Community structure of denitrifying and total bacteria during nitrogen accumulation in an ammonia-loaded biofilter.

AIMS: To obtain insight into the complex behaviour of denitrifying and total bacterial groups during the nitrogen accumulation process in an ammonia-loaded biofiltration system. METHODS AND RESULTS: Denitrifying and total bacterial communities in a laboratory-scale rockwool biofilter with intermittent water recirculation were analysed by using denaturing gradient gel electrophoresis targeting nosZ and metabarcoding sequencing of the 16S rRNA gene. Gene abundance was evaluated by quantitative PCR. The nosZ number increased from 6·59 × 106 to 3·33 × 108 copies per gram dry sample over the 436 days of operation, during which nitrogen mass balance errors increased to 39%. The nosZ sequences associated with the genera Castellaniella, Hyphomicrobium and Pseudomonas were detected. Metabarcoding sequencing analysis indicated that the proportions of the genera for which at least one denitrifying strain or species possessing nosZ had been characterized corresponded well to the nitrogen loss. In addition, the genus Nitrosococcus (γ-proteobacteria) increased its relative abundance at days 317 and 436. CONCLUSIONS: The increased proportion of denitrifying bacteria in this ammonia-loaded biofiltration system could be related to the nitrogen loss. SIGNIFICANCE AND IMPACT OF THE STUDY: These results will help to clarify the complex behaviour of nitrifiers and denitrifiers within ammonia-loaded biofiltration systems.

Gemcitabine-based regimen for primary ovarian angiosarcoma with MYC amplification.

Angiosarcoma is a rare and aggressive type of sarcoma, and primary angiosarcoma of the ovary is extremely rare. We report the case of a 29-year-old woman who was diagnosed with ovarian angiosarcoma and possible bone metastases. We treated this patient with a gemcitabine-based regimen as postoperative adjuvant chemotherapy, after which she achieved at least 7 years of progression-free survival, an extremely long duration given the aggressive features of this tumour. We retrospectively performed immunohistochemical analyses and fluorescence in situ hybridization to make a pathology diagnosis and to investigate the tumour features. MYC amplification and c-Myc protein overexpression were positively detected. It might be possible to correlate the effectiveness of the gemcitabine-based chemotherapeutic regimen with MYC gene amplification and c-Myc protein overexpression.

Responses of community structure of amoA-encoding archaea and ammonia-oxidizing bacteria in ammonia biofilter with rockwool mixtures to the gradual increases in ammonium and nitrate.

AIMS: To investigate community shifts of amoA-encoding archaea (AEA) and ammonia-oxidizing bacteria (AOB) in biofilter under nitrogen accumulation process. METHODS AND RESULTS: A laboratory-scale rockwool biofilter with an irrigated water circulation system was operated for 436 days with ammonia loading rates of 49-63 NH(3) g m(-3) day(-1). The AEA and AOB communities were investigated by denaturing gradient gel electrophoresis, sequencing and real-time PCR analysis based on amoA genes. The results indicated that changes in abundance and community compositions occurred in a different manner between archaeal and bacterial amoA during the operation. However, both microbial community structures mainly varied when free ammonia (FA) concentrations in circulation water were increasing, which caused a temporal decline in reactor performance. Dominant amoA sequences after this transition were related to Thaumarchaeotal Group I.1b, Nitrosomonas europaea lineages and one subcluster within Nitrosospira sp. cluster 3, for archaea and bacteria, respectively. CONCLUSIONS: The specific FA in circulation water seems to be the important factor, which relates to the AOB and AEA community shifts in the biofilter besides ammonium and pH. SIGNIFICANCE AND IMPACT OF THE STUDY: One of the key factors for regulating AEA and AOB communities was proposed that is useful for optimizing biofiltration technology.

A small molecule CXCR4 inhibitor that blocks T cell line-tropic HIV-1 infection.

