Antigen presentation by the BCL1 murine B cell line: in vitro stimulation by LPS

We examined the antigen-presenting capacity of BCL1 tumor cells, which are capable of differentiating in vitro with respect to immunoglobulin synthesis/secretion under the influence of LPS. In vivo passaged BCL1 cells depleted of host cell contamination either by positive selection employing panning with anti-lambda reagents, or by elimination of latex-ingesting adherent cells, are capable of MHC-restricted antigen presentation to a GAT-immune T cell line. The BCL1 cells act as antigen-presenting cells when freshly explanted, but gradual loss of this function occurs, and cells cultured for 3.5 days cannot present antigen unless LPS is included during the culture period. BCL1 cells are equivalently Ia+ after the culture period with or without LPS stimulation. Other B cell lines capable of antigen presentation appear to express this trait constitutively, and the in vivo passaged BCL1 line is therefore unique among B cell lines in having antigen-presenting cell function that can be modulated. The data suggest that freshly explanted or LPS-cultured BCL1 cells are heterogeneous with respect to antigen-presenting capacity, and the basis for this heterogeneity is being sought. BCL1 offers an opportunity to study requirements for antigen presentation by B cells.     

Separation of metabolically radiolabeled O-methylmannitols and O-methylfucitol by reverse-phase high-performance liquid chromatography: applications to animal cell oligosaccharide structural analysis

Reverse-phase high-performance liquid chromatography was utilized to separate efficiently and rapidly a standard mixture of various radiolabeled O-methylated mannitols and O-methylfucitol commonly encountered when vertebrate asparagine-linked oligosaccharides are subjected to permethylation, hydrolysis, and reduction with NaBH4. The following reduced, radioactive O-methylhexitols were resolved: 2,4-, 3,4-, and 3,6-di-O-methylmannitols; 3,4,6-tri-O-methylmannitol, 2,3,4-tri-O-methylfucitol, and 2,3,4,6-tetra-O-methylmannitol. To demonstrate the utility of this separation method in the analysis of metabolically radiolabeled asparagine-linked oligosaccharides, mouse lymphoma BW 5147 cells were metabolically radiolabeled with [2-3H]mannose and their glycopeptides prepared by Pronase digestion and fractionated by serial chromatography on immobilized lectins. Each fraction was subjected to methylation and hydrolysis, the released monosaccharides were reduced, and the radioactive O-methylhexitols were separated by reverse-phase HPLC. The relative amounts of the O-methylhexitols in each glycopeptide fraction analyzed were similar to those values determined by a combination of other separation systems.     

Determination of antibody affinity by using antigenic and antibody immunosorbents

To determine the affinity of the active centers of antibodies, cellulose immunosorbents for antibodies and antigens have been used. The fixation of serum proteins on the sorbent, the interaction of fixed antibodies with a monovalent antigen and the graphic analysis of the results thus obtained allows one to assess not only the concentration of the effective active centers on the sorbent, but also all known characteristics of antibody affinity: the average association constant K0, the common association constant Kt, the geometric association constant Kg, the average association constants which determine the affinity of different antibody groups. The use of antigenic immunosorbent permits one to determine the value of the average internal association constant K0. The determination of antibody affinity in hyperimmune antiplague sera by means of immunosorbents and red blood cells coated with capsular antigen has resulted in obtaining similar values of affinity indices.     

Algal single cell protein from wastewater treatment and renovation process

A Source of high-quality protein for animal feed, based upon algae recovered in the process of upgrading waste oxidation pond effluents and promising to be particularly economical, is being developed at the Technion. Unlike other types of single cell protein(SCP), the algal protein does not have to return the full production cost but only that of concentration and final processing. The balance is shared by the value of waste disposal and the reclaimed water. Whereas such systems as activated sludge require considerable mechanical energy to supply the oxygen needed for aerobically degrading organics in wastewater, oxidation ponds utilize solar energy for that purpose. The sludge obtained when their effluents are clarified consists largely of algae, bacteria, fungi, and zooplankton in relative proportions varying with operating conditions, and contains 40–60%(dry basis) high-quality protein. The high rate oxidation pond (a particularly intensive type of pond) produces on the average 34 g/m²7sol;day solids, or over 100 tons/ha (hectare) annually. Two clarification routes have been found promising: centrifugation and alum flocculation followed by frothflotation. The latter route is less expensive in terms of both fixed and operating cost, and gives clarified effluent of higher quality, which can be seasonally stored with minimal eutrophication because the aluminum removes most of the phosphate from the effluent. A good product has been obtained by drum-drying the concentrate, and preliminary feeding tests have indicated that it can replace at least 1/4 of the soymeal in broiler rations and 2/3 of the fishmeal in carp feed. No ill effect of the aluminum in the product recovered by alum flocculation has been found so far a process for removing and recycling the aluminum has been developed nonetheless, in case ill effects do show up in further tests. The combined value of the benefits derived from a system centered around the high-rate oxidation pond with clarification by flocculation–flotation, in terms of waste treatment by alternative means, potable water saved, and soymeal replaced, significantly exceeds estimated cost.     

Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by ribulose-1,5-bisphosphate epimerization and degradation products

Xylulose-1,5-bisphosphate in preparations of ribulose-1,5-bisphosphate (ribulose-P₂) arises from non-enzymic epimerization and inhibits the enzyme. Another inhibitor, a diketo degradation product from ribulose-P₂, is also present. Both compounds simulate the substrate inhibition of ribulose-P₂ carboxylase/oxygenase previously reported for ribulose-P₂. Freshly prepared ribulose-P₂ had little inhibitory activity. The instability of ribulose-P₂ may be one reason for a high level of ribulose-P₂ carboxylase in chloroplasts where the molarity of active sites exceeds that of ribulose-P₂. Because the K_D of the enzyme/substrate complex is ≤1 μM, all ribulose-P₂ generated in situ may be stored as this complex to prevent decomposition.     

Development of a yeast system to assay mutational specificity

We have developed a system wherein DNA alterations occurring in a target gene in the yeast Saccharomyces cerevisiae can be determined by DNA sequencing. The target gene, SUP4-o, an ochre suppressor allele of a yeast tyrosine tRNA gene, has been inserted into a shuttle vector (YCpMP2) which is maintained in yeast at a copy number of one per cell Mutations in SUP4-o are selected by virtue of their inactivation of suppressor activity. Rapid DNA preparations from these mutants are used to transform an appropriate bacterial strain. Since YCpMP2 also carries the M13 phage replication origin, superinfection of bacterial cells containing the plasmid with wild-type M13 phage yields single stranded YCpMP2 DNA suitable for dideoxynucleotide chain termination sequencing. We have used this system to examine mutations arising spontaneously in the SUP4-o gene. The spontaneous mutants occurred at a frequency of 3.2 X 10(-6)/viable cell, corresponding to a rate of 2.7 X 10(-7) events/cell division. Following bacterial transformation, 16% of the recovered plasmids tested displayed altered gel mobility consistent with loss of significant portions of the plasmid. Hybridization analysis of total yeast DNA and use of purified YCpMP2 revealed that these very large deletions were not generated in yeast but were associated with bacterial transformation. Among the SUP4-o mutants analyzed by DNA sequencing, we identified each type of single base pair substitution (transitions and transversions), small deletions of varying length (1-32 base pairs) and more extensive deletions of undetermined size. These results demonstrate that the SUP4-o system can be used to detect various types of mutation at numerous sites in a single eukaryotic gene and to characterize the DNA sequence changes responsible for the mutations selected.