Microbial response to environmental changes in an Anaerobic Baffled Reactor (ABR)

Two 10 litre Anaerobic Baffled Reactors (ABR), with 8 separate compartments, were used to examine the effect of Hydraulic Retention Time (HRT), effluent recycle and temperature changes on the trophic groups in anaerobic digestion. A synthetic carbohydrate (sucrose)-protein substrate was used, and the reactors run at 20 h HRT, and 35 °C. Changing the HRT from 40 to 20 hours doubled the organic loading which caused accumulation of reduced intermediates. The pattern of Volatile Fatty Acids (VFA) at steady state due to an increase in recycle ratios led to the breakage of microbial flocs, and a reduced overall microbial activity. However, the quantity of reduced intermediates was substantially reduced. Decreasing the temperature to 25 °C had differing degrees of influence on reactors I &II, but the same pattern of microbial response occurred; that is the slower growing microorganisms were more affected by the temperature drop. It was found that the unique structure of the ABR brings about the partial separation of acidogenesis and methanogenesis.     

The consequence of passive administration of an anti-human immunodeficiency virus type 1 neutralizing monoclonal antibody before challenge of chimpanzees with a primary virus isolate.

The anti-gp41 virus neutralizing monoclonal antibody 2F5 was infused into chimpanzees, which were then given an intravenous challenge with a primary human immunodeficiency virus type I (HIV-1) isolate. In two control animals, the infection was established immediately, as evidenced by positive cell-associated DNA PCR and serum RNA PCR tests within 1 week, seroconversion by 4 weeks, and development of lymphadenopathy in this acute phase. Serum RNA PCR tests were negative in one of the two antibody-infused animals until week 8 and in the other antibody-infused animal until week 12; both animals seroconverted at week 14. The peak of measurable virus-specific serum RNA was delayed until week 16 in one antibody-infused animal. Virus-specific RNA in the other animal did not reach levels comparable to those in the other animals through 1 year of follow-up studies. Virus was isolated from the week 16 blood sample from one infused animal. Virus was not isolated from peripheral blood of the second animal but was isolated from lymph node cells taken at week 36. The infection of untreated chimpanzees with this primary isolate appears robust. Use of this isolate should widen the scope of possible experiments in the chimpanzee model. This antibody infusion study indicates that neutralizing antibody, when present at the time of challenge, affects the timing and level of infection and remains influential after it can no longer be detected in the peripheral circulation. It is possible that preexisting, neutralizing antibodies (passively administered or actively elicited) affect the course of acute-phase virus replication in humans. It remains to be established whether these immunologically mediated early effects will influence the course of HIV-1 disease.     

A model of the platelet factor 4 complex with heparin.

A model of heparin bound to bovine platelet factor 4 (BPF4) was completed using a graphically designed heparin molecule and the crystallographic coordinates of the native bovine platelet factor 4 tetramer. The oligosaccharides had a chain length of at least eight disaccharide units with the major repeating disaccharide unit consisting of (1----4)-O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1----4)-(2-deoxy-2-sulfamino-2-D-glucopyranosyl 6-sulfate). Each disaccharide unit carried a -4.0 charge. The structure of BPF4 was solved to 2.6 A resolution with R = 0.237. Each monomer of BPF4 contains an alpha-helix lying across 3 strands of antiparallel beta-sheet. Each helix has four lysines, which have been implicated in heparin binding. These lysine residues are predominantly on one side of the helix and are solvent accessible. Electrostatic calculations performed on the BPF4 tetramer show a ring of strong, positive charge which runs perpendicularly across the helices. Included in this ring of density is His-38, which has been shown by NMR to have a large pKa shift when heparin binds to BPF4. Our model of heparin bound to PF4 has the anionic polysaccharide perpendicular to the alpha-helices, wrapped about the tetramer along the ring of positive charge, and salt linked to all four lysines on the helix of each monomer.     