Several members of the chemokine receptor family have been shown to function in association with CD4 to permit human immunodeficiency virus type 1 (HIV-1) entry and infection. The CXC chemokine receptor CXCR4/fusin is a receptor for pre-B cell growth stimulating factor (PBSF)/stromal cell-derived factor 1 (SDF-1) and serves as a coreceptor for the entry of T cell line-tropic HIV-1 strains. Thus, the development of CXCR4 antagonists or agonists may be useful in the treatment of HIV-1 infection. T22 ([Tyr5,12,Lys7]-polyphemusin II) is a synthesized peptide that consists of 18 amino acid residues and an analogue of polyphemusin II isolated from the hemocyte debris of American horseshoe crabs (Limulus polyphemus). T22 was found to specifically inhibit the ability of T cell line-tropic HIV-1 to induce cell fusion and infect the cell lines transfected with CXCR4 and CD4 or peripheral blood mononuclear cells. In addition, T22 inhibited Ca2+ mobilization induced by pre-B cell growth stimulating factor (PBSF)/SDF-1 stimulation through CXCR4. Thus, T22 is a small molecule CXCR4 inhibitor that blocks T cell line-tropic HIV-1 entry into target cells.

Anti-chymotrypsin and anti-elastase activities of a synthetic bicyclic fragment containing a chymotrypsin-reactive site of soybean Bowman-Birk inhibitor.

A bicyclic hexadecapeptide, which corresponds to the sequence 36-51 and contains the chymotrypsin-reactive Leu-43-Ser-44 bond of soybean Bowman-Birk inhibitor, has been synthesized. This peptide consists of two loops formed by disulfide bridges between Cys-36 and Cys-51 and between Cys-41 and Cys-49. The bicyclic peptide showed a strong anti-chymotryptic activity with a Ki of 7.1.10(-7) M. Comparison of inhibitory activity and digestive stability against chymotrypsin with other hexadecapeptides having the same sequence but lacking one or both disulfide bridges suggested that the compact bicyclic structure increases the activity and protects the Leu-Ser bond from chymotryptic digestion. Interestingly, the bicyclic peptide was found to inhibit porcine pancreatic elastase with a Ki of 4.3.10(-5) M, indicating the broad specificity of this ring system.

Altered function and structure of low-density lipoprotein receptor in compactin (ML236B)-resistant mutants of Chinese hamster cells.

Mutants resistant to compactin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, have been previously isolated from the Chinese hamster V79 cell line. Two compactin-resistant mutants, MF-1 and MF-2, show altered responses to human low-density lipoprotein (LDL). Accumulation of fluorescent-labeled LDL was much reduced. Ligand blotting showed LDL receptor activity in MF-1 and MF-2 cells of about one half to one third that of V79. Internalization and degradation of LDL in MF-1 or MF-2 cells were about one tenth those in V79 cells, suggesting that the LDL binding as well as the LDL internalization of the compactin-resistant clones was altered. Down-regulation of LDL receptor activity as well as hydroxymethylglutaryl CoA reductase was observed in V79 cells treated with LDL, while there appeared to be much less down-regulation in MF-1 and MF-2 cells. Using anti-LDL receptor antibody, MF-1 and MF-2 cells were found to produce smaller-sized mature forms of LDL receptor: the molecular mass of the mutant LDL receptor was 3-5 kDa smaller than that of the parental LDL receptor. Altered O-linked oligosaccharides or amino acid sequence might account for the decreased molecular mass and aberrant properties of the LDL receptor in MF-1 and MF-2.

A comparative study of the solution structures of tachyplesin I and a novel anti-HIV synthetic peptide, T22 ([Tyr5,12, Lys7]-polyphemusin II), determined by nuclear magnetic resonance.

The solution structure of tachyplesin I, which was isolated from membrane acid extracts of the hemocytes from the Japanese horseshoe crab (Tachypleus tridentatus), was determined by nuclear magnetic resonance (NMR) and distance geometry calculation. Tachyplesin I takes an antiparallel beta-sheet structure with a type-II beta-turn. Recently, among more than 20 synthetic peptides associated with tachyplesin and its isopeptide (polyphemusin), we found that a novel compound, which we designated as T22 ([Tyr5,12, Lys7]-polyphemusin II), strongly inhibited the human immunodeficiency virus (HIV)-1-induced cytopathic effect and viral antigen expression. The solution structure of T22 was investigated using NMR, and its secondary structure was confirmed to be similar to that of tachyplesin I. The anti-parallel beta-sheet structure and the several amino-acid side chains on the plane of the beta-sheet of T22 are thought to be associated with the expression of anti-HIV activity.

Analysis of the interaction of an anti-HIV peptide, T22 ([Tyr5, 12, Lys7]-polyphemusin II), with gp120 and CD4 by surface plasmon resonance.

