Intratumoural immune cell landscape in germinoma reveals multipotent lineages and exhibits prognostic significance.

AIMS: Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown. METHODS: We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases. RESULTS: Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression-free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in-situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte-lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD-L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%). CONCLUSIONS: The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies.

Expression and characterization of recombinant human antithrombin III in Pichia pastoris.

Antithrombin III (ATIII) is a member of the serpin superfamily and a major regulator of the blood coagulation cascade. To express recombinant human ATIII (rATIII) in the methylotrophic yeast Pichia pastoris, we constructed an rATIII expression plasmid which contained the ATIII cDNA encoding mature protein region connected with the truncated mAOX2 promoter and the SUC2 secretion signal, introduced it into the P. pastoris genome, and screened for a single copy transformant. The secretion of rATIII from the transformant reached a level of 320 IU/L in the culture broth at 169 h. From the culture-supernatant, rATIII was purified to over 99% by heparin-affinity chromatography and other column chromatography methods. We characterized rATIII and compared it with human plasma-derived ATIII (pATIII). The purified rATIII possessed correct N-terminal amino acid sequence, and its molecular weight by SDS-PAGE of 56,000 Da was slightly different from the 58,000 Da of pATIII. Sequence and mass spectrometry analysis of BrCN fragments revealed that posttranslational modifications had occurred in rATIII. O-linked mannosylation was found at Ser 3 and Thr 9, and in some rATIII molecules, modification with O-linked mannosyl-mannose had probably occurred at Thr 386, close to the reactive center. Although the heparin-binding affinity of rATIII was 10-fold higher than that of pATIII, its inhibitory activity against thrombin was only half. As the conformation of rATIII and pATIII by circular dichroism spectroscopy was similar, O-glycosylation in the reactive center loop was assumed to be mainly responsible for the decreased inhibitory activity. pATIII can inactivate thrombin through formation of a stable thrombin-ATIII complex, but rATIII modified with O-glycosylation in the reactive center loop may act as a substrate rather than an inhibitor of thrombin.

Cdc6 expression as a marker of proliferative activity in brain tumors.

Antibodies against human proteins that regulate DNA replication such as Cdc6 and Mcm5 became available as a new marker of proliferation. We performed immunohistochemical analysis with MIB-1 and antibody against Cdc6 on 35 brain tumors, including tumors of neuroepithelial tissue, vestibular schwannomas, meningiomas, and pituitary adenomas. Median reactivity for MIB-1 was 8.8%, and that for Cdc6 was 55%. Reactivity in most brain tumors was significantly higher for Cdc6 than for MIB-1, but reactivity of Cdc6 was independent of tumor grade. Detection of Cdc6 expression might be useful for the estimation of proliferative activity in brain tumors.

Incidentally identified syringomyelia associated with Chiari I malformations: is early interventional surgery necessary?

OBJECTIVE: The purpose of this study was to analyze clinical data and magnetic resonance imaging (MRI) findings for patients with asymptomatic, incidentally identified syringomyelia associated with Chiari I malformations who were monitored for more than 10 years, and to clarify the natural history of these lesions. METHODS: The clinical records of nine patients who had not been surgically treated and were regularly subjected to neurological and MRI examinations were analyzed. In MRI studies, the axial diameter of the syrinx at the widest level, the longitudinal extent of the syrinx, and the extent of tonsillar herniation into the spinal canal were analyzed. As a control, MRI findings for 11 patients with symptomatic syringomyelia associated with Chiari I malformations who had been surgically treated were also analyzed, and these MRI parameters were statistically compared between the asymptomatic and symptomatic groups. RESULTS: One patient underwent surgery, because of neurological changes, 7 years after the first visit. None of the remaining patients demonstrated any neurological change during the follow-up period (11.2+/-0.7 yr), and all of them have been faring well without surgery. No statistically significant differences in MRI findings between the asymptomatic and symptomatic groups were observed. CONCLUSION: The long-term clinical courses of patients with asymptomatic, incidentally identified syringomyelia associated with Chiari I malformations were observed to be benign. MRI parameters did not provide predictable values to recommend interventional surgery. Unless changes in neurological or MRI findings are detected, early interventional surgery is not necessary.

Purification and comparison of triosephosphate isomerases from ammonia-oxidizing bacteria isolated from terrestrial and marine environments.

