Identification and comparison of aerobic and denitrifying polyphosphate-accumulating organisms

Two laboratory-scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic-aerobic or alternating anaerobic-anoxic modes, respectively. Polyphosphate-accumulating organisms (PAOs) were enriched in the anaerobic-aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic-aerobic SBR. Fluorescence in situ hybridization (FISH) demonstrated that the well-known PAO, "Candidatus Accumulibacter phosphatis" was abundant in both SBRs, and post-FISH chemical staining with 4,6-diamidino-2-phenylindol (DAPI) confirmed that they accumulated polyphosphate. When the anaerobic-anoxic SBR enriched for DPAOs was converted to anaerobic-aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately. However, when the anaerobic-aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5-h lag period elapsed before phosphorus uptake proceeded. This anoxic phosphorus-uptake lag phase was not observed in the subsequent anaerobic-aerobic cycle. These results demonstrate that the PAOs that dominated the anaerobic-aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic-anoxic conditions.     

Generic/matrix evaluation of SV40 clearance by anion exchange chromatography in flow-through mode

The potential of viral contamination is a regulatory concern for continuous cell line-derived pharmaceutical proteins. Complementary and redundant safety steps, including an evaluation of the viral clearance capacity of unit operations in the purification process, are performed prior to registration and marketing of biotechnology pharmaceuticals. Because process refinement is frequently beneficial, CBER/FDA has published guidance facilitating process improvement by delineating specific instances where the bracketing and generic approaches are appropriate for virus removal validation. In this study, a generic/matrix study was performed using Q-Sepharose Fast Flow (QSFF) chromatography to determine if bracketing and generic validation can be applied to anion exchange chromatography. Key operational parameters were varied to upper and lower extreme values and the impact on viral clearance was assessed using simian virus 40 (SV40) as the model virus. Operational ranges for key chromatography parameters were identified where an SV40 log(10) reduction value (LRV) of >or=4.7 log(10) is consistently achieved. On the basis of the apparent robustness of SV40 removal by Q-anion exchange chromatography, we propose that the concept of "bracketed generic" validation can be applied to this and potentially other chromatography unit operations.     

Bracketed generic inactivation of rodent retroviruses by low pH treatment for monoclonal antibodies and recombinant proteins

Viral safety is a predominant concern for monoclonal antibodies (mAbs) and other recombinant proteins (RPs) with pharmaceutical applications. Certain commercial purification modules, such as nanofiltration and low-pH inactivation, have been observed to reliably clear greater than 4 log(10) of large enveloped viruses, including endogenous retrovirus. The concept of "bracketed generic clearance" has been proposed for these steps if it could be prospectively demonstrated that viral log(10) reduction value (LRV) is not impacted by operating parameters that can vary, within a reasonable range, between commercial processes. In the case of low-pH inactivation, a common step in mAb purification processes employed after protein A affinity chromatography, these parameters would include pH, time and temperature of incubation, the content of salts, protein concentration, aggregates, impurities, model protein pI, and buffer composition. In this report, we define bracketed generic clearance conditions, using a prospectively defined bracket/matrix approach, where low-pH inactivation consistently achieves >or=4.6 log(10) clearance of xenotropic murine leukemia virus (X-MLV), a model for rodent endogenous retrovirus. The mechanism of retrovirus inactivation by low-pH treatment was also investigated.     

Model-based analysis of anaerobic acetate uptake by a mixed culture of polyphosphate-accumulating and glycogen-accumulating organisms

