability.

Changes in DNA superhelicity during DNA replication are mediated primarily by the activities of DNA helicases and topoisomerases. If these activities are defective, the progression of the replication fork can be hindered or blocked, which can lead to double-strand breaks, elevated recombination in regions of repeated DNA, and genome instability. Hereditary diseases like Werner's and Bloom's Syndromes are caused by defects in DNA helicases, and these diseases are associated with genome instability and carcinogenesis in humans. Here we report a Saccharomyces cerevisiae gene, MGS1 (Maintenance of Genome Stability 1), which encodes a protein belonging to the AAA(+) class of ATPases, and whose central region is similar to Escherichia coli RuvB, a Holliday junction branch migration motor protein. The Mgs1 orthologues are highly conserved in prokaryotes and eukaryotes. The Mgs1 protein possesses DNA-dependent ATPase and single-strand DNA annealing activities. An mgs1 deletion mutant has an elevated rate of mitotic recombination, which causes genome instability. The mgs1 mutation is synergistic with a mutation in top3 (encoding topoisomerase III), and the double mutant exhibits severe growth defects and markedly increased genome instability. In contrast to the mgs1 mutation, a mutation in the sgs1 gene encoding a DNA helicase homologous to the Werner and Bloom helicases suppresses both the growth defect and the increased genome instability of the top3 mutant. Therefore, evolutionarily conserved Mgs1 may play a role together with RecQ family helicases and DNA topoisomerases in maintaining proper DNA topology, which is essential for genome stability.

Role of walker motif A of RuvB protein in promoting branch migration of holliday junctions. Walker motif a mutations affect Atp binding, Atp hydrolyzing, and DNA binding activities of Ruvb.

Escherichia coli RuvB protein, an ATP-dependent hexameric DNA helicase, acts together with RuvA protein to promote branch migration of Holliday junctions during homologous recombination and recombinational repair. To elucidate the role of the Walker motif A of RuvB (GXGKT; X indicates a nonconserved residue) in ATP hydrolysis and branch migration activities, we constructed four ruvB mutant genes by site-directed mutagenesis, altering the highly conserved Lys(68) and Thr(69). K68R, K68A, and T69A mutants except T69S failed to complement UV-sensitive phenotype of the ruvB strain. These three mutant proteins, when overexpressed, made the wild-type strain UV-sensitive to varying degrees. K68R, K68A, and T69A were defective in ATP hydrolysis and branch migration activities in vitro. In the presence of Mg(2+), K68R showed markedly reduced affinity for ATP, while K68A and T69A showed only mild reduction. K68A and T69A could form hexamers in the presence of Mg(2+) and ATP, while K68R failed to form hexamers and existed instead as a higher oligomer, probably a dodecamer. In contrast to wild-type RuvB, K68R, K68A, and T69A by themselves were defective in DNA binding. However, RuvA could facilitate binding of K68A and T69A to DNA, whereas it could not promote binding of K68R to DNA. All of the three mutant RuvBs could physically interact with RuvA. These results indicate the direct involvement in ATP binding and ATP hydrolysis of the invariant Lys(68) and Thr(69) residues of Walker motif A of RuvB and suggest that these residues play key roles in interrelating these activities with the conformational change of RuvB, which is required for the branch migration activity.

Mutational analysis on structure-function relationship of a holliday junction specific endonuclease RuvC.

BACKGROUND: Escherichia coli RuvC protein is a specific endonuclease that resolves Holliday junctions during homologous recombination. For junction resolution, RuvC undergoes distinct steps such as dimerization, junction-specific binding and endonucleolytic cleavage. The crystal structure of RuvC has been revealed. RESULTS: To identify functionally important residues, we isolated a large number of mutant ruvC genes created by random mutagenesis and characterized their properties in vivo and in vitro. The mutations which were isolated most frequently were mapped to the four acidic residues constituting the catalytic centre. Amongst the several mutant proteins affected in the dimer interface, only one could not form a dimer. The others were able to form a dimer but were defective in cleavage. F69L and K118R mutant proteins could not cleave the junction, but they were able to form a dimer and bind the junction DNA. CONCLUSIONS: Random mutagenesis highlighted many structurally and functionally important residues of RuvC, most of which are highly conserved among RuvC homologues. Dimer formation and also conservation of intact interface interactions between the subunits are important for junction binding and subsequent cleavage. Phe-69 and Lys-118 are critically important for the interactions which lead to junction cleavage.