We have previously found that T22 ([Tyr5, 12, Lys7]-polyphemusin II) exhibits strong anti-human immunodeficiency virus (HIV) activity comparable to that of 3'-azido-2', 3'-dideoxythymidine (AZT). The inhibition mechanism of T22 on HIV-replication has not been elucidated precisely yet, and hence the target molecules of T22 have not been identified. However, our recent research suggested that T22 exerts its effect by blocking virus-cell fusion at an early stage of HIV infection and that T22 might interact with an HIV envelope protein and/or a T-cell surface protein, both of which are critical for HIV infection. In this paper we demonstrated that T22 binds specifically to both gp120 (an envelope protein of HIV) and CD4 (a T-cell surface protein) and that both bindings can be inhibited by an anti-T22 antibody, using biosensor technology (BIAcoreTM) based on the principles of surface plasmon resonance. Linearization by the BIAcoreTM system (BIAlogue software) and nonlinear least squares analysis by curve fitting with exponential equations showed that both interactions have close dissociation constants (approximately 10(-7) M). The present study suggests that T22 inhibits the virus-cell fusion process through binding to both gp120 and CD4.

Antisense src expression inhibits tyrosine phosphorylation of Shc and its association with Grb2 and Sos which leads to MAP kinase activation in U937 human leukemia cells.

We constructed a recombinant plasmid which expresses antisense src RNA after dexamethasone (Dexa) treatment, and transfected it into U937 human monoblastic leukemia cells (U937-ASRC). Induction of antisense src RNA expression diminished the amounts of c-Src and its protein tyrosine kinase (PTK) activity in U937-ASRC cells. The declines in c-Src and its PTK activity subsequently reduced the proliferation of U937-ASRC cells. To elucidate the growth signal transduction pathway downstream of c-Src, tyrosine phosphorylation of Shc was examined in U937-ASRC cells treated with Dexa. The decline in c-Src by induction of antisense src RNA expression decreased the level of tyrosine phosphorylation of Shc. Immunoprecipitated c-Src directly phosphorylated immunoprecipitated Shc on tyrosine residues in vitro. The amounts of Grb2 and Sos co-immunoprecipitated with Shc were decreased after Dexa treatment. However, the amount of Sos co-immunoprecipitated with Grb2 was apparently not affected by Dexa treatment. These results indicate that Grb2 and Sos constitutively associate with each other in U937 cells. Furthermore, the level of phosphorylation on tyrosine (204) essential for MAP kinase activation was decreased after Dexa treatment. Taken together with all these findings, it is suggested that c-Src directly phosphorylates Shc on tyrosine residues, which in turn binds to Grb2 constitutively associated with Sos to form a Shc-Grb2-Sos complex, and that the complex formation is coupled with MAP kinase activation mediated by Ras activation in U937 cells.

Methane-dependent denitrification by a semi-partitioned reactor supplied separately with methane and oxygen.

Methane (CH4) can be used as an alternative carbon source for denitrification with added oxygen (O2). However, the off-gas of denitrification reactors using a CH4-O2 mixture contains unused CH4 and O2 in proportions that make it unusable for fuel, carry explosion risks, and, if released into the atmosphere, contribute to the greenhouse effect. This study tested a novel reactor with a partition dividing the headspace completely and extending partly into the liquid layer. When CH4 and O2 were supplied separately to the liquid layer on opposite sides of the partition, the methane-dependent denitrification (MDD) activity was similar to that when the two gases were supplied as a mixture. In reactors with separate gas supplies, the off-gas from the CH4 supply side was high in CH4 and low in O2, and was usable for fuel, and that from the O2 supply side was very low in CH4, and might be released into the atmosphere. MDD activity increased with the O2 supply rate, and separate discharge of CH4 and O2 was maintained. The concentration of dissolved methane in the effluent was decreased by lowering the CH4/O2 supply ratio to 1.0 and drawing the effluent from the O2 supply side. This novel reactor enhances the safety of MDD, allows reuse of methane as fuel, and reduces methane leakage to the atmosphere.

Revised structure of the peptide lactone antibiotic, TL-119 and/or A-3302-B.

The peptide lactone antibiotic TL-119 and/or A-3302-B was chemically synthesized in order to confirm the proposed structure. The synthetic compound was different from both natural TL-119 and A-3302-B in their physicochemical properties and in biological activity. Re-examination of the configuration of the constituent amino acid residues in natural TL-119 and/or A-3302-B indicated that natural TL-119 and A-3302-B contains D-aThr instead of the original L-Thr. We tentatively propose a revised structure for TL-119 and/or A-3302-B.

