Co-fermentation of xylose and cellobiose by an engineered Saccharomyces cerevisiae

We have integrated and coordinately expressed in Saccharomyces cerevisiae a xylose isomerase and cellobiose phosphorylase from Ruminococcus flavefaciens that enables fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. The native xylose isomerase was active in cell-free extracts from yeast transformants containing a single integrated copy of the gene. We improved the activity of the enzyme and its affinity for xylose by modifications to the 5′-end of the gene, site-directed mutagenesis, and codon optimization. The improved enzyme, designated RfCO*, demonstrated a 4.8-fold increase in activity compared to the native xylose isomerase, with a K_m for xylose of 66.7?mM and a specific activity of 1.41?μmol/min/mg. In comparison, the native xylose isomerase was found to have a K_m for xylose of 117.1?mM and a specific activity of 0.29?μmol/min/mg. The coordinate over-expression of RfCO* along with cellobiose phosphorylase, cellobiose transporters, the endogenous genes GAL2 and XKS1, and disruption of the native PHO13 and GRE3 genes allowed the fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. Interestingly, this strain was unable to utilize xylose or cellobiose as a sole carbon source for growth under anaerobic conditions, thus minimizing yield loss to biomass formation and maximizing ethanol yield during their fermentation.     

Differences among immune complexes: association of C1q in SLE immune complexes with renal disease

Studies that made use of multiple assay systems demonstrated increased levels of immune complexes (IC) in patients with systemic lupus erythematosus (SLE), but no consistent correlations of IC concentration to patterns or activity of disease have been observed. Furthermore, consistent associations between qualitative differences in IC and disease manifestations have been elusive. IC interaction with erythrocytes and mononuclear phagocytic cells is another variable in SLE that may also mediate some of the biological effects of IC. The present report concerns studies of the composition of purified IC obtained from individuals with SLE and other rheumatic diseases; a 64,000 dalton component identified as the A-B subunit of C1q was detected in purified IC from 27 of 51 SLE patients (53%). The presence of this 64,000 dalton component was not related to either IC concentration or to the serum C1q level. However, the presence of the C1q component in isolated SLE IC did correlate with the presence of renal disease (p less than 0.02). These observations are interpreted relative to a recently described kinetic model of IC clearance.     

Application of the Fröhlich theory to the modelling of rouleau formation in human erythrocytes

The details of Fröhlich's theory and some recent experiments on the rouleau formation of human erythrocytes which exhibit a strong interaction that appears to satisfy the prerequisites of the Fröhlich theory, are summarized. To verify whether the Fröhlich theory of long-range coherence in biological systems is applicable to the phenomenon of rouleau formation in human erythrocytes, the interactions between erythrocytes are modelled as those between two large, coupled oscillating dipoles. Relevant expressions for the resonant long-range and the van der Waals interaction are then derived. Using the available numerical data, the eigenfrequencies and the interaction energies corresponding to the experimental conditions are then derived. In the range of postulated frequencies (10¹¹–10¹² Hz) the effective interaction coefficient due to the resonant long-range forces is, indeed, found to agree with its experimental value of 3.0. However, the same value of can also be achieved through the ordinary van der Waals interactions between dipoles oscillating at lower frequencies. It is concluded that the resonant long-range interaction between erythrocytes may be responsible for the onset of rouleau formation. However, other mechanisms cannot be ruled out at this stage, especially since the Fröhlich mechanism requires a number of unconfirmed preconditions.     

Immunological relationship among hydrogenases.

We examined the immunological cross-reactions of 11 different hydrogenase antigens with 9 different hydrogenase antibodies. Included were antibodies and antigens of both subunits of the hydrogenases of Bradyrhizobium japonicum and Thiocapsa roseopersicina. The results showed a strong relationship among the Ni-Fe dimeric hydrogenases. The two subunits of Ni-Fe dimeric hydrogenases appeared immunologically distinct: specific interactions occurred only when antibodies to the 60- and 30-kilodalton subunits reacted with the 60- and 30-kilodalton-subunit antigens. The interspecies cross-reactions suggested that at least one conserved protein region exists among the large subunits of these enzymes, whereas the small subunits are less conserved. Antibodies to the Fe-only bidirectional hydrogenase of Clostridium pasteurianum reacted with the Desulfovibrio vulgaris bidirectional hydrogenase. Surprisingly, antibodies to the clostridial uptake hydrogenase did not react with any of the Fe-only bidirectional hydrogenases but did react with several of the Ni-Fe dimeric hydrogenases. The two hydrogenases from C. pasteurianum were found to be quite different immunologically. The possible relationship of these findings to the structure and catalytic functions of hydrogenase are discussed.     

A phase II trial of murine monoclonal antibody 17-1A and interferon-γ: Clinical and immunological data

Summary A group of 15 patients with metastatic colorectal adenocarcinoma received a combination of interferon (0.1 mg/m², days 1–15) and the murine monoclonal antibody 17-1A (400 mg, days 5, 7, 9 and 12). The treatment was tolerated with minimal toxicity. Of the 14 evaluable patients, 13 developed human antibody to murine 17-1A, with 11 patients demonstrating antibody to the variable region of 17-1A (anti-idiotype). Antibody to the variable region was inhibited by 17-1A but not by mouse immunoglobulin. Sera from patients with substantial anti-idiotype reactivity were capable of inhibiting the binding of murine 17-1A to antigen expressing LS174-T cells thus indicating the presence of antibody directed against the 17-1A combining site (mirror-image anti-idiotype). The median survival of the whole group was 56 weeks and there was no correlation between clinical response/survival and the development of anti-idiotype antibody.Supported by the Veterans Administration Medical Center and by Public Health Services grant CA 45 232 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services     

Aerobic, inactive forms of Azotobacter vinelandii hydrogenase: activation kinetics and insensitivity to C2H2 inhibition

Azotobacter vinelandii hydrogenase (EC class 1.12), either purified or membrane-associated, was obtained aerobically in an inactive state. The kinetics of activation by treatment with a reductant (H2 or dithionite) were determined. Three distinct phases of the activation were observed. Aerobically prepared, inactive hydrogenase was insensitive to acetylene inhibition, but could be rendered acetylene-sensitive by reduction with dithionite. These findings indicate that acetylene inhibition of hydrogenase requires catalytically active enzyme.     