Migration of Gianturco expandable metallic stents in the upper trachea.

Endotracheal expandable metallic stents have been shown to be useful in treating malignant tracheobronchial stenosis. We report two cases of early stent migration in the upper trachea after what appeared to be a successful stent placement. We conclude that care should be taken when placing Gianturco stents across short, extrinsic, stenotic lesions with smooth mucosa located in the upper trachea because they have a tendency to migrate.

Molecular analysis of the Pseudomonas aeruginosa genes, ruvA, ruvB and ruvC, involved in processing of homologous recombination intermediates.

In Escherichia coli, the products of the ruvA, ruvB and ruvC genes are all involved in the processing of recombination intermediates (Holliday structures) into recombinant molecules. We cloned a 9.4-kb DNA fragment from Pscudomonas aeruginosa PAO1 in a plasmid by functional complementation of the UV sensitivity of an E. coli strain with ruvABC deleted. In P. aeruginosa, the ruv region seemed to form a non-SOS regulated single operon consisting of orf26-ruvC-ruvA-ruvB, while in this region of E. coli, ruvA and ruvB form an SOS-regulated operon, orf26 and ruvC form a non-SOS operon, and these two operons are split by orf23. The deduced amino acid sequences of P. aeruginosa RuvA, RuvB and RuvC proteins were 55, 72 and 55% identical to those of the corresponding E. coli Ruv proteins. The individual ruv genes of P. aeruginosa complemented the corresponding single ruv mutations of E. coli, suggesting that the P. aeruginosa Ruv proteins can interact functionally with their E. coli Ruv partners in forming heterologous complexes. The sequence alignments of the Ruv proteins were extended by incorporation of data about the putative ruv genes obtained from data banks, and the RuvB sequences were conspicuously more conserved than the RuvA and RuvC sequences.

[Brain CT and MRI findings in fat embolism syndrome].

To elucidate brain CT and MRI findings in fat embolism syndrome (FES), we retrospectively analyzed images from 5 patients with FES during the acute and subacute stages. Brain CT examinations demonstrated brain edema in 2 patients and transient spotty low density lesions in 2 patients. Three patients showed no abnormalities. Brain MRI, however, showed brain abnormalities in all patients during the acute stages. These were revealed as spotty high signal intensity lesions on T2WI, and some showed low intensity on T1WI. These spotty lesions were considered to reflect edematous fluid occurring as a result of the unique pathophysiological condition of FES. While the spotty high signal intensity lesions on T2WI were distributed in the cerebrum, cerebellum, brain stem, thalamus, basal ganglia, internal capsule and corpus callosum, cerebral and cerebellar spotty lesions were characteristically located along the boundary zones of the major vascular territories. This characteristic location might be induced by a hypoxic brain condition in FES because the numerous fat globules present in this condition can block entire brain capillaries. This characteristic signal location on T2WI is a useful indicator for differentiating FES from the primary intra-axial brain injury in patients with multifocal trauma.

[Stage IIIa endometrial carcinoma: MR findings].

[PURPOSE]: An attempt was made to evaluate the ability of magnetic resonance (MR) imaging to diagnose stage IIIa endometrial carcinoma. [MATERIALS AND METHODS]: Thirty-three patients with endometrial carcinoma underwent MR imaging and surgery. Surgical staging was classified as I in 21 patients, II in 3 patients and III in 9 patients. The MR images of each patient were retrospectively reviewed by three radiologists. Only the clinical diagnosis of endometrial carcinoma was previously notified. Segmental disruption of the full thickness of the myometrium was considered serosal invasion. Intraperitoneal metastasis was diagnosed according to three criteria (intraperitoneal solid mass of isointensity compared with endometrial lesion, cystic mass excluding benign ovarian cysts, ascites). These evaluations were compared with the surgical findings and analyzed by the kappa statistic. [RESULTS]: The rates of sensitivity and positive predictive value (PPV) for serosal invasion were 33% and 6%, respectively. False positive evaluation frequently occurred when thickness of the intact myometrium was less than 5mm. The rates of sensitivity and PPV for intraperitoneal metastasis were 86% and 72%, respectively. The reason for false negative evaluation was small foci of intraperitoneal metastasis. Overall, sensitivity and PPV for stage IIIa were 86% and 69%, respectively. [CONCLUSION]: MRI was useful in detecting intraperitoneal metastasis of endometrial carcinoma with the exception of diagnosing serosal invasion. It is difficult to detect small foci of peritoneal metastasis. It is necessary to differentiate adnexal metastasis from benign adnexal masses.

