The majority of independently transformed BHK cell clones share a single functional lesion which determines anchorage independence and influences tumorigenicity

Summary Expression of the anchorage-independent transformed phenotype in BHK 21/13 cells generally behaves as a recessive trait. When chemically induced and spontaneously arising transformants are fused to the nontransformed parent line, transformation is initially suppressed, reappearing after extended growth of the hybrids. In this paper, complementation for the expression of anchorage independence was sought among a large group of such transformants, all independently derived from BHK 21/13 cells. Tumorigenicity studies on selected hybrids and parental lines indicated that the in vitro trait of anchorage independence is an accurate indicator of in vivo neoplasia for these cells. Seventeen of the 18 clones tested did not complement one or more of three tester strains. This result indicates that anchorage independence arose in these clones as a result of lesions in the same genetic function and suggests that the final step in the progressive changes of carcinogenesis may frequently be restricted to lesions at a single locus. This investigation was supported by National Institutes of Health grant CA27306.     

The influence of natural short photoperiodic and temperature conditions on plasma thyroid hormones and cholesterol in male Syrian hamsters

Adult male Syrian hamsters were subjected to 1, 3, 5, 7 or 11 weeks of either natural winter conditions or rigorously controlled laboratory conditions (LD 1014; 22 ± 2C). Although both groups of hamsters gained weight over the course of the experiment, hamsters housed indoors were significantly heavier after 5 weeks of treatment compared to their outdoors counterparts. Animals housed under natural conditions exhibited a significant decrease in circulating levels of thyroxine (T₄) and a rapid rise in triiodothyronine (T₃) levels; the free T₄ and free T₃ index (FT₄I and FT₃I) mirrored the changes in circulating levels of the respective hormones. Laboratory-housed animals had a slight rise in T₄ and FT₄I at 3 weeks followed by a slow steady decline in these values; T₃ and FT₃I values did not change remarkably in these animals. Plasma cholesterol declined steadily over the course of the experiment in laboratory-maintained animals but increased slightly during the first 5 weeks in animals under natural conditions. Since the photoperiodic conditions were approximately of the same duration in these 2 groups, it is concluded that the major differences in body weight, thyroid hormone values and plasma cholesterol are due to some component (possibly temperature) in the natural environment.     

Chromatographic behavior of cyclic AMP dependent protein kinase and its subunits from Dictyostelium discoideum

An adenosine cyclic 3',5'-monophosphate (cAMP) dependent protein kinase has recently been shown to exist in Dictyostelium discoideum and to be developmentally regulated. In this report we have followed the chromatographic behavior of both the holoenzyme and its subunits. A cAMP-dependent holoenzyme could be obtained from the 100000 g soluble fraction after passage through DE-52 cellulose (pH 7.5) and Sephacryl S300. Under conditions of low pH the holoenzyme could be further purified by flat-bed electrofocusing (pI = 6.8). Application of the holoenzyme to electrofocusing at high pH resulted in dissociation of the holoenzyme into a cAMP binding component (pI = 6.1) and a cAMP-independent catalytic activity (pI = 7.4). Dissociation of the holoenzyme into subunits also occurred during histone affinity chromatography and gel filtration chromatography (S300) in the presence of a dissociating buffer. Although the subunit structure was clearly evident during chromatography, the holoenzyme could not be dissociated by simple addition of cAMP to the extract. The catalytic subunit could be purified further by CM-Sephadex, DE-52 cellulose (pH 8.5), histone affinity, and hydrophobic chromatography. The regulatory subunit was further purified by DE-52 cellulose (pH 8.5) and cAMP affinity chromatography. Proof that the cAMP binding activity and the cAMP-independent catalytic activity were in fact the regulatory and catalytic subunits was shown by reconstitution of the cAMP-dependent holoenzyme from the purified subunits. By using these separation procedures, one can obtain from extracts of Dictyostelium the subunits that are free of each other as well as free of any endogenous protein substrates.     

Induction of congenital malformations in the offspring of male mice treated with X-rays at pre-meiotic and post-meiotic stages