Triosephosphate isomerases [TIMs, EC 5.3.1.1] were purified from two ammonia-oxidizing bacteria: Nitrosomonas sp. K1 (K1), Nitrosomonas sp. TNO632 (TNO). The molecular masses of the native enzymes were estimated to be about 53.6 (K1-TIM) and 51.9 kDa (TNO-TIM) by gel filtration, whereas SDS-PAGE produced one band for each enzyme with M(r) values of 27.1 (K1-TIM) and 26.4 kDa (TNO-TIM), respectively, suggesting that the enzymes consist of identical subunits. The apparent K(m) for d-glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) were about 1.19 and 4.78 mM (K1-TIM), and 0.41 and 6.01 mM (TNO-TIM), respectively. The two TIMs had different pH-activity curves with an optimum pH range of 6.5 (K1-TIM) and 8.0 (TNO-TIM). The temperature optima of K1-TIM and TNO-TIM were 50-60 and 60-65 degrees C, respectively. Both enzymes were strongly inhibited by 5,5'-ditiobis at 1.0 mM. The N-terminal amino acid sequences of K1-TIM and TNO-TIM were MRAGFVAGNWKMHG (K1-TIM) and MVRTGLVAGNWKMNG (TNO-TIM). A homology of 74.1% was observed between K1-TIM and TNO-TIM.

Characteristics of an ammonia-oxidizing bacterium with a plasmid isolated from alkaline soils and its phylogenetic relationship.

An ammonia-oxidizing bacterium, strain TCH716, was isolated from alkaline soil at Harbin city, China. The cells of strain TCH716 are lobate (0.8-1.5 x 1.0-2.0 microm), gram-negative, obligately aerobic, and nonmotile. Colonies (1-2 mm in diameter) on gellan gum plate culture are reddish, circular, and smooth. The G + C content of DNA is 54.78 mol%. Its percentage of 16S rRNA gene sequence similarity (%) to Nitrosolobus multiformis ATCC 25196T (type strain) is 98.56%. This bacterium has an optimal growth temperature and pH at 30 degrees C and 8.0-8.5, respectively. The concentration of ammonium sulfate in the HEPES medium for optimum growth of this bacterium is 38 mM. Strain TCH716 was found to have a plasmid (approximately 6.5 kbp) that possessed a plasmid-linked gene for sulfonamide resistance. Phosphoglycerate kinase, RubisCO and PEPC were found to possess high specific activities compared to the activities of these enzymes in strain ATCC 25978T. In identification of strain TCH716, both morphological characteristics (compartmentalized cells) and the phylogenetic relationship based on 16S rRNA gene sequence are important. Based on results obtained, strain TCH716 belongs to the genus Nitrosolobus, and designated as Nitrosolobus sp. TCH716.

Effects of haemodialysis on diabetic macular leakage.

AIM: To evaluate the effects of haemodialysis on macular oedema by fluorescein angiography in patients with diabetic retinopathy and end stage renal disease. METHODS: In this prospective study, fluorescein angiography was performed on 40 eyes of 22 non-insulin dependent diabetic patients with end stage renal disease just before (baseline) and 4 weeks after the beginning of haemodialysis. The change of macular leakage was determined by evaluating the same phase of the angiograms. RESULTS: Fluorescein angiograms obtained at 4 weeks showed that macular leakage was unchanged in 28/40 eyes (70%), decreased in 4/40 eyes (10%), and increased in 8/40 eyes (20%) when compared with the baseline appearance. CONCLUSIONS: These results indicate that haemodialysis does not benefit macular leakage in diabetic patients receiving haemodialysis for end stage renal disease.

A novel degradative pathway of 2-nitrobenzoate via 3-hydroxyanthranilate in Pseudomonas fluorescens strain KU-7.