Environment-dependent conformation and antimicrobial activity of a gramicidin S analog containing leucine and lysine residues.

An analog of gramicidin S, cyclo(-L-Leu-L-Lys-L-Leu-D-Leu-L-Leu-)2, in which four out of five amino acid components of gramicidin S were substituted, has been synthesized. This analog assumes a conformation similar to that of gramicidin S in acidic liposomes and a random conformation in neutral liposomes. The antimicrobial activity of this analog corresponded to one-fourth of that of gramicidin S. A possible mechanism for conformational changes in acidic liposomes is discussed.

[4,4'-D-diaminopropionic acid]gramicidin S: a synthetic gramicidin S analog with antimicrobial activity against Gram-negative bacteria.

Gramicidin S is especially active against Gram-positive bacteria; e.g., Staphylococcus aureus. An analog, [4,4'-D-diaminopropionic acid]gramicidin S, which contains D-diaminopropionic acid residues instead of D-phenylalanine residues, has been synthesized. This analog is active against some of the Gram-negative bacteria; e.g., Escherichia coli and Salmonella typhosa. Activities of several related analogs are discussed.

Enzyme inhibition by dipeptides containing 2,3-methanophenylalanine, a sterically constrained amino acid.

Both isomers of (E)-2,3-methanophenylalanine (delta EPhe), a sterically restricted amino acid, were incorporated into peptides in order to examine their possible enzyme inhibitory activity. Both (2R,3S)- and (2S,3R)- delta EPhe-Phe(or Leu)-OMe were found to inhibit effectively the hydrolysis of Ac-Tyr-OEt by chymotrypsin in a competitive manner. The ester groups of these dipeptides were quite resistant to chymotrypsin hydrolysis, and the delta EPhe-Phe peptide bond was also entirely stable. The inhibition constant (Ki) of the most potent dipeptide of H-(2R,3S)-delta EPhe-Phe-OMe was 0.16 mM at 25 degrees C. The inhibitory action of delta Phe-containing peptides was found to depend on the configuration of the delta Phe residue. The electrophilic nature of the cyclopropane ring which is conjugated with both the phenyl ring and the ester carbonyl group appears to be relevant to the inhibitory activity. Fully irreversible inactivation of chymotrypsin was achieved by its incubation with H-(2R,3S)- delta EPhe-Leu-OMe. An enzyme carboxylate group is thought to be responsible for nucleophilic attack on the cyclopropane ring leading to irreversible inactivation.

[4,4'-(Z)-dehydrophenylalanine]gramicidin S with stabilized bioactive conformation and strong antimicrobial activity.

Dehydrophenylalanine (delta Phe) was incorporated into an antibiotic peptide gramicidin S (GS) in place of D-Phe4,4' to prepare an unsaturated analog. Conformational analysis with 1H-NMR indicated that the unsaturated analog has much the same backbone conformation as that of natural gramicidin S as shown by NOE experiments. Studies on temperature dependences and on the chemical shift differences showed that the hydrogen bonds between Val-NH and Leu-CO in the unsaturated analog are strengthened by the incorporation of delta Phe4,4'. This resulted in the reinforcement of the beta-sheet structure which is the most important structural element for GS bioactivity. [delta Phe4,4']gramicidin S exhibited indeed very strong antimicrobial activities against Gram-positive bacteria as well as the natural peptide.

Differences in skeletal muscle and bone mineral mass between black and white females and their relevance to estimates of body composition.

This study tested the hypothesis that black females have an increase in skeletal muscle and bone mineral mass compared with white females matched for age (+/- 5 y), weight (+/- 2 kg), height (+/- 3 cm), and menstrual status. Conventional [underwater weighing, whole body 40K counting (WBC), 3H2O dilution] and newly developed (dual-photon absorptiometry) techniques were used to provide ethnicity-independent estimates of body composition in 28 pairs of matched subjects. Black females had greater appendicular skeletal muscle (P less than 0.001), bone mineral (P less than 0.001), and total body potassium (TBK) (P = 0.05) compared with white females. Two classic coefficients used in body composition research [density of fat-free mass (FFM) for underwater weighing and TBK/FFM for WBC] differed significantly (P less than 0.05) between black and white females; currently applied coefficients underestimated fat in black females. This study confirms that black and white females differ in body composition and that errors in fat estimates occur when ethnicity is not accounted for in body composition models.