An increasing number of studies shows that the glycogen-accumulating organisms (GAOs) can survive and may indeed proliferate under the alternating anaerobic/aerobic conditions found in EBPR systems, thus forming a strong competitor of the polyphosphate-accumulating organisms (PAOs). Understanding their behaviors in a mixed PAO and GAO culture under various operational conditions is essential for developing operating strategies that disadvantage the growth of this group of unwanted organisms. A model-based data analysis method is developed in this paper for the study of the anaerobic PAO and GAO activities in a mixed PAO and GAO culture. The method primarily makes use of the hydrogen ion production rate and the carbon dioxide transfer rate resulting from the acetate uptake processes by PAOs and GAOs, measured with a recently developed titration and off-gas analysis (TOGA) sensor. The method is demonstrated using the data from a laboratory-scale sequencing batch reactor (SBR) operated under alternating anaerobic and aerobic conditions. The data analysis using the proposed method strongly indicates a coexistence of PAOs and GAOs in the system, which was independently confirmed by fluorescent in situ hybridization (FISH) measurement. The model-based analysis also allowed the identification of the respective acetate uptake rates by PAOs and GAOs, along with a number of kinetic and stoichiometric parameters involved in the PAO and GAO models. The excellent fit between the model predictions and the experimental data not involved in parameter identification shows that the parameter values found are reliable and accurate. It also demonstrates that the current anaerobic PAO and GAO models are able to accurately characterize the PAO/GAO mixed culture obtained in this study. This is of major importance as no pure culture of either PAOs or GAOs has been reported to date, and hence the current PAO and GAO models were developed for the interpretation of experimental results of mixed cultures. The proposed method is readily applicable for detailed investigations of the competition between PAOs and GAOs in enriched cultures. However, the fermentation of organic substrates carried out by ordinary heterotrophs needs to be accounted for when the method is applied to the study of PAO and GAO competition in full-scale sludges.     

Prevention of myosin-induced autoimmune myocarditis in mice by anti-L3T4 monoclonal antibody

This study was aimed at studying the effect of the induction of immune tolerance to swine cardiac myosin from anti-L3T4 monoclonal antibody injection and whether the immune tolerance could protect mice with myosin-induced myocarditis from myocardial injury. Twenty-four Balb/c mice were divided into two groups at random. All of the mice were immunized with swine cardiac myosin on the 1st day, 14th, 28th, 42nd, and 52nd day. Immune tolerance was induced by triplicate injections of 400 microg anti-L3T4 McAb on the 0 day (intravenous), 1st day, and 2nd day (intraperitoneal) in McAb-treated group. In the saline-treated group, saline of the same volume as anti-L3T4 monoclonal antibody was used as a control. The sera and hearts biopsies of all mice were collected on the 58th day. The anti-cardiac myosin antibody was examined with ELISA, and pathological changes of heart were observed by light microscope. It was shown that mice immunized with swine cardiac myosin could produce anti-myosin antibody and the anti-cardiac myosin antibody was positive in most of the saline-treated group but negative in the McAb-treated group. Morphologically, myocardial degeneration, necrosis, and infiltration of inflammatory cells were found in the saline-treated group but not in the McAb-treated group. In conclusion, this study indicated that the immune tolerance to cardiac myosin was induced by the anti-L3T4 monoclonal antibody, and accordingly myocardial injury could be prevented by induction of immune tolerance.     

Morphological manipulation of PC liposome by controlled bolaamphiphile doping

The morphological characterization of aqueous dispersions of PC amphiphile and bolaamphiphile AEC was observed by transmission electron microscopy, the measurement of the liposomal membrane fluidity, differential scanning calorimetry, 5(6)-CF release from liposome and zeta potential measurement. Results indicate that the bolaamphiphile AEC can be included within conventional egg-PC liposome bilayer, which leads to the decrease of liposomal membrane fluidity (P) and the release behavior of 5(6)-CF. This behavior could be due to the property of bolaamphiphile AEC and the good miscibility of bolaamphiphile AEC with PC.     

