Murine polyspecific antibodies. I. Monoclonal and serum anti-DNA antibodies cross-reactive with 2,4,6-trinitrophenyl derivatives

Six anti-DNA hybridoma autoantibodies were prepared by fusing spleen cells from unimmunized MRL/MpJ/lpr/lpr female mice with BALB/c myeloma cells. The monoclonal antibodies were analyzed by solid-phase ELISA for antigen-binding specificities. Three antibodies (62A2, 85A5, and 43B2) bound ssDNA, TNP-KLH, and recognized an epitope(s) present on insolubilized proteins such as BSA, KLH, ferritin, and insulin. The antibodies bound, with a marked preference, TNP-KLH, either soluble or insoluble. The other three antibodies (35A1, 32C5, and 39D2) bound only ssDNA. However, this binding was inhibited by free flavinic acid. None of the six antibodies bound either cardiolipin or proteoglycans, indicating that they do not recognize the repeating negatively charge units common to cardiolipin, proteoglycans, and DNA. All six monoclonal antibodies were purified by affinity chromatography with TNP-Sepharose. Moreover, both anti-DNA and anti-TNP antibodies from sera of nonautoimmune and autoimmune mice were purified easily on TNP-Sepharose.     

Chronic Giardia muris infection in anti-IgM-treated mice. I. Analysis of immunoglobulin and parasite-specific antibody in normal and immunoglobulin-deficient animals

To investigate the role of B cells and antibody in the immune response of mice to the murine intestinal parasite Giardia muris, we used mice treated from birth with rabbit anti-IgM antisera (aIgM). Such mice developed in serum and in gut secretions extreme Ig deficiency (IgM, IgA, and IgG) relative to control animals. The aIgM-treated mice showed no anti-G. muris antibody in serum or in gut wash material. Infections of G. muris in these mice were chronic, with a high load of parasite present in the small bowel, as reflected by prolonged cyst excretion (greater than 11 wk) and high trophozoite counts. In contrast, normal, untreated mice or NRS-treated animals developed anti-parasite IgA and IgG antibody in serum, demonstrated IgA antibody against the parasite in gut washings, and expelled the parasite within 9 wk. These effects of aIgM treatment on the murine response to primary infection with G. muris were demonstrated in two strains of mice: BALB/c and (C57BL/6 X C3H/He) F1. It was also observed that the response to G. muris infection in untreated animals was characterized by higher than normal total secretion of IgA into the gut and a concomitant increase in the serum polymeric IgA level. Mice treated with aIgM had a marked decrease of both monomeric and polymeric IgA in serum, and little detectable IgA in the intestinal lumen. These experiments provide the first demonstration that anti-IgM treatment suppresses a specific intestinal antibody response to antigen, and provide evidence that B cells and antibody play a role in the development of an effective response to a primary infection with G. muris in mice.     

Spin probe clustering in human erythrocyte ghosts

Summary A model has been developed for 5-nitroxide stearate, I(12,3), distribution in human erythrocyte ghosts which accurately predicts ESR spectral alterations observed with increased probe/total lipid (P/L) at 37°C. This spin probe occupies a class of high-affinity, noninteracting sites at low loading. Saturation occurs with increasing probe concentration, and, at higher loading, the probe inserts itself at initially dilute sites to form membranebound clusters of variable size. No low probe remains at high P/L where all I(12,3) clusters in a concentrated phase. This model allows determination of the dilute/clustered probe ratio, and shows that I(12,3) segregates in erythrocytes at what might otherwise be considered low P/L (e.g., 1/359). These findings validate the earlier use of empirical parameters to estimate probe sequestration in biological membranes.     

