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     

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     

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.     

Elements of the rat tropoelastin gene associated with alternative splicing

Multiple isoforms of tropoelastin, the soluble precursor of elastin, are the products of translation of splice-variant mRNAs derived from the single-copy tropoelastin gene. Previous data had demonstrated DNA sequence heterogeneity in three domains of rat tropoelastin mRNA, indicating alternative splicing of several exons of the rat tropoelastin gene. Rat tropoelastin genomic clones encompassing the sites of alternative splicing were isolated and sequenced. Two sites of alternative splicing identified in rat tropoelastin mRNA sequences corresponded to exons 13-15 and exon 33 of the rat tropoelastin gene. Furthermore, the variable inclusion of an alanine codon in exon 16 resulted from two functional acceptor sites separated by three nucleotides. DNA sequences flanking exons subject to alternative splicing were analyzed. These exons contained splicing signals that differed from consensus sequences and from splicing signals of constitutively spliced exons. Introns immediately 5' of exons 14 and 33, for example, lacked typical polypyrimidine tracts and had weak, overlapping branch point sequences. Further, a region of secondary structure encompassing the acceptor site of exon 13 may influence alternative splicing of this exon. These results demonstrate that multiple cis-acting sequence elements may contribute to alternative splicing of rat tropoelastin pre-mRNA.     

Cyclic behavior of plum curculio,Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae), within caged dwarf apple trees in spring

From 12 to 19 May 1987, during Morspur apple bloom, 21 radioactively labeled (⁶⁵ Zn) adult plum curculios, Conotrachelus nenuphar (Herbst), were released within a field cage containing four dwarf apple trees and located three times a day. A technique was developed for quickly obtaining (x, y, z)coordinates of location for adults foraging within apple trees. Cyclic patterns of behavior were detected using spectral analysis procedures. Over 70% of plum curculios exhibited diel periodicity with respect to activity and rate of movement, 36% exhibited such periodicity with respect to presence in the trees, and 27% with respect to movements from the center to the periphery of the canopy. Presence in fruit clusters, height in the trees, and movements along east-west and north-south axes showed little or no periodicity. Factors triggering cyclic behavior and practical implications of the results are discussed.