Species specificity of iron delivery in hybridomas

Summary Studies with Human x Human (HxH), Human x Mouse (HxM), and Mouse x Mouse (MxM) hybridomas have enabled us to define specific factors that affect hybridoma growth in a species-specific manner. Three transferrins and three lipophilic iron chelates have been tested for their ability to support hybridoma proliferation and antibody production. The results of these studies demonstrate that HxH hybridomas do not respond to bovine transferrin a⁺ concentrations up to 100 μg/ml and are approximately 100-fold less responsive to mouse transferrin than to human transferrin. HxM and MxM hybridomas respond equally to human or mouse transferrin but are 100-fold less sensitive to bovine transferrin. An antibody to the human transferrin receptor inhibited the growth-promoting activity of human or mouse transferrin on HxH hybridomas but was ineffective on HxM hybridomas. This semonstrated the functionality of the human transferrin receptor in HxH hybridomas and that human, mouse, and bovine transferrin were interacting through the mouse transferrin receptor in HxM hybridomas. HxH and HxM hybridomas respond similarly to three different iron chelates exhibiting 80 to 110% of the growth response to human transferrin. MxM hybridomas fail to respond to the iron chelates at similar concentrations, suggesting that the human genome present in the other hybridoma species confers a unique ability for utilizing iron when delivered in this form.     

Some mathematical aspects of mapping DNA cosmids

A number of experimental and mathematical problems must be solved before high resolution physical maps of mammalian chromosomes can be reliably determined. Such a map might consist of an ordered set of nonsequenced, overlapping DNA fragments 20,000-40,000 bases long, produced by digestion of a chromosome, using two restriction enzymes. Map construction requires assigning a signature to each fragment that differentiates it unambiguously from every other fragment, and then devising a computationally efficient algorithm that will provide a unique ordering of the fragments. In the first part of this paper we present a polynomial time algorithm that yields a unique map, and is largely independent of the method for assigning signatures. In the next section we analyze the distribution of lengths of restriction digest fragments and discuss the implications for the algorithm, including the expected number of map gaps. Finally, we discuss a specific method for assigning signatures proposed by Hans Lehrach, based on which of a panel of probes binds to a given fragment. In particular we examine the effects of fragment length heterogeneity on the theoretical optimum length and number of probes, and the extent to which false signatures might be obtained by nonspecific binding. We conclude that the Lehrach strategy is effective provided the number of probes is >-150, but that each fragment will need testing with at most 25 probes.     

Definition and identification of homology domains

A method is described for identifying and evaluating regionsof significant similarity between two sequences. The notionof a ‘homology domain’ is employed which definesthe boundaries of a region of sequence homology containing noinsertions or deletions. The relative significance of differentpotential homology domains is evaluated using a non-linear similarityscore related to the probability of finding the observed levelof similarity in the region by chance. The sensitivity of themethod is demonstrated by simulating the evolution of homologydomains and applying the method to their detection. Severalexamples of the use of homology domain identification are given. Received on July 29, 1987; accepted on November 15, 1987     

Unstable amplification of two extrachromosomal elements in alpha-difluoromethylornithine-resistant Leishmania donovani.

We describe the first example of unstable gene amplification consisting of linear extrachromosomal DNAs in drug-resistant eukaryotic cells. alpha-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with an amplified ornithine decarboxylase (ODC) gene copy number contained two new extrachromosomal DNAs, both present in 10 to 20 copies. One of these was a 140-kb linear DNA (ODC140-L) on which all of the amplified copies of the odc gene were located. The second was a 70-kb circular DNA (ODC70-C) containing an inverted repeat but lacking the odc gene. Both ODC140-L and ODC70-C were derived from a preexisting wild-type chromosome, probably by a conservative amplification mechanism. Both elements were unstable in the absence of DFMO, and their disappearance coincided with a decrease in ODC activity and an increase in DFMO growth sensitivity. These results suggest the possibility that ODC70-C may play a role in DFMO resistance. These data expand the diversity of known amplification mechanisms in eukaryotes to include the simultaneous unstable amplification of both linear and circular DNAs. Further characterization of these molecules will provide insights into the molecular mechanisms underlying gene amplification, including the ability of linear amplified DNAs to acquire telomeres and the determinants of chromosomal stability.     

Microtubule-associated protein 2 (MAP2)-immunoreactive neurons in the retina of Bufo marinus: colocalisation with tyrosine hydroxylase and serotonin in amacrine cells

Summary Neuron populations in the retina of the toad, Bufo marinus, were labelled with a monoclonal antibody raised against microtubule-associated protein 2 (MAP2). A subpopulation of cones, probably corresponding to the blue-sensitive small single cones, large diameter amacrine cells in the most proximal row of the inner nuclear layer and some large ganglion cells in the ganglion cell layer were labelled. Double labelling experiments were carried out to establish the colocalisation of MAP2 with known putative transmitter substances of the anuran amacrine cells. MAP2 was colocalised in a subpopulation of serotonin-immunoreactive and in all tyrosine hydroxylase-immunoreactive amacrine cells. The results indicate, that the MAP2 content in the neurons of the anuran retina can be correlated with other well-defined neurochemical and/or physiological properties.On leave from Department of Zoology, Attlia József University, Szeged, Hungary     