A bacterial strain KU-7, identified as a Pseudomonas fluorescens by 16S rDNA sequencing, was one of the 12 new isolates that are able to grow on 2-nitrobenzoate as a sole source of carbon, nitrogen, and energy. Resting cells of KU-7 were found to accumulate ammonia in the medium indicating that degradation of 2-NBA proceeds through a reductive route. Metabolite analyses by thin layer chromatography and high pressure liquid chromatography indicated that 3-hydroxyanthranilate is an intermediate of 2-nitrobenzoate metabolism in KU-7 cells. This offers an alternative route to 2-nitrobenzoate metabolism since anthranilate (2-aminobenzoate) or catechol were detected as intermediates in other bacteria. Crude extracts of KU-7 cells converted 2-nitrobenzoate to 3-hydroxyanthranilate with oxidation of 2 mol of NADPH. Ring cleavage of 3-hydroxyanthranilate produced a transient yellow product, identified as 2-amino-3-carboxymuconic 6-semialdehyde, that has a maximum absorbance at 360 nm. The initial enzymes of the 2-nitrobenzoate degradation pathway were found to be inducible since succinate-grown cells produced very low enzyme activities. A pathway for 2-nitrobenzoate degradation in KU-7 was proposed.

Physical model-based indirect measurements of blood pressure and flow using a centrifugal pump.

This article describes a technique offering indirect measurements of pump pressure differential and flow with certain accuracy independent of changes in blood viscosity. This technique is based on noninvasive measurements of the motor current and rotation speed using the physical model equations of the centrifugal pump system. Blood viscosity included in the coefficients of the dynamic equations is first estimated, and then substitution of the estimated viscosity into the steady equations of the model provides pump flow and pressure differential. In vitro tests using a Capiox pump showed a sufficient linear correlation between actual values and their estimates for pressure differential and pump flow. An in vivo test using a 45 kg sheep showed that the proposed algorithm needs robustness for the convergence of estimates of viscosity. An overall evaluation, however, of the developed algorithm/model showed indications of success in terms of efficient computation and modeling.

Purification and characterization of cyclohexanone 1,2-monooxygenase from Exophiala jeanselmei strain KUFI-6N.

Baeyer-Villiger cyclohexanone 1,2-monooxygenase (CHMO) was purified 17.1-fold from cell extracts of the fungus Exophiala jeanselmei grown on cyclohexanol to electrophoretically homogeneity by serial chromatographies. The molecular mass of the native enzyme was approximately 74 kDa by gel filtration and SDS-PAGE. Some enzymic characterizations were studied. The NH2-terminal amino acid residues were Ala-Lys-Ser-Leu-Asp-Val-Leu-Ile-Val-Gly-Ala-Gly-Phe-Gly-Gly-Ile-Tyr-Gln-Leu-, with similarity to the bacterial CHMOs of FAD-binding and NADPH-dependent type Baeyer-Villiger monooxygenases.

Ribulose-1,5-bisphosphate carboxylase/oxygenase from an ammonia-oxidizing bacterium, Nitrosomonas sp. K1: purification and properties.

The ammonia-oxidizing chemoautotrophic Nitrosomonas sp. strain K1 exhibited marked ribulose-1,5-bisphosphate carboxylase (RubisCO) activity. The RubisCO [EC 4.1.1.39] was purified as an electrophoretically homogeneous protein. The molecular mass of the enzyme was estimated to be about 460 kDa by gel filtration, and it consists of two subunits [large (L): 52.2 kDa; small (S): 13.3 kDa] as demonstrated by SDS-PAGE. This confirmed that the enzyme has an L(8)S(8) structure. The K(m) values of the enzyme for RuBP, NaHCO3, and Mg2+ were estimated to be 0.112, 0.415, and 1.063 mM, respectively. The optimum pH and temperature for its activity were approximately 7.0 and 45 degrees C. The enzyme was stable up to 45 degrees C and in a pH range from 7.0-9.0 (4 degrees C, 48 h). The enzyme activity was inhibited by Cu2+, Hg2+, N-ethylmaleimide, p-chloromercuribenzoate, and SDS (0.1 mM). The activity was also inhibited by ammonium sulfate at high concentrations (38-303 mM) but the stability of the enzyme showed no inhibition at the same ammonium sulfate concentrations. The N-terminal amino acid sequences of the large and small subunits are AIKTYQAGVKEYRQTYW QPDYVPL and AIQAYHLTKKYETFSYLPQM, respectively.

Identification of a transcriptional activator (ChnR) and a 6-oxohexanoate dehydrogenase (ChnE) in the cyclohexanol catabolic pathway in Acinetobacter sp. Strain NCIMB 9871 and localization of the genes that encode them.

