C1 binding by mouse IgM. The effect of abnormal glycosylation at position 402 resulting from a serine to asparagine exchange at residue 406 of the mu-chain.

We have previously shown that IgM-Asn406, a mutant IgM which has asparagine in place of the serine which is normally found at position 406, also has an abnormally glycosylated mu-chain and is defective in complement-dependent cytolysis. Here we show by analyzing cyanogen bromide fragments from normal and mutant mu-chains that the site of abnormal glycosylation is at the neighboring position, Asn402. The cytolytic defect was shown to be due to impaired C1 binding.

Effect of epidural and subarachnoid injections of a 10% butamben suspension.

Three groups of dogs were each given repeat epidural injections of a 10% butamben suspension. A fourth group received a single subarachnoid injection of the butamben suspension. All dogs were later sacrificed and the spinal cord, meninges and spinal nerves were examined.

Patterns and extent of isotype-specificity in the murine H chain switch DNA rearrangement.

We have analyzed the configuration of the H chain locus of 41 hybridomas by Southern blot analysis. Each H chain switch region was determined to be germ line, rearranged, or deleted. Including 13 previously analyzed hybridomas, 60% of those with rearrangements on both alleles showed a correlation of the two alleles, i.

Intermolecular disulfide bonding in IgM: effects of replacing cysteine residues in the mu heavy chain.

The conventional model of polymeric IgM depicts a unique structure in which the mu heavy chains and J chain are joined by well defined disulfide bonds involving cysteine residues at positions 337, 414 and 575 of the mu chain. To test this model, we have used site directed mutagenesis to produce IgM in which these cysteines have been replaced by serine. In each case the single mutants were able to assemble polymeric IgM, which was analyzed for its size, morphology, J chain content and activity in complement dependent cytolysis.

Mutations of the mouse mu H chain which prevent polymer assembly.

Earlier work has shown that truncated mu-chains lacking the carboxy-terminal C mu 4-tail region are secreted as monomeric rather than polymeric IgM and that the monomer phenotype is not due to the lack of a disulfide bond at Cys-575 in the tail. In order to define with greater precision, the molecular requirements for IgM polymer assembly, we have isolated several mutant hybridomas which produce monomeric IgM. For three such mutants, we synthesized cDNA clones of their mu mRNA and identified a mutation in the mu-chain which was responsible for the failure to assemble polymers.

IgM--molecular requirements for its assembly and function.

The conventional model of IgM structure depicts a unique, array of mu, L and J chains, held together by well-defined disulfide bonds and other interactions. Some, but not all, recent data support this model. Here Ann Davis and Marc Shulman review recent, as well as older, studies of IgM and consider their implications for our understanding of IgM structure and function.

Differential glycosylation of polymeric and monomeric IgM.

A small fraction of normal IgM is secreted as monomers rather than polymers. We show here that the mu chains of monomeric IgM are glycosylated differently from the mu chains of polymeric IgM and are comparable in their glycosylation to the mu chains from mutant hybridoma cell lines which produce predominantly monomeric IgM. The difference in glycosylation between monomer and polymer mu chains is due to differences in the terminal processing of their oligosaccharides.

Structural requirements for IgM assembly and cytolytic activity. Effects of mutations in the oligosaccharide acceptor site at Asn402.

Glycosylation of IgG occurs at asparagine 297 of the gamma H chain and is necessary for the normal capacity of IgG to activate the classical pathway of complement-dependent cytolysis. IgM is glycosylated at five sites in the constant region of the mu H chain, of which glycosylation at asparagine 402 seems analogous to the glycosylation of IgG. In order to assess the importance of glycosylation at asparagine 402 for IgM cytolytic activity, we have used site-directed mutagenesis to produce IgM which is not glycosylated at this position.

Immunoglobulin molecular genetics: the prospects for mutational analysis of the chromosomal immunoglobulin genes.

Homologous recombination between transferred and chromosomal DNA can be used to effect precise, predetermined modifications of the chromosomal genes. Ultimately this phenomenon should allow the assessment of genetic regulatory elements as they function in the normal chromosomal environment. We have previously described a system for isolating mutant hybridoma cells that are defective in immunoglobulin (Ig) production, with a view toward using these mutants to define cis-acting elements that influence Ig gene expression.

Homologous recombination between transferred and chromosomal immunoglobulin kappa genes.