Protective effect of dipyridamole against lethality and lipid peroxidation in liver and spleen of the ddY mouse after whole-body irradiation.

The effects of dipyridamole on radiation damage in the mouse were investigated. Dipyridamole (i.p. 2 mg/mouse) administered 1 h before exposure, protected against gamma-irradiation. Pretreatment significantly decreased the death rate at 30 days from 89 to 33% (p<0.001) after 9 Gy whole-body irradiation. LD50 at 30 days was increased from 6.67 to 7.65 Gy in the dipyridamole pretreated group. The level of thiobarbituric acid reactive substances (TBARS) in the liver and spleen, a measure of free radical initiated liver peroxidation, increased 155, 193, 195, and 236% of control (without irradiation) in liver, and 132, 146, 168, and 276% of control (without irradiation) in spleen on days 2, 4, 7, and 10 after 9 Gy of whole-body irradiation respectively. The TBARS levels in both liver and spleen 2 days after irradiation were reduced to 73 +/- 7 and 60 +/- 19% respectively after dipyridamole treatment (2 mg/mouse, i.p. injection 1 h before exposure). In electron microscopic studies, mitochondria and endoplasmic reticulum in the irradiated mouse liver were swollen, but otherwise appeared normal after dipyridamole treatment. These results suggest that dipyridamole has a protective effect on animal survival 30 days after 60Co gamma-irradiation and inhibits lipid peroxidation - which is thought to play a part in the radiation injury in mouse liver and spleen.

Hepatocyte growth factor stimulates liver regeneration and elevates blood protein level in normal and partially hepatectomized rats.

The effects of recombinant human hepatocyte growth factor (HGF) on liver growth and function of normal and partially hepatectomized rats have been examined. HGF was continuously administered into the jugular vein because it was rapidly eliminated from the plasma (t1/2 alpha; approximately 4.5 min) and degraded. In normal rats, the labeling index of hepatocytes was increased about 6 times by the administration of HGF. HGF also decreased the prothrombin time and increased the hepaplastin and serum albumin content. In 70%-hepatectomized rats, HGF stimulated liver regeneration and increased the level of blood proteins such as hepaplastin in a dose-dependent manner. The stimulation of serum protein level seemed to result from not only the increase of hepatic cell number but also the direct effect of HGF on the protein production in hepatocytes, because HGF rapidly enhanced the protein synthesis prior to the increase of cell number and increased the mRNA content of albumin in the liver in vivo. In addition, a combination of heparin with HGF further accelerated the effects of HGF described above, possibly due to the decrease of HGF clearance. These findings suggest that HGF accelerates both the hepatic regeneration and function in vivo, and that rhHGF is clinically expected to be a potent therapeutic agent in hepatectomy and liver injury.

[US and CT findings of mucinous carcinomas of the gallbladder].

Mucinous carcinomas of the gallbladder are relatively uncommon. Their radiological findings have not been described previously. We describe the CT and US findings of mucinous carcinoma of the gallbladder in 3 cases. Tumors (thickened wall and/or intraluminal polypoid mass) showed hyperechogeneity or isoechogeneity on US and water density on CT. US clearly detected large polypoid lesions, but CT was unable to detect these lesions in 2 cases. Therefore, we stressed the discrepancy between the findings of US and CT. These features can be explained by the fact that a tumor containing a large amount of mucin produces a mass of near-water density in the gallbladder on CT. It is of value to know the radiological findings of these tumors because the diagnosis is easily missed by CT study alone.

[Evaluation of usefulness of 99mTc-GSA scintigraphy in hepatitis C treated with alpha interferon].

