Immunochemical studies on human monoclonal macroglobulins with specificities for 3,4-pyruvylated D-galactose and 4,6-pyruvylated D-glucose

Four of six human monoclonal IgM proteins were found to react best with Klebsiella polysaccharides containing 3,4py beta DGal (pyruvic acetalated D-galactopyranose), one with Klebsiella polysaccharides with 4,6pyDGlc; the sixth is uncharacterized. The combining sites of two of these (IgMWEA and IgMNAE) were essentially indistinguishable by quantitative precipitin studies at varying pH and by quantitative precipitin inhibition assays, but the other two differed in specificity of their combining sites from these and from each other. These differences were detected by precipitin inhibition assays with 3,4py beta DGal-containing oligosaccharide alditols, the R and S isomers of methyl 4,6py alpha DGal, the R isomer of methyl 4,6py beta DGal, or the R and S isomers of methyl 4,6py alpha DGlc, and -beta DGlc. In all of these except the S isomer of methyl 4,6pyDGal and R isomer of methyl 4,6pyDGlc, the carboxyl group is axial to the plane of the acetal ring. Their specificity appears to be determined by the nonreducing ends of chains and is considered to be cavity-type.     

The membrane glycoprotein of Friend spleen focus-forming virus: evidence that the cell surface component is required for pathogenesis and that it binds to a receptor.

The leukemogenic membrane glycoprotein of Friend spleen focus-forming virus (SFFV) has an apparent Mr of 55,000 (gp55), is encoded by a recombinant env gene, and occurs on cell surfaces and in intracellular organelles. There is evidence that the amino-terminal region of gp55 forms a dualtropic-specific domain that is connected to the remainder of the glycoprotein by a proline-rich linker (C. Machida, R. Bestwick, B. Boswell, and D. Kabat, Virology 144:158-172, 1985). Using the colinear form of a cloned polycythemic strain of SFFV proviral DNA, we constructed seven in-phase env mutants by insertion of linkers and by a deletion. The mutagenized SFFVs were transfected into fibroblasts and were rescued by superinfection with a helper murine leukemia virus. Four of the mutants cause erythroblastosis. These include one with a 6-base-pair (bp) insert in the ecotropic-related sequence near the 3' end of the gene, two with a 12- or 18-bp insert in the region that encodes the proline-rich linker, and one with a 6-bp insert in the dualtropic-specific region. The other mutants (RI, Sm1, and Sm2) are nonpathogenic and contain lesions in dualtropic-specific region. The other mutants (RI, Sm1, and Sm2) are nonpathogenic and contain lesions in dualtropic-specific sequences that are highly conserved among strains of SFFV. A pathogenic revertant (RI-rev) was isolated from one mouse that developed erythroblastosis 3 weeks after infection with RI. RI-rev contains a second-site env mutation that affects the same domain as the primary mutation does and that increases the size of the encoded glycoprotein. All pathogenic SFFVs encode glycoproteins that are expressed on cell surfaces, whereas the nonpathogenic glycoproteins are exclusively intracellular. The pathogenic SFFVs also specifically cause a weak interference to superinfection by dualtropic MuLVs. These results are compatible with the multidomain model for the structure of gp55 and suggest that processing of gp55 to plasma membranes is required for pathogenesis. The amino-terminal region of gp55 binds to dualtropic murine leukemia virus receptors, and this interaction is preserved in the SFFV mutants that cause erythroblastosis.     

Lampetra (Eudontomyzon) gracilis,a synonym of Eudontomyzon danfordi

Synopsis Evidence is presented which suggests that the nonparasitic lamprey, Lampetra (Eudontomyzon) gracilis Kux, 1965, is conspecific with the parasitic lamprey Eudontomyzon danfordi Regan, 1911. The diagnostic characters of the holotype and of the non-type material of E. gracilis are features found in E. danfordi specimens in their second and final year of adult life, thereby making the former a junior synonym of the latter.     