Molecular and genetic analysis of factors controlling host range in Agrobacterium tumefaciens

Summary We have investigated the factors which contribute to the host specificity of a tumor inducing plasmid of Agrobacterium, pTiAg162, which confers a narrow host range. Determinants both within the T-DNA and virulence regions contribute to host specificity. Within the T-DNA a defective cytokinin biosynthetic gene limits host range. Nucleotide sequence analysis revealed a large deletion in the 5 coding region of this gene when compared with the homologous gene from the wide host range tumor inducing plasmid, pTiA6. Introduction of the wide host range cytokinin biosynthesis gene into the T-DNA of the limited host range strain expanded the host range and suppressed the rooty morphology of tumors incited by the limited host range strain. Two genes from the virulence region of the wide host range plasmid, designated virA and virC, must also be introduced into the limited host range strain in order to restore a wide host range phenotype. The wide host range strain is avirulent on some cultivars of Vitis plants on which the limited host range strain induces tumors. This avirulence is apparently due to a hypersensitive response in which infected plant cells are killed at the site of inoculation. Mutations within the virC locus of the wide host range plasmid prevented the hypersensitive response and allowed the formation of tumors by the wide host range strain.     

Collagenous substrata regulate the nature and distribution of glycosaminoglycans produced by differentiated cultures of mouse mammary epithelial cells

We have investigated the influence of culture substrata upon glycosaminoglycans produced in primary cultures of mouse mammary epithelial cells isolated from the glands of late pregnant mice. Three substrata have been used for experiments: tissue culture plastic, collagen (type I) gels attached to culture dishes, and collagen (type I) gels that have been floated in the culture medium after cell attachment. These latter gels contract significantly. Cells cultured on all three substrata produce hyaluronic acid, heparan sulfate, chondroitin sulfates and dermatan sulfate but the relative quantities accumulated and their distribution among cellular and extracellular compartments differ according to the nature of the culture substratum. Notably most of the glycosaminoglycans accumulated by cells on plastic are secreted into the culture medium, while cells on floating gels incorporate almost all their glycosaminoglycans into an extracellular matrix fraction. Cells on attached collagen gels secrete approx. 30% of their glycosaminoglycans and assemble most of the remainder into an extracellular matrix. Hyaluronic acid is produced in significant quantities by cells on plastic and attached gels but in relatively reduced quantity by cells on floating gels. In contrast, iduronyl-rich dermatan sulfate is accumulated by cells on floating gels, where it is primarily associated with the extracellular matrix fraction, but is proportionally reduced in cells on plastic and attached gels. The results are discussed in terms of polarized assembly of a morphologically distinct basal lamina, a process that occurs primarily when cells are on floating gels. In addition, as these cultures secrete certain milk proteins only when cultured on floating gels, we discuss the possibility that cell synthesized glycosaminoglycans and proteoglycans may play a role in the maintenance of a differentiated phenotype.     

Autophagic sequestration of [14C]sucrose introduced into isolated rat hepatocytes by electrical and non-electrical methods

Isolated rat hepatocytes were found to become permeable to [14C]sucrose at 0 degree C under three different conditions: Immediately following their liberation from the collagenase-perfused liver. Following a short incubation under hypoxic conditions. After electropermeabilisation. All three conditions were characterised by the formation of small protuberances (blebs) indicative of localised cell surface damage, and it is possible that the stretched plasma membrane of such blebs acted as a high-permeability region. Disappearance of blebs and restoration of normal plasma membrane impermeability could be achieved by a short (15 min) incubation at 37 degrees C. It could be shown that [14C]sucrose introduced into rat hepatocytes by non-electrical means was autophagically sequestered at the same rate as [14C]sucrose introduced electrically. In both cases the sequestration was inhibited by the specific autophagy inhibitor 3-methyladenine to a similar extent. The subcellular distribution of sequestered isotope in metrizamide/sucrose density gradients was found to be independent of the conditions of its introduction into cells.     

Limited-host-range plasmid of Agrobacterium tumefaciens: molecular and genetic analyses of transferred DNA.

A tumor-inducing (Ti) plasmid from a strain of Agrobacterium tumefaciens that induces tumors on only a limited range of plants was characterized and compared with the Ti plasmids from strains that induce tumors on a wide range of plants. Whereas all wide-host-range Ti plasmids characterized to date contain closely linked oncogenic loci within a single transferred DNA (T-DNA) region, homology to these loci is divided into two widely separated T-DNA regions on the limited-host-range plasmid. These two plasmid regions, TA-DNA and TB-DNA, are separated by approximately 25 kilobases of DNA which is not maintained in the tumor. The TA-DNA region resembles a deleted form of the wide-host-range TL-DNA and contains a region homologous to the cytokinin biosynthetic gene. However, a region homologous to the two auxin biosynthetic loci of the wide-host-range plasmid mapped within the TB-DNA region. These latter genes play an important role in tumor formation because mutations in these loci result in a loss of virulence on Nicotiana plants. Furthermore, the TB-DNA region alone conferred tumorigenicity onto strains with an intact set of vir genes. Our results suggest that factors within both the T-DNA and the vir regions contribute to the expression of host range in Agrobacterium species. There was a tremendous variation among plants in susceptibility to tumor formation by various A. tumefaciens strains. This variation occurred not only among different plant species, but also among different varieties of plants within the same genus.     