Serial studies of autologous antibody reactivity to squamous cell carcinoma of the head and neck

Summary In previous studies we evaluated the incidence and specificity of autologous antibody reactivity against squamous cell carcinoma of the head and neck (SCCHN). We were able to demonstrate that autologous antibody reactivity is present in native sera but was usually of too low a titer to allow further analysis. Dissociation of immune complexes by acidification and ultrafiltration of serum augmented autologous antibody reactivity in nine out of nine autologous systems tested. Native antibody and antibody derived from immune complexes produced by the host and reactive with autologous tumor cells may be directed against physiologically relevant antigens. Therefore, correlations of antibody titers with clinical course may provide insight into the nature of the host response to cancer. In the present analysis, serological studies of six patients with SCCHN were performed with serum samples obtained over many months. Results of serial serological assays were correlated to tumor progression and clinical course. Fluctuations in autologous antibody reactivity were noted over time. In four cases, rises in autologous antibody titers preceded the clinical diagnosis of recurrence by several months. Drops in autologous antibody reactivity were noted in two cases following surgery or radiation therapy. In two cases of long-term survivors, no correlation between antibody reactivity and clinical course was noted. Specificity analysis of the six autologous systems demonstrated reactivity against autologous and allogeneic SCCHN as well as melanoma cell lines. These sera did not react with glioma, neuroblastoma, renal cell, breast, bladder and colon carcinoma cell lines nor with fetal calf serum, pooled lymphocytes, red blood cells and platelets. Autologous serial serological studies may provide a means by which to evaluate the host/tumor relationship in patients with SCCHN.     

Bacterial flagellar diversity and significance in pathogenesis

Bacterial flagella are structurally diverse, ranging from the thoroughly investigated model examples found in Escherichia coli and Salmonella typhimurium to the more exotic sheathed flagella of, for example, Helicobacter pylori, and the complex multi-flagellin endoflagella found in many spirochaetes. We summarize some of the emerging structural and genetic findings relating to these more novel flagellar types, and outline their possible significance in the pathogenicity of some medically important bacteria.     

Passive immunization protects guinea pigs from lethal toxoplasma infection

Abstract The cellular and humoral interactions that contribute to protective immunity in toxoplasmosis were studied by adoptive transfer of selective cell populations or immune serum and its fractions into normal syngeneic strain 2 guinea pigs. The results of this study with the RH strain of Toxoplasma gondii confirm and extend the findings of previous studies by showing that the passive transfer of parasite-sensitized T cells or of immune serum from previously infected donors protected recipient guinea pigs against lethal toxoplasmosis. An additional key finding was that similar levels of complete protection against lethal infection occurred in guinea pigs receiving partially purified anti- Toxoplasma immunoglobulins or immune cells that had been enriched for B cells prior to transfer. Cells residing in the spleen, lymph nodes and peritoneal cavity, but not the thymus, were equally effective in conferring immunity to challenged recipients. In addition, cell titration experiments revealed that guinea pigs could survive T. gondii infection by infusing them with as little as 2 × 10⁷ sensitized T cells or B cells. Unlike protection mediated by T cells, protection against lethal disease occurring in the B cell recipients was associated with the formation of Toxoplasma antibodies. These findings illustrate the major role of both humoral and cell-mediated immunity in affording protection against toxoplasmosis based on a guinea pig model of the human disease.     

Leukoregulin, a T-cell derived cytokine, upregulates stromelysin-1 gene expression in human dermal fibroblasts: evidence for the role of AP-1 in transcriptional activation.

Leukoregulin (LR), a product of activated T-cells, has been recently shown to modulate the metabolism of extracellular matrix components in human skin fibroblast cultures (Mauviel et al., J Cell Biol 113:1455-1462, 1991). In this study we focused our attention on the effects of LR on the expression of stromelysin-1 gene. This matrix metalloprotease has a broad spectrum of degradative activity and it is also required for maximal activation of interstitial collagenase. Incubation of skin fibroblast cultures with LR resulted in a dose- and time-dependent elevation of stromelysin-1 mRNA levels, the maximum enhancement being up to approximately sevenfold. This effect was abolished by cycloheximide, suggesting a requirement for ongoing protein synthesis. Transient cell transfections with a promoter/reporter gene construct containing 1.3 kb of 5' flanking DNA of the human stromelysin-1 gene linked to the chloramphenicol acetyl transferase (CAT) gene, indicated enhancement of promoter activity by LR. This enhancement was abolished by a single base substitution in the AP-1 binding site of the promoter. Furthermore, gel mobility shift assays demonstrated enhanced AP-1 binding activity in nuclear extracts from cells incubated with LR. However, LR did not alter the activity of a construct containing three AP-1 sequences in front of the thymidine kinase promoter linked to the CAT gene. These results collectively suggest that activation of stromelysin-1 gene expression by LR is mediated by AP-1 regulatory elements which are necessary, but not sufficient, for gene response.