Performance of the anaerobic baffled reactor under steady-state and shock loading conditions

The anaerobic baffled reactor (ABR) contains a granulated, mixed anaerobic culture segregated into compartments. Operation of four reactors under a range of hydraulic retention times showed that this novel reactor design offers highly efficient performance in the conversion of carbon in the feed stream to methane and carbon dioxide. The design parameter varied was the number of compartments. COD removal at 20 h retention time was routinely over 95% in all reactors, with low washout of biomass. Very high specific reaction rates were achievable (although with a loss of efficiency) at low biomass concentrations and high loading rates. In order to optimize volumetric reaction rates, a tradeoff has to be made between high biomass concentration, granule size, and the resulting mass transfer limitations. Formate is shown to be an important intermediate in the process under conditions of high loading.     

Expression, characterization, and crystallization of a member of the novel phospholipase D family of phosphodiesterases.

A family of phospholipase D (PLD) proteins has recently been identified (Koonin, 1996; Ponting & Kerr, 1996) based upon amino acid sequence identity. This family includes human and plant PLDs, proteins encoded by open reading frames in pathogenic viruses and bacteria, as well as an endonuclease. The endonuclease, known as Nuc, is encoded by the IncN plasmid, pKM101, present in Salmonella typhimurium. The recombinant Nuc protein has been expressed and purified from Escherichia coli. The amino-terminal sequencing of the purified protein indicated that the mature protein started from the 23rd residue of the predicted sequence, suggesting that the protein is proteolytically processed during export to the periplasmic space. The recombinant enzyme was able to hydrolyze both double and single-strand DNA and an artificial substrate, bis(4-nitrophenyl) phosphate, which contains a phosphodiester bond. The enzyme activity was not inhibited in the presence of EDTA and was not regulated by divalent cations. The purified protein has been crystallized by hanging drop vapor diffusion methods, and those crystals diffract to 1.9 A resolution.     

A reassessment of decreased amino acid accumulation by ehrlich ascites tumor cells in the presence of metabolic inhibitors

This study was undertaken to examine the mechanism by which metabolic inhibition reduces amino acid active transport in ehrlich ascites tumor cells. At 37 degrees C the metabolic inhibitor combination 0.1 mM 2,4-dinitrophenol (DNP) + 10 mM 2- deoxy-D-glucose (DOG) reduced the cell ATP concentration to 0.10- 0.15 mM in less than 5 min. This inhibition was associated with a 20.6 percent +/- 6.4 percent (SD) decrease in the initial influx of α-aminoisobutyric acid (AIB), and a two- to fourfold increase in the unidirectional efflux. These effects could be dissociated from changes in cell Na(+) or K(+) concentrations. Cells incubated to the steady state in 1.0-1.5 mM AIB showed an increased steady-state flux in the presence of DNP + DOG. Steady- state fluxes were consistent with trans-inhibition of AIB influx and trans-stimulation of efflux in control cells, but trans- stimulation of both fluxes in inhibited cells. In spite of the reduction of the cell ATP concentration to less than 0.15 mM and greatly reduced transmembrane concentration gradients of Na(+) and K(+), cells incubated to the steady state in the presence of the inhibitors still established an AIB distribution ration 13.8 +/- 2.6. The results are interpreted to indicate that a component of the reduction of AIB transport produced by metabolic inhibition is attributable to other actions in addition to the reduction of cation concentration gradients. Reduction of cell ATP alone is not responsible for the effects of metabolic inhibition, and both the transmembrane voltage and direct coupling to substrate oxidation via plasma-membrane-bound enzymes must be considered as possible energy sources for amino acid active transport.     

Hemolytic Transfusion Reactions

After receiving apparently compatible blood three patients suffered hemolytic reactions. The compatibility tests were by saline and indirect Coombs technique including a screening tube of group 0 cells. The antibodies responsible for these reactions were not clearly demonstrable for several days following the transfusion. In two instances the antibody was anti-E.These case reports point up the following. (a) Currently used cross-matching procedures will occasionally fail to demonstrate an incompatibility. (b) In two of the cases the direct Coombs test was negative on an immediate post-transfusion specimen, when it could have been of great aid in diagnosis. (c) When a transfusion reaction of hemolytic type is suspected, a follow-up study several days after the reaction may clarify the diagnosis; when possible, transfusions should be avoided in the interim.