HTLV-III env gene products synthesized in E. coli are recognized by antibodies present in the sera of AIDS patients

The envelope gene of HTLV-III, the retrovirus directly linked to AIDS, encodes a protein of 856 amino acids. Our sequence analysis of the cloned HTLV-III (HXB-3) env gene and its comparison with other isolates reveal significant divergence, especially in the external portion of this protein. A large segment of the env gene (1800 bp) was inserted into the expression vector pEV-vrf3, and a corresponding 68 kd protein, which encompasses both the extracellular and the membrane-associated regions of the native protein, was produced in E. coli. Several smaller polypeptides, which appear to be internal initiation products, were also produced. All 50 AIDS patient sera obtained from different locations in the United States specifically recognized the bacterially synthesized envelope proteins, as judged by Western blots. This suggests that these proteins will be useful for the diagnosis of HTLV-III infection and possibly as a vaccine against AIDS.     

Transposon-like properties of the major, long repetitive sequence family in the genome of Physarum polycephalum

A family of long, highly-repetitive sequences, referred to previously as `HpaII-repeats', dominates the genome of the eukaryotic slime mould Physarum polycephalum. These sequences are found exclusively in scrambled clusters. They account for about one-half of the total complement of repetitive DNA in Physarum, and represent the major sequence component found in hypermethylated, 20-50 kb segments of Physarum genomic DNA that fail to be cleaved using the restriction endonuclease HpaII. The structure of this abundant repetitive element was investigated by analysing cloned segments derived from the hypermethylated genomic DNA compartment. We show that the `HpaII-repeat' forms part of a larger repetitive DNA structure, ~8.6 kb in length, with several structural features in common with recognised eukaryotic transposable genetic elements. Scrambled clusters of the sequence probably arise as a result of transposition-like events, during which the element preferentially recombines in either orientation with target sites located in other copies of the same repeated sequence. The target sites for transposition/recombination are not related in sequence but in all cases studied they are potentially capable of promoting the formation of small `cruciforms' or `Z-DNA' structures which might be recognised during the recombination process.     

Effect of beta-adrenoceptor blockade on thermoregulation during prolonged exercise

The effect of clinically used equipotent doses of nonselective (beta 1/beta 2; propranolol) and selective (beta 1; atenolol) beta-adrenoceptor blockers on thermoregulation was studied during prolonged exercise in the heat. Oral propranolol (160 mg/day), atenolol (100 mg/day) or matching placebo were taken for 6 days each by 11 healthy young adult caucasian males. Subjects participated in 2 h of block-stepping at a work rate of 54 W in an environmental chamber with a temperature of 33.2 +/- 0.3 degree C dry bulb and 31.7 /+- 0.3 degree C wet bulb, 2 h after ingestion of the final dose of each drug. Both active agents produced similar marked (P less than 0.001) increases in subjective perception of effort, the mechanism of which was not immediately evident from changes in serum electrolytes, blood glucose, blood lactate, or ventilatory parameters. Propranolol did, however, cause a greater rise in serum K+ than placebo (P less than 0.02) and atenolol (P = NS) after exercise. Although rectal and mean skin temperatures were insignificantly altered by beta-adrenoceptor blockade, an increased total sweat production was noted with propranolol (P less than 0.01 vs. placebo) and to a lesser degree atenolol (P = NS vs. placebo) therapy. Analysis of the time course of sweat production showed the propranolol-mediated enhancement of sweating to ensue largely during the initial hour of block-stepping and to be transient in nature. The scientific and clinical implications of this observation will be dependent upon the precise underlying mechanism, a factor not identified by the present study.