Sequences of variable regions of hybridoma antibodies to alpha (1----6) dextran in BALB/c and C57BL/6 mice

The variable region sequences of light and heavy chains of three hybridoma antibodies to alpha (1----6) dextran, two from BALB/c and one from C57BL/6 mice, were determined by cloning and sequencing their cDNA. The three kappa-light chains are identical in nucleotide and amino acid sequences, except for the use of different J by BALB/c and C57BL/6; all three had the germ-line sequence of antibodies to 2-phenyloxazolone (20). Nevertheless, 2-phenyloxazolone BSA did not cross-react in gel with antidextrans, nor did dextran react with anti-2-phenyloxazolone ascitic fluids. The heavy chains differed, the BALB/c hybridomas having only three amino acid differences in CDR2 and two in CDR3; the C57BL/6 hybridoma differed throughout the variable region. All three VH are members of the J558 family. The three identical V kappa sequences suggest a significant role in dextran binding, with the differences in CDR of VH and the various J mini-genes of VL and VH being responsible for only fine differences in specificity. Alternatively, the role of V kappa might be minor, with most of the complementarity ascribable to VH. Additional sequences are needed to evaluate whether these data are typical of the repertoire of anti-alpha (1----6) dextran-combining sites.     

The binding site of chicken hepatic lectin

The binding site of the chicken hepatic lectin involved in the clearance of N-acetylglucosamine-terminated serum glycoproteins was explored by a competitive binding assay using 3H-labeled agalacto-orosomucoid and various glycoproteins, polysaccharides, monosaccharides, and glycosides as inhibitors. The binding site is relatively small, involving a terminal nonreducing DGlcNAc structure with an equatorial N-acetamido group on carbon 2 and an equatorial hydroxyl group on carbon 4. Among the mono- and oligosaccharides tested, benzyl alpha DGlcNAc was the best inhibitor, being three times as effective as DGlcNAc; and in general, all alpha-anomeric glycosides were better than beta-glycosides. All oligosaccharides with terminal nonreducing beta DGlcNAc have almost the same inhibitory power, whereas those with nonreducing DGlc or DGal were relatively inactive. Among the serum and blood group glycoproteins, a Smith degraded human H substance with several exposed terminal nonreducing beta DGlcNAc residues was the most active and twice as effective as agalacto-orosomucoid and an A substance, Hog 75 10% precipitate. Almost all hog preparations, some with A or with H activity, were equally effective. A glycopeptide with terminal DGlcNAc was twice as active as one with terminal nonreducing DMan and DGlcNAc residues and almost three times as potent as one with terminal nonreducing DGal; a glycopeptide with terminal sialic acid was inactive. The slopes of the inhibition lines differed, reflecting the heterogeneity of the various determinant groups on the glycoproteins.     

Ping-pong amplification of a retroviral vector achieves high-level gene expression: human growth hormone production.

Retroviral vectors offer major advantages for gene transfer studies but have not been useful for producing proteins in large quantities. This deficiency has resulted in part from interference to superinfection, which limits the numbers of active proviruses in cells. Recently, we found that these vectors amplify when they are added as calcium phosphate precipitates to cocultures of cells that package retroviruses into ecotropic and amphotropic host range envelopes. Helper-free virions from either cell type can infect the other without interference, resulting in theoretically limitless back-and-forth (ping-pong) vector replication. In initial studies, however, amplifications of a vector that contained the human growth hormone gene ceased when the hormone produced was 0.3% or less of cellular protein synthesis. This limit was caused by two factors. First, recombinant shutoff viruses that are replication defective and encode envelope glycoproteins form at a low probability during any round of the vector replication cycle and these spread in cocultures, thereby establishing interference. Single cells in shutoff cocultures therefore synthesize both ecotropic and amphotropic envelope glycoproteins, and they release promiscuous (presumably hybrid) virions. The probability of forming shutoff viruses before the vector had amplified to a high multiplicity was reduced by using small cocultures. Second, cells with large numbers of proviruses are unhealthy and their proviral expression can be unstable. Stable expresser cell clones were obtained by selection. Thereby, cell lines were readily obtained that stably produce human growth hormone as 4 to 6% of the total protein synthesis. A ping-pong retroviral vector can be used for high-level protein production in vertebrate cells.     

Splenic accumulation of interleukin-3-dependent hematopoietic cells in Friend erythroleukemia.

Interleukin-3-dependent hematopoietic stem cells commonly accumulate in spleens of mice infected with leukemia viruses. To study their origins, a molecularly tagged helper-free Friend spleen focus-forming virus was used to produce erythroleukemias. Uninfected interleukin-3-dependent basophil-mast cell progenitors coproliferated amidst the spleen focus-forming virus-infected leukemic cells. Splenic proliferation of normal stem cells is apparently a host response to leukemogenesis, and we propose that it may contribute to certain retroviral diseases.