Inhibition of Abeta production and APP maturation by a specific PKA inhibitor

Alzheimer's disease is characterized pathologically by extracellular amyloid beta protein (Abeta) deposition in the brain. The Abeta peptide, a 39-42 amino acid fragment, is derived from defined proteolysis of the amyloid precursor protein (APP) [Glenner et al., Appl. Pathol. 2 (1984) 357-369; Selkoe, Neuron 6 (1991) 487-498] and is the primary component of senile plaques. Although it is known that intracellular APP is subjected to posttranslational modification, the molecular mechanism that regulates the APP processing is not completely clear. In the present study, we demonstrates that H89, a specific inhibitor for cAMP dependent protein kinase A (PKA), inhibits Abeta production and APP secretion in a dose dependent manner in cells stably transfected with human APP bearing a 'Swedish mutation'. Concurrent with the effect, H89 inhibits C-terminal fragment of the APP. We also found that the PKA inhibitor abolishes the mature form of intracellular APP and accumulates the immature form. Finally, direct administration of H89 into brains of transgenic mice overexpressing human APP shows that the compound inhibits Abeta production in the hippocampal region. Our data suggests that PKA plays an important role in the maturation of APP associated with APP processing.     

Negative regulation of T cell antigen receptor-mediated Crk-L-C3G signaling and cell adhesion by Cbl-b

It was previously reported that Cbl-b associates with Crk-L in Jurkat T cells. However, the physiological significance of such association remains unclear. Here we examined a regulatory role of Cbl-b in Crk-L-C3G signaling pathway. We found that Cbl-b associates with, and induces, ubiquitin conjugation to Crk-L, which requires a functional RING finger. Cbl-b deficiency does not affect Crk-L stability, but its association with C3G. In Cbl-b-/- T cells, the interaction between Crk-L and C3G, and the activity of the small GTPase Rap1, are increased. Cbl-b-/- T cells also display increased adhesion and cell surface binding to ICAM-1, a finding that is supported by the enhanced clustering of LFA-1 in Cbl-b-/- T cells in response to TCR stimulation. Thus, Cbl-b plays a negative role in Crk-L-C3G-mediated Rap1 and LFA-1 activation in T cells.     

Regulation of the cyclin D3 promoter by E2F1

We have previously demonstrated that ectopic expression of E2F1 is sufficient to drive quiescent cells into S phase and that E2F1 expression can contribute to oncogenic transformation. Key target genes in this process include master regulators of the cell cycle, such as cyclin E, which regulates G(1) progression, and cyclin A, which is required for the initiation of DNA synthesis. In the present work, we present novel evidence that a second G(1) cyclin, cyclin D3, is also potently activated by E2F1. First, an estrogen receptor-E2F1 fusion protein (ER-E2F1) potently activates the endogenous cyclin D3 mRNA upon treatment with 4-hydroxytamoxifen, which induces nuclear accumulation of the otherwise cytosolic fusion protein. Furthermore, trans-activation of cyclin D3 by ER-E2F1 occurs even in the presence of the protein synthesis inhibitor cycloheximide and thus appears direct. Second, all of the growth-stimulatory members of the E2F family (E2F1, -2, and -3A) potently activate a cyclin D3 promoter reporter, whereas growth-restraining members of the family (E2F4, -5, and -6) have little effect. Third, recombinant E2F1 binds with high affinity to the cyclin D3 promoter in vitro. Fourth, chromatin immunoprecipitation assays demonstrate that endogenous E2F1 is associated with the cyclin D3 promoter in vivo. Finally, mapping experiments localize the essential E2F regulatory element of the cyclin D3 promoter to a noncanonical E2F site in the promoter between nucleotides -143 and -135 relative to the initiating methionine codon. We conclude that in addition to cyclins E and A, E2F family members can also activate one member of the D-type cyclins, further contributing to the ability of the stimulatory E2F family members to drive cellular proliferation.     

Indirect photometric ion chromatographic analysis of anions in Haematococcus pluvialis culture media

An indirect photometric ion chromatographic method for the simultaneous determination of chloride, nitrate and sulfate ions was developed and applied to the determination of anions, mainly nitrate, in the alga Haematococcus pluvialis culture media. Using phthalic acid/sodium tetraborate aqueous solution as the mobile phase, anions can be detected indirectly by a UV detector. The calibration curves for these anions gave good linearity from 1 to 1000 g ml^–1.