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.     

Preparative electrophoresis on agarose submerged gels of two aggregating proteoglycan monomers from articular cartilage

Analytical electrophoresis on polyacrylamide-agarose gels of aggregating proteoglycan monomers from baboon articular cartilage produces two distinct bands, corresponding to two different aggregating monomer populations. A preparative electrophoresis procedure is described for isolating the two monomers. Proteoglycans were extracted from young baboon articular cartilage in 4 M guanidinium chloride containing proteolysis inhibitors and aggregated after hyaluronic acid addition. The aggregates were separated from non-aggregated proteoglycans by isopycnic centrifugation, followed by gel chromatography on Sepharose CL-2B. The monomers of the aggregates were obtained by isopycnic centrifugation under dissociative conditions. Two monomers were separated by preparative electrophoresis on 0.8 % agarose submerged gels. Approximately 60 % of the proteoglycans were recovered from the gel using a freeze-squeeze procedure. Aliquots of the separated monomers gave single bands when submitted to analytical polyacrylamide-agarose gel electrophoresis. Their migration and appearance were similar to that of the two bands present in the non separated preparation of monomers.     

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.     

DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators.

The DNA unwinding effects of some 9-aminoacridine derivatives were compared under reaction conditions that could be used to study drug-induced topoisomerase II inhibition. An assay was designed to determine drug-induced DNA unwinding by using L1210 topoisomerase I. 9-aminoacridines could be ranked by decreasing unwinding potency: compound C greater than or equal to 9-aminoacridine greater than o-AMSA greater than or equal to compound A greater than compound B greater than m-AMSA. Ethidium bromide was more potent than any of the 9-aminoacridines. This assay is a fast and simple method to compare DNA unwinding effects of intercalators. It led to the definition of a drug intrinsic unwinding constant (k). An additional finding was that all 9-aminoacridines and ethidium bromide inhibited L1210 topoisomerase I. Enzyme inhibition was detectable at low enzyme concentrations (less than or equal to 1 unit) and when the kinetics of topoisomerase I-mediated DNA relaxation was studied. Topoisomerase I inhibition was not associated with DNA swivelling or cleavage.     

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     

Comparative effects of some 3-amino 4,6-diarylpyridazines on the biosynthesis in vitro of TXA2 and PGI2- and on the TXA2- and PGI2-synthesizing activities of cardiac tissue

Some 3-amino 4,6-diarylpyridazine derivatives were tested for their effects on TXA2 and PGI2 biosyntheses in vitro and on the TXA2- and PGI2-synthesizing activities of cardiac tissue. Horse platelet and aorta microsomes were used as sources of thromboxane and prostacyclin synthetases respectively. The TXA2- and PGI2-synthesizing activities of cardiac tissue were studied on isolated perfused rabbit hearts (the heart microsomes being used both as TXA2 synthetase and PGI2 synthetase sources). TXB2 and 6-keto PGF1 alpha were determined by RIA. Among the compounds under study, 3-morpholino 4,6-diphenylpyridazine (III) was shown to inhibit specifically the TXA2 synthetase. Substitution of the morpholino group by a dimethylamino one (I) reinforced the inhibiting effects on TXA2 synthetase but it also revealed a slight anti-prostacyclin synthetase action of the molecule. Replacement of 3-morpholino moieties by either a 3-hydrazino (IV), or a 2-dimethylaminoethylamino (V), or a 2-morpholinoethylamino group (VI) abolished completely the effects of the molecule on TXA2 and PGI2 synthetases. Likewise the addition of chlorine on the para-position on the phenyl ring of I neutralized all its inhibitory effects both on TXA2 and PGI2 synthetases in vitro. None of the 3-amino 4,6-diarylpyridazine derivatives was active on either the TXA2- or PGI2-synthesizing activities of cardiac tissue.     

A T cell surface molecule different from CD2 is involved in spontaneous rosette formation with erythrocytes

Increasing evidence indicates that rosettes which spontaneously from between human T cells and E might be of physiologic relevance. We show here that another T cell-surface molecule than CD2 is involved in rosette formation. Four mAb have been obtained reacting with human T cells that block rosettes with E from many species, including autologous cells. They react with a molecule, we termed E2, which is actively synthetized by T and monocytic cells. Immunoprecipitation revealed a major 32-kDa band. Immunoblots revealed a major 32-kDa band and a minor 20-kDa band. This molecule was detected on all T cells tested--and present at high densities on corticothymocytes, but at low densities on medullary thymocytes. It was also found on monocytes but not on B cells. However B-CLL cells did carry this molecule. E2 molecules were also detected on nonhematologic cells. Together with the recent evidence that 3 molecules from the erythrocyte surface are also involved for rosettes, intricate molecular interactions would account for adhesion of T cells to autologous E and possibly autologous nucleated cells.     

A selective inhibitor of thromboxane synthetase activity of rabbit heart tissue: a pyridazinic derivative

The effects of 2-(2 dimethylaminoethyl) 5-benzylidene 6-methyl (2H,4H)-3-pyridazinone (III) were studied on the biosynthesis of TXA2 and PGI2 in vitro the TXA2 and PGI2 synthetase activity of heart tissue. Biosyntheses of TXA2 and PGI2 were carried out using arachidonic acid as a substrate and horse platelet and aorta microsomes as sources of TXA2 and PGI2 synthetases respectively. TXB2 and 6-keto PGF1 alpha were determined by RIA. III--did not significantly modify either the biosynthesis of PGI2 in vitro or the PGI2 synthetase activity of heart tissue. did not significantly inhibit TXA2 biosynthesis in vitro but markedly reduced the TXA2 synthetase activity of heart tissue: for a microsomal fraction concentration of 100 micrograms protein, the ID50 was 6.37 X 10(-5) M +/- 1.29 X 10(-8) M. Thus III behaves as a specific inhibitor of the TXA2 synthetase activity of heart tissue and could have a beneficial use in therapeutics.