To evaluate hepatic function before and after treatment with alpha interferon, 99mTc-GSA scintigraphy was performed in seven patients with hepatitis C. The ratios of HH 15 and LHL 15 in 99mTc-GSA scintigraphy were well correlated with the blood laboratory test(GPT) before and after treatment. The uptake of 99mTc-GSA depends on the number of hepatic cells. 99mTc-GSA scintigraphy was useful for evaluating potential liver function after treatment.

[A case of mediastinal malignant lymphoma; 16 years follow up after surgery].

A 32-year-old female was admitted to our hospital on September 1976 because of left mediastinal mass shadows on chest roentgenogram. Needle biopsy studies provided no definitive diagnosis, and exploratory operation was carried out through left postero-lateral thoracotomy. Two large masses were seen in the mediastinum and five small tumors were seen on the diaphragm. All of these masses were removed. Histopathological examination of the tumors indicated non-Hodgkin's lymphoma, diffuse small cell type. Radiation therapy was carried out postoperatively, but chemotherapy could not continue because of side effect. Eight years after surgical therapy, recurrence was seen at left parietal pleura, ten years at peritoneum, twelve years at left parietal pleura, thirteen years at upper mediastinal lymph node, 16 years at post-peritoneal space. These tumors disappeared after radiation therapy. She is doing well seventeen years after the surgery.

[CT findings of pulmonary inflammatory pseudotumors (plasma cell granulomas)].

Pulmonary inflammatory pseudotumor (plasma cell granuloma) is not a true neoplastic lesion, but is composed of a variety of inflammatory cells, predominantly plasma cells. The chest X-ray features resemble those of malignant lung tumors; therefore, CT is often necessary for further evaluation. We report the CT features of five cases with histologically proved pulmonary inflammatory pseudotumor, which can be summarized as follows: a solitary round or oval parenchymal mass with regular or irregular margin, and with or without calcifications. The calcifications are useful for differential diagnosis if present, but they are usually non-specific in shape and configuration. The mean CT attenuation value of the major portion of the mass was increased from 41 HU to 78 HU by the injection of contrast material. In one case, a linear extension of the lesion was seen from the mass to the lung hilum. In three cases, satellite lung nodules were seen. One of these nodules was also proved histopathologically to be inflammatory pseudotumor. The biopsy specimens obtained by using a 20 gauge cutting-needle and an automated biopsy gun were satisfactory for histopathological diagnosis.

Heparin modulates the receptor-binding and mitogenic activity of hepatocyte growth factor on hepatocytes.

Hepatocyte growth factor (HGF) is a mitogen for hepatocytes, having high affinity for heparin. In this study, we examined the function of cell surface heparin-like molecules in HGF/receptor interaction and HGF-induced mitogenic activity using hepatocytes. Binding studies using 125I-HGF showed that more than 85% of bound HGF was released from the cell surface by washing with heparin. This procedure also released HGF from the c-Met protein, which is a component of the high-affinity receptor. In addition, heparitinase digestion of hepatocytes reduced the HGF bound to c-Met protein. Furthermore, excess heparin added during the binding of 125I-HGF to hepatocytes, significantly diminished HGF bound to c-Met protein. Moreover, when DNA synthesis of hepatocytes was repressed and retarded by excess HGF, exogenous heparin restored it. These results suggest that HGF is bound to c-Met protein and that its mitogenic activity is regulated by heparin-like molecules on hepatocytes.

Effect of dimethyl sulfoxide pretreatment on activities of lipid peroxide formation, superoxide dismutase and glutathione peroxidase in the mouse liver after whole-body irradiation.

We investigated the effects of dimethyl sulfoxide (DMSO) on radiation damage in the mouse. DMSO (i.p. 0.11 g/mouse) administered 30 min before exposure protected the mice from the gamma-whole body irradiation: the 30 days lethality was significantly decreased from 44% to 16% (P < 0.05). The contents of thiobarbituric acid reactive substances(TBA-RS) in the mouse liver increased linearly between days 2 and 10 after 9 Gy gamma ray irradiation. The TBA-RS contents in the liver on days 2 to 10 after irradiation were reduced by DMSO pretreatment. The irradiation decreased superoxide dismutase (SOD) activity in the liver on day 10. Decrease in SOD activity was prevented by DMSO pretreatment. In the electron microscopic study, the mitochondria in the irradiated mouse liver were swollen, but we could observe no change after DMSO pretreatment. The results suggest that DMSO has radioprotective effects, probably due to inhibition of lipid peroxidation.