Noninvasive quantitative evaluation of early atherosclerosis and the effect of monatepil, a new antihypertensive agent. An interim report.

In two studies on the same group of patients we evaluated noninvasive methods of assessing atherosclerosis and determined the effect of the new calcium channel-blocking agent monatepil on the progression of early atherosclerosis in humans. Computed tomography (CT) of the lower abdominal aorta and ultrasonography of the carotid arteries were used as noninvasive methods to determine the extent of atherosclerosis. To evaluate the CT images, we developed a new medical image analysis program. This enabled aortic calcification volume (ACV) to be quantified using plain CT images, and aortic wall volume (AWV) and aortic wall and calcification volume (AWCV) to be quantified from contrast CT images. Interobserver coefficients of variation of ACV, AWV, and AWCV (n = 8) were 4.7, 2.4, and 5.0%, respectively. In the monatepil study, the effect of the drug on serum lipid profiles was evaluated. Preliminary results show that shortly after monatepil administration, total serum cholesterol levels decreased significantly from 253.8 +/- 35.6 to 244.8 +/- 38.6 mg/dL (P < .009) and triglyceride levels tended to decrease. A positive correlation between the change in total cholesterol and changes in mean platelet volume was found (P = .028). Fasting immunoreactive insulin levels decreased in the four patients in which they were determined. Although this is a preliminary study, results indicate that CT of the lower abdominal aorta in combination with our new analysis program may be a precise, reproducible means of assessing early atherosclerosis. We have also shown that monatepil significantly decreases total cholesterol levels. However, the long-term effects of monatepil on the progression of atherosclerosis remain to be determined.

Purification and characterization of a coagulant enzyme, okinaxobin I, from the venom of Trimeresurus okinavensis (Himehabu snake) which releases fibrinopeptide B.

A coagulant enzyme, named okinaxobin I, has been purified to homogeneity from the venom of Trimeresurus okinavensis (Himehabu) by chromatographies on Sephadex G-100 and CM-Toyopearl 650M columns. The enzyme was a monomer with a molecular weight of 37,000 and its isoelectric point was 5.4. The enzyme acted on fibrinogen to form fibrin clots with a specific activity of 77 NIH units/mg. Fibrinopeptide B was released at a rate much faster than fibrinopeptide A. The enzyme exhibited 2 to 3 times higher activity toward tosyl-L-arginine methyl ester and benzoyl-L-arginine p-nitroanilide than bovine thrombin. The esterase activity was strongly inhibited by diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and to a lesser extent by tosyl-L-lysine chloromethyl ketone, indicating that the enzyme is a serine protease like thrombin. The N-terminal sequence was highly homologous to those of coagulant enzymes from T. flavoviridis and Bothrops atrox, moojeni venoms which preferentially release fibrinopeptide A. In order to remove most, if not all, of the bonded carbohydrates, the enzyme was treated with anhydrous hydrogen fluoride (HF), thereby reducing the molecular weight to 30,000. The protein contained approximately 260 amino acid residues when computation was based on this value. The HF-treated enzyme retained about 50% of the clotting and esterolytic (TAME) activities and preferentially released fibrinopeptide B from fibrinogen. The carbohydrate moiety is not crucial for enzyme activity but might be necessary for eliciting full activity.

Body composition in humans: advances in the development of multicompartment chemical models.

Until recently it was practical to divide body weight into only two or three chemical compartments in living subjects due to an inability to quantify directly total body mineral, protein, and fat in vivo. The six-compartment chemical model is now the cornerstone of research in human body composition. Advanced technologies, including neutron activation analysis systems and dual photon absorptiometry, now enable investigators to extend body composition estimates and to construct near-complete chemical models in vivo. These new or refined approaches will advance our knowledge of human body composition and will also improve our accuracy in calibrating simpler epidemiologic and bedside body-composition techniques.

Dimerization of neurokinin A and B COOH-terminal heptapeptide fragments enhanced the selectivity for tachykinin receptor subtypes.

We have synthesized dimeric analogues of neurokinin A and B COOH-terminal heptapeptide fragments and evaluated their biological activities to contract isolated smooth muscle preparations of the guinea-pig ileum and rat vas deferens. The dimers were fairly active and showed two-fold increased selectivity for receptors in the guinea-pig ileum as compared with monomers. Extremely slow dissociation indicated a possible bivalent interaction between dimer and receptor.