Homologous recombination between transferred and chromosomal DNAs provides a means of introducing well-defined, predetermined changes in the chromosomal genes. Here we report that this approach can be used to specifically modify the immunoglobulin genes in mouse hybridoma cells. The test system is based on the Sp6 hybridoma, which synthesizes immunoglobulin M (kappa) specific for the hapten 2,4,6-trinitrophenyl (TNP).

Expression of an immunoglobulin kappa light-chain gene in lymphoid cells using a bovine papillomavirus-1 (BPV-1) vector.

Bovine papillomavirus-1 (BPV-1) replicates extrachromosomally in certain murine cell lines, suggesting that vectors based on the BPV-1 replicon might provide a means of obtaining more uniform gene expression among independent transformants. We have tested such a vector for the expression in hybridoma cells of the immunoglobulin kappa light-chain gene, but found that the level of expression varies greatly among transformants. Our results also indicate that in these transformants the vector has probably been incorporated into chromosomal DNA.

Homologous recombination can restore normal immunoglobulin production in a mutant hybridoma cell line.

We report here the occurrence of homologous recombination between transferred and chromosomal immunoglobulin genes. Specifically, we have corrected a chromosomal immunoglobulin gene mutation by transferring pSV2neo vectors encoding the constant region of the immunoglobulin mu heavy chain to mutant hybridoma cells that bear a 2-base-pair deletion in the third constant region exon of their chromosomal mu gene. After DNA transfer, we detected G418-resistant transformants that produce normal IgM.

C1 binding by murine IgM. The effect of a Pro-to-Ser exchange at residue 436 of the mu-chain.

We have examined a defect in complement activation in a mutant trinitrophenyl-binding pentameric murine monoclonal IgM which has serine replacing the proline normally found at position 436 in the protein. The mutant protein showed equivalent hapten binding but a 100-fold decreased ability to initiate complement-dependent lysis of trinitrophenyl-coupled erythrocytes at physiological ionic strength (mu = 0.15).

Novel application of video image processing to biochemical and antimicrobial susceptibility testing.

ALADIN (Analytab Products, Plainview, N.Y.) is an automated instrument that uses video imaging (computer-assisted guided video camera) for the determination of biochemical and antimicrobial susceptibility test reactions.

On the structure of polymeric IgM.

The cysteine at position 575 of the immunoglobulin mu heavy chain is thought to provide the only disulfide bonds joining the monomer subunits of mouse polymeric IgM. The importance of this cysteine in the assembly of polymeric IgM was investigated by using site-directed mutagenesis to produce mu chains with serine at position 575. Thirty percent of the secreted mutant IgM was covalently assembled polymer implying that cysteines other than Cys575 can form inter-subunit disulfide bonds.

Complement activation is required for IgM-mediated enhancement of the antibody response.

The ability of IgM antibodies to specifically enhance the thymus-dependent humoral immune response to particulate antigens is well documented. We have used two approaches to test whether complement factors play a role in this process. First, mice were depleted of C3 by treatment with cobra venom factor (CVF) and then immunized with SRBC with or without IgM-anti-SRBC.

Stimulation of rabbit lacrimal gland secretion with biologically active peptides.

To determine whether biologically active peptides can stimulate lacrimal gland secretion, we measured fluid and protein secretion from the cannulated lacrimal gland excretory duct of anesthetized rabbits after arterial injection of various peptides. Vasoactive intestinal peptide (VIP, 0.003-3 nmol) and porcine histidine isoleucine-containing peptide (PHI, 0.

Complement activation by IgM: evidence for the importance of the third constant domain of the mu heavy chain.

We have isolated and analyzed the DNA encoding the mu heavy chain constant region of a mutant IgM which is defective in initiating complement-dependent cytolysis. By assaying the expression of mu genes which were constructed in vivo from mutant and normal gene segments, we have mapped the mutation into a 555-base pair segment. In this segment there is one nucleotide change, such that the mutant mu gene encodes serine rather than the normal proline at amino acid position 436 in the third constant domain.

Fibreoptic bronchoscopy for tracheal and endobronchial intubation with a double-lumen tube.

A 68-year-old patient was scheduled for a thoracotomy. A double-lumen endobronchial tube was requested by the surgeon to facilitate operating conditions. Initial attempts at intubation by conventional methods were unsuccessful.