We identified chnR, a gene encoding an AraC-XylS type of transcriptional activator that regulates the expression of chnB, the structural gene for cyclohexanone monooxygenase (CHMO) in Acinetobacter sp. strain NCIMB 9871. The gene sequence of chnE, which encodes an NADP(+)-linked 6-oxohexanoate dehydrogenase, the enzyme catalyzing the fifth step of cyclohexanol degradation, was also determined. The gene arrangement is chnB-chnE-chnR. The predicted molecular masses of the three polypeptides were verified by radiolabeling by using the T7 expression system. Inducible expression of cloned chnB in Escherichia coli depended upon the presence of chnR. A transcriptional chnB::lacZ fusion experiment revealed that cyclohexanone induces chnB expression in E. coli, in which a 22-fold increase in activity was observed.

A clinicopathological study of lung cancer patients with occupational exposure to chrysotile asbestos fibers.

OBJECTIVE: To summarize the features of asbestos-related lung cancer. PATIENTS: Thirty-one lung cancer patients with occupational exposure to chrysotile asbestos fibers. They worked or had worked in one asbestos factory or its subcontracters. RESULT: All patients were male with mean age of 60.6 when diagnosed, and all except one were current or ex-'heavy' smokers. Histological types were fairly evenly divided into adeno-, squamous and small cell carcinoma and 24 (78%) of patients showed 'peripheral type' lung cancers. Regarding clinical stages, 20 patients (65%) were classified as III or IV (advanced stage). Tumor shadow(s) was detected on chest X-ray in 22 patients (71%), and in 5 patients with 'negative' chest X-ray, chest CT was necessary to recognize a primary tumor. Seventeen patients (55%) did not undergo periodical check-ups. CONCLUSION: Occupational asbestos exposure is interpreted as one of the important risks for lung cancer and frequent and accurate observation is necessary.

Biodegradation of cyclohexylamine by Brevibacterium oxydans IH-35A.

A bacterial strain capable of growing on cyclohexylamine (CHAM) was isolated by using enrichment and isolation techniques. The strain isolated, strain IH-35A, was classified as a member of the genus Brevibacterium. The results of growth and enzyme studies are consistent with degradation of CHAM via cyclohexanone (CHnone), 6-hexanolactone, 6-hydroxyhexanoate, and adipate. Cell extracts obtained from this strain grown on CHAM contained CHAM oxidase, and the model for CHAM oxidation by this enzyme was similar to the model for deamino oxidation of amine by amine oxidase.

Occurrence and significance of decahydroquinolines from dendrobatid poison frogs and a myrmicine ant: use of 1H and 13C NMR in their conformational analysis

Structures for 2,5-disubstituted decahydroquinolines (DHQs) are reported for the two diastereomeric pairs cis-275B (14) and cis-275B' (15) and 5-epi-trans-269AB (18) and trans-269AB (19), all isolated from skin extracts of dendrobatid frogs, and for 5-epi-cis-275B' (16) and 5-epi-trans-275B (17) found in the extracts of virgin queens of a myrmicine ant [Solenopsis (Diplorhoptrum) azteca]. Detection of such DHQs in an ant, their first reported occurrence, strengthens a dietary hypothesis for the origin of the approximately 30 DHQs that have been detected in extracts of frog skin. NMR data on the two conformers of cis-decahydroquinoline permit assignment of ring conformations and stereochemistry to cis-DHQs of the "N-endo" type or the "N-exo" type. These conformations are also assigned on whether H-8a is equatorial or axial as determined with E-COSY or 1D-HOHAHA spectra.

Cloning and expression of the N,N-dimethylformamidase gene from Alcaligenes sp. strain KUFA-1.

N,N-Dimethylformamidase (DMFase) from Alcaligenes sp. strain KUFA-1, a bacterium that can grow on N,N-dimethylformamide (DMF) as the sole carbon and nitrogen source, catalyzes the first step of the DMF degradation. The DMFase gene dmfA1A2 was cloned in Escherichia coli, and its nucleotides were sequenced. The deduced amino acid sequence of the enzyme consisted of two alpha- and two beta-subunits with 132 and 762 amino acids, respectively, and had little similarity to sequences in protein databases, including various amidases. The protein may be a new kind of amidase. DMFase activity was detected in E. coli cells transformed with an expression plasmid of the cloned DMFase gene. The properties of recombinant DMFase purified from E. coli were identical to those of Alcaligenes DMFase.

Characteristics of a high-concentration-ammonium sulfate-requiring ammonia-oxidizing bacterium isolated from deodorization plants of chicken farms.

