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.     

Involvement of Carrot Cell Surface Proteins in Attachment of Agrobacterium tumefaciens

The initial step in tumor formation by Agrobacterium tumefaciens is the site-specific attachment of the bacteria to plant cells. A similar attachment to plant tissue culture cells has been observed. Binding to carrot suspension culture cells was not dependent on the presence of divalent cations and was not inhibited by the addition of mannose, α-methyl mannoside, galactose, arabinose, glucosamine, 2-deoxyglucose, or 0.25 molar NaCl to the culture medium. The ability of the carrot cells to bind A. tumefaciens was markedly reduced by elution of the cells with dilute detergent or CaCl₂ or by incubation of the cells with proteolytic enzymes. The carrot cells were not killed by these treatments and recovered the ability to bind A. tumefaciens within 3 to 6 hours. A. tumefaciens did not bind to carrot cells which had been induced to form embryos (AG Matthysse, RHG Gurlitz 1982 Physiol Plant Pathol 21: 381-387). A comparison of the peptides eluted from embryos and from uninduced cells using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that there were several changes in extractable polypeptides after embryo induction. One or more of the polypeptides present before embryo induction and absent from embryos may be involved in the binding of A. tumefaciens to the carrot cell surface.     

Identification of somatic hybrids in plant protoplast fusions with monoclonal antibodies to plasma-membrane antigens

Monoclonal antibodies generated by immunization with a plasma-membrane preparation from suspension-cultured cells of Nicotiana glutinosa L. were used in combination with fluoresceinor rhodamine-labeled goat anti-mouse immunoglobulins to identify heterokaryons in protoplast fusion procedures. Antibody labeling did not inhibit callus formation nor plantlet regeneration. The antibodies are non-invasive and surface labeling provides clear optical discrimination of true heterokaryons from unfused aggregates as well as from parental protoplasts and homokaryons. Labeling is stable throughout fusion and hence by pre-labeling parental protoplast populations the strategy is both versatile and of general applicability.     

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.     

Mutational analysis of the Bradyrhizobium japonicum common nod genes and further nod box-linked genomic DNA regions

Summary By insertional and deletional marker replacement mutagenesis the common nod region of Bradyrhizobium japonicum was examined for the presence of additional, essential nodulation genes. An open reading frame located in the 800 bp large intergenic region between nodD1 and nodA did not appear to be essential for nodulation of soybean. Furthermore, a strain with a deletion of the nodI- and nodJ-like genes downstream of nodC had a Nod⁺ phenotype. A mutant with a 1.7 kb deletion immediately downstream of nodD1 considerably delayed the onset of nodulation. This region carried a second copy of nodD (nodD2). A nodD1-nodD2 double mutant had a similar phenotype to the nodD2 mutant. Using a 22-mer oligonucleotide probe partially identical to the nod box sequence, a total of six hybridizing regions were identified in B. japonicum genomic DNA and isolated from a cosmid library. Sequencing of the hybridizing regions revealed that at least three of them represented true nod box sequences whereas the others showed considerable deviations from the consensus sequence. One of the three nod box sequences was the one known to be associated with nodA, whereas the other two were located 60 to 70 kb away from nif cluster I. A deletion of one of these two sequences plus adjacent DNA material mmutant 308) led to a reduced nodulation on Vigna radiata but not on soybean. Thus, this region is probably involved in the determination of host specificity.Dedicated to Prof. Giorgio Semenza on the occasion of his 60th birthday     

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.     

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