Evidence for titratable gating charges controlling the voltage dependence of the outer mitochondrial membrane channel,VDAC

Summary A voltage-dependent anion-selective channel, VDAC, is found in outer mitochondrial membranes. VDAC's conductance is known to decrease as the transmembrane voltage is increased in either the positive or negative direction. Charged groups on the channel may be responsible for this voltage dependence by allowing the channel to respond to an applied electric field. If so, then neutralization of these charges would eliminate the voltage dependence. Channels in planar lipid bilayers which behaved normally at pH 6 lost much of their voltage dependence at high pH. Raising the pH reduced the steepness of the voltage dependence and raised the voltage needed to close half the channels. In contrast, the energy difference between the open and closed state in the absence of a field was changed very little by the elevated pH. The groups being titrated had an apparent pK of 10.6. From the pK and chemical modification, lysine epsilon amino groups are the most likely candidates responsible for VDAC's ability to respond to an applied electric field.     

Defective mononuclear phagocyte function in systemic lupus erythematosus: dissociation of Fc receptor-ligand binding and internalization

Fc receptor-mediated mononuclear phagocyte system (MPS) clearance is impaired in systemic lupus erythematosus (SLE) and may contribute to the pathogenesis of the immune complex disease. To investigate the basis of MPS dysfunction, we have examined concurrent in vivo and in vitro Fc receptor function in 22 patients with SLE and 23 disease-free adults. Blood monocyte Fc receptor binding was increased rather than decreased as predicted by the saturation hypothesis of MPS blockade. Rosette formation of IgG-sensitized bovine erythrocytes (EA) with monocytes demonstrated increased Fc receptor-ligand binding in SLE (percent rosettes: 40 +/- 12 vs 27 +/- 8, p less than 0.001). Scatchard analysis of the binding of radiolabeled IgG oligomers to SLE monocytes indicated a mean receptor number 30% higher than control, although this did not reach statistical significance. Despite enhanced Fc receptor-ligand (EA) binding, Fc-mediated phagocytosis of EA was decreased in SLE (1.7 +/- 0.7 erythrocytes/monocytes/hour vs 2.6 +/- 1.0, p less than 0.004). This decrease in phagocytosis by blood monocytes from SLE patients was significantly greater than that attributable to the predominance in SLE of individuals with certain HLA B cell alloantigens and intrinsically lower phagocytic rates (p less than 0.05 for all groups). This decrease therefore represents a disease-acquired characteristic. Furthermore, the phagocytic rate of the four SLE patients with marked prolongation in MPS clearance was significantly lower than that of the eight patients with near normal clearance values (p less than 0.01). Saturation of Fc receptors by immune complexes does not explain impaired immune clearance in SLE. Our results indicate that despite increased binding of the EA ligand, Fc receptor-mediated phagocytosis is markedly impaired in SLE monocytes. This impairment cannot be explained on the basis of HLA-related differences in phagocytosis among lupus patients. The defect in phagocytosis of EA is most profound in those patients with the most significantly impaired MPS clearance. Thus, the dissociation of receptor-ligand binding and receptor-mediated internalization may contribute significantly to the in vivo clearance defect in SLE.     

Carbohydrates on human Fc gamma receptors. Interdependence of the classical IgG and nonclassical lectin-binding sites on human Fc gamma RIII expressed on neutrophils

To explore the molecular basis for the ability of aggregated IgG to block the phagocytosis by human polymorphonuclear leukocytes of Con A-opsonized E and of nonopsonized Escherichia coli with mannose-binding adhesins, we examined specific aspects of the glycoprotein structure of both the 40- to 43-kDa receptor for the Fc portion of IgG (Fc gamma RII) and the 50- to 78-kDa receptor for the Fc portion of IgG (Fc gamma RIIIPMN) from human polymorphonuclear leukocytes. Fc gamma RIIIPMN isolated by both mAb and ligand affinity chromatography, but not Fc gamma RII, binds Con A in Western blots. This binding is specifically inhibitable by alpha-methylmannoside. Digestion of Fc gamma RIIIPMN by recombinant endoglycosidase H, which is specific for high mannose-type (Con A-binding) oligosaccharides, alters the epitope recognized by mAb 3G8 in or near the IgG ligand-binding site of the receptor. Similarly, the ability of Fc gamma RIIIPMN to bind human IgG ligand is sensitive to endoglycosidase H digestion. Our data indicate that ligands other than the classical IgG opsonins can bind to human Fc gamma RIIIPMN per se through lectin-carbohydrate interactions. Furthermore, Fc gamma RIIIPMN contains a high mannose type oligosaccharide chain which contributes importantly to the integrity of the classical IgG ligand-binding site. Thus, specific glycosylations of the receptor are important for both classical and nonclassical engagement of Fc gamma RIII and may play a role in determining the properties of the ligand-binding site.