Molecular cloning of silkworm (Bombyx mori) antichymotrypsin. A new member of the serpin superfamily of proteins from insects.

The cDNA of silkworm (Bombyx mori) antichymotrypsin (sw-Achy) was cloned from larval fat body and its nucleotide sequence was determined. The deduced amino acid sequence of mature sw-Achy begins with Phe1 and ends with Phe384, with a preceding 16-amino-acid signal peptide. The amino-acid sequence similarities of sw-Achy with the serine-proteinase inhibitors (serpins) silkworm antitrypsin, tobacco hornworm alaserpin, human alpha-1-antitrypsin and human alpha-1-antichymotrypsin were 29.6%, 30.3%, 26.1%, and 25.0%, respectively. The highly conserved amino acids in other serpins are also conserved in sw-Achy. sw-Achy is thought to be a new member of the serpin family. Multiple alignment of sw-Achy with 23 other kinds of serpin by the progressive method produced a phylogenetic tree in which all four insect serpins are grouped separately within one branch. The reactive site of sw-Achy with alpha-chymotrypsin was identified as Thr343-Ser344 by direct amino-acid sequence analysis of cleaved and purified protein.

[CT and MR imaging of desmoid tumors--comparison of two modalities].

As desmoid tumors invade locally and postoperative recurrence is common, accurate diagnosis of the extent of the tumor is needed prior to surgery. CT and/or MRI evaluation of tumor extension was retrospectively studied in eight patients with desmoid tumors, and the results were correlated with the histopathological findings. All tumors were completely resected even in patients who were evaluated by CT alone. However, the delineation of tumor and local invasion were not clearly demonstrated by CT. On the other hand, the delineation of tumor and local invasion were well visualized on MRI. The MRI picture of desmoid tumors was mainly composed of two different areas of signal intensity. The area of hypointensity in both T 1- and T 2-weighted images was found to have abundant collagen fibers, while the area of isointensity or slight hyperintensity in T 1-weighted images and hyperintensity in T 2-weighted images was found to have fibroblasts. In conclusion, MRI is better suited to the evaluation of patients with desmoid tumors than CT.

Amino acid sequence of alkaliphilic serine protease from silkworm, Bombyx mori, larval digestive juice.

Alkaliphilic protease, P-IIc, from silkworm, Bombyx mori, larval midgut digestive juice consists of 232 amino acids. It has a catalytic triad, Asp-His-Ser, invariably found in a serine protease. A shift of optimal pH value towards the alkaline side diminished at mu = 1.0. This suggests the existence of an electrostatic interaction that affects the proteolytic activity. The higher Arg content may be responsible for this phenomenon. Two cysteine residues probably exist unpaired in a novel position among serine proteases.

Human intrahepatic biliary epithelial cells proliferate in vitro in response to human hepatocyte growth factor.

In previous studies, intrahepatic human biliary epithelial cells (BEC) were isolated in high purity. However, these cells demonstrated only limited growth responses. Here we report that human BEC proliferate in response to human hepatocyte growth factor (hHGF), retain BEC-specific phenotype, and can be serially passaged. BEC showed dose-dependent growth in response to 0.01-100 ng/ml hHGF. The maximum S-phase labeling index reached 40% with half-maximal stimulation at 1 ng/ml. The response of cells from normal and primary biliary cirrhotic liver to hHGF was similar. Cultures were immunostained with specific antibodies and then processed for [3H]thymidine autoradiography. Proliferating cells expressed BEC-specific markers (HEA125 and CK-19), but were negative for desmin and factor VIII-related antigen. Occasional vimentin-positive cells were observed, but these were nonproliferative. In conclusion, cells responding to hHGF were clearly BEC in origin. The observation that HGF is mitogenic for BEC as well as hepatocytes has important implications. First, greater yields of intrahepatic BEC are available for subsequent studies of the pathogenesis and etiology of diseases of the biliary epithelium. Secondly, some means of regulating the cellular response to HGF in vivo must operate, in that HGF levels rise early after partial hepatectomy and yet BEC proliferate 24 h later than hepatocytes.