A high-concentration-ammonium sulfate-requiring, ammonia-oxidizing bacterium, strain K1, was newly isolated from packed tower biological deodorization plants of chicken farms. The cells of strain K1 are rods (0.1-1.0 x 1.0-2.0 microm), gram negative, obligately aerobic, and nonmotile. Colonies (1-2 mm in diameter) on a plate culture are reddish, circular, and smooth. Intracytoplasmic membranes characteristic of nitrifying bacteria are present. The G+C content of the total DNA is 48.5 mol%. The similarity of 16S rRNA (%) to N. europaea ATCC 25978T (type strain) is 93.77%. This bacterium has a higher optimal growth temperature (35 degrees C) than is usually the case and tolerance up to 40 degrees C. The optimum concentration of ammonium sulfate in the medium is 303 mM, which should make it applicable for use in deodorization plants for enhancing the efficiency of deodorization. Phosphoglycerate kinase (PGK) and triosephosphate isomerase (TIM) were found to possess high specific activities (5700 and 4 x 10(5) U/mg, respectively) compared to the activities of these enzymes in strain ATCC 25978T (300 and 14 U/mg).

Purification and characterization of a novel cyclohexylamine oxidase from the cyclohexylamine-degrading Brevibacterium oxydans IH-35A.

Cyclohexylamine oxidase (CHAO) from a cell extract of Brevibacterium grown on cyclohexylamine was purified 50.2-fold, to electrophoretic homogeneity, by serial chromatographies. The molecular mass of the native enzyme was estimated to be approximately 50 kDa by gel filtration and SDS-PAGE. The optimum pH was 7.4 and the stable pH range was 6.0 to 7.0. The enzyme was thermostable up to 30 degrees C. The enzyme was found to be highly specific for the deamination of alicyclic monoamines such as cyclopentylamine, cycloheptylamine, and N-methylcyclohexylamine and aliphatic monoamines, such as sec-butylamine. The apparent K(m) value for cyclohexylamine was 1.23 mM. The enzyme was inhibited by flavin enzyme inhibitors such as quinine and quinacrine. The N-terminal 27 amino acid residues were determined as Gly-Ser-Val-Thr-Pro-Asp-Pro-Asp-Val-Asp-Val-Ile-Ile-His-Gly-Ala-Gly-Ile-Ser-Gly-Ser-Ala-Ala-Ala-Lys-Ala-Leu-, revealing homology to conventional flavin-containing amine oxidases (EC 1.4.3.4).

Estimation of regional lung function in interstitial pulmonary disease using 99mTc-technegas and 99mTc-macroaggregated albumin single-photon emission tomography.

For quantitative evaluation of the regional lung function in patients with interstitial pulmonary disease (IP) in the sitting position, 99mTc-Technegas and 99mTc-macroaggregated albumin (MAA) single-photon emission tomography (SPET) studies were performed in 12 healthy controls (HC) and 42 IP patients. Four transverse images were prepared from the data obtained and designated as slices no. 1-4 from the top downward. Regions of interest (ROIs) were determined in the anterior and posterior parts of the lung in each slice, and the ratio of the count per voxel in the ROIs to the count in the entire lung was calculated as the regional Technegas index (T). The regional perfusion index (Q) was calculated by a similar procedure using the data of 99mTc-MAA SPET. The ratios between T and Q (T/Q) in the anterior and posterior regions of the lung, and the ratios of T and Q between the anterior and posterior regions of the lung (Tp/Ta and Qp/Qa) were examined. In the HC group, T/Q decreased but Tp/Ta and Qp/Qa increased from the upper to the lower lung fields. When IP patients were classified into (I) those in whom T/Q decreased from the upper to the lower lung fields, (II) those in whom it was similar in all slices, (III) those in whom it increased from slice 3 to slice 4, and (IV) those in whom it increased from slice 2 to slices 3 and 4, this classification was more closely correlated with %VC than with %DLCO or PaO2. When the patients were classified according to Tp/Ta and Qp/Qa into (A) those in whom the values were greater in the lower than the upper lung field, (B) those in whom the values were similar in all slices, and (C) those in whom the values were smaller in lower than in upper lung fields, categories B and C were observed frequently even in patients whose %VC was in the normal range. This method is considered to be an effective means to evaluate the progression and pathology of IP and to detect early impairment of lung function.