The induction of congenital malformations among the offspring of male mice treated with X-rays at pre-meiotic and post-meiotic stages has been studied in two experiments. Firstly, animals were exposed to varying doses (108–504 cGy) of X-rays and mated at various time intervals (1–7, 8–14, 15–21 and 64–80 days post-irradiation), so as to sample spermatozoa, spermatids and spermatogonial stem cells. In the second experiment, only treated spermatogonial stem cells were sampled. One group of males was given a single 500-cGy dose, a second group a fractionated dose (500 + 500 cGy, 24 h apart) and a third group was left unexposed.In the first experiment, induced post-implantation dominant lethality increased with dose, and was highest in week 3, in line with the known greater radiosensitivity of the early spermatid stage. Preimplantation loss also increased with dose and was highest in week 3. There was no clear induction of either pre-implantation or post-implantation loss at spermatogonial stem cell stages.There was a clear induction of congenital malformations at post-meiotic stages, the overall incidence being 2.0 ± 0.32% in the irradiated series and 0.24 ± 0.17% among the controls. The induction was statistically significant at each dose. At the two highest doses the early spermatids (15–21 days) appeared more sensitive than spermatozoa, and at this stage the incidence of malformations increased with dose. The data from Expt. 1 on the induction of malformations by irradiation of spermatogonial stages were equivocal. In contrast, Expt. 2 showed a statistically significant induction of malformations at both dose levels (2.2 ± 0.46% after 500 cGy and 3.1 ± 0.57% after 500 + 500 cGy). The relative sensitivities of male stem cells, post-neiotic stages and mature oocytes to the induction of congenital malformations were reasonably similar to their sensitivities for specific-locus mutations, except that the expected enhancing effect of the fractionation regime used was not seen.Dwarfism and exencephaly were the two most commonly observed malformations in all series.     

Experimental studies of pollen carryover: effects of floral variability in Ipomopsis aggregata

Summary In the montane herb Ipomopsis aggregata, size and placement of stamens and pistils vary substantially among flowers within plants, among nearby plants, and among groups of plants separated by 50–100 m. We trained captive hummingbirds to feed from flowers of this species in a flight cage, and explored the effects of different degrees of floral variability on carryover of fluorescent dyes that act as pollen mimics. We found that the slopes of linear dye carryover functions generally became more shallow as floral variability increased; this led to substantially longer carryover in the treatment with greatest variability. On the other hand, total amounts of dye transferred did not appear to be sensitive to the degree of variability. Floral variability may have a subtle but important effect on plant fitness by influencing the distance of pollen transfer.     

Lymphatic endothelial celline (CH3) from a recurrent retroperitoneal lymphangioma

Summary An endothelial cell line derived from a massive recurrent chyle-containing retroperitoneal lymphangioma was isolated in monolayer culture. Scanning and transmission electron microscopy and immunohistochemistry confirmed a close resemblance to blood vascular endothelium with typical cobblestone morphology, positive immunofluorescence staining for endothelial marker Factor VIII-associated antigen and fibronectin, and prominent Weibel-Palade bodies. The endothelial cells also exhibited other ultrastructural features characteristic of lymphatic endothelium, including sparse microvillous surface projections, overlapping intercellular junctions, and abundant intermediate filaments. This endothelial cell line represents a new source of proliferating lymphatic endothelium for future study, including structural and functional comparison to blood vascular endothelium. Supported in part by Arizona Disease Control Research Commission contracts 8277-000000-1-1-AT-6625 and ZB-7492. Presented in part at the 10th International Congress of Lymphology in Adelaide, Australia, August 1985.     

Molecular genotyping of human T-cell antigen receptor variable gene segments

The gene complex encoding the chain of the T-cell antigen receptor (Tcr) in man was previously reported to contain a restriction fragment length polymorphism (RFLP) involving a single Bgl II site adjacent to the second constant region gene. This RFLP allowed assignment of Tcr genotypes in certain human families. In the present study, two different RFLP in a V gene family were detected using the murine probe V8.1 in genomic DNA samples digested with the restriction endonucleases Hind III and Bam HI. Use of these RFLP to mark the V gene complex allowed complete haplotype assignment in four of seven families studied and provided support for linkage of the V gene complex to the constant region genes. Different combinations of the C and two V region markers can result in eight possible distinct haplotypes. The observation of all but one of the eight possible haplotypes in parents of the families studied suggests that recombination events occur between the C and V region and among members of the V region subfamily marked by the V8.1 probe. These markers can be used for mapping studies of the V gene complex in man and will allow an appraisal of possible associations between Tcr genes and disease susceptibility.Abbreviations used in this paper: Tcr T-cell antigen receptor - RFLP restriction fragment length polymorphism - C2 second Tcr constant region gene - V Variable - C constant - J joining - D diversity     

Sex-dependent effects of non-H-2 loci on lethal GVH reaction induced across an H-2 barrier

We have studied the influence of DBA/2 non-H-2 antigens on the lethal graft-versus-host reaction (GVHR) developed across an H-2 barrier. (DBA/2 x B10.D2)F₁ x B10.D2 (H-2 ^d) backcross (BC) mice were typed for their allelic constitution at nine genetically independent chromosome markers and used as individual cell donors simultaneously for two to three (DBA/2 X B10.D2)F₁ recipients incompatible for DBA/2 non-H-2 antigens alone and two to three (DBA/2 x B10.BR)F₁ recipients incompatible for DBA/2 non-H-2 antigens and H-2^k. The results showed that, when compared with that developed in a control group incompatible for H-2 ^kalone [B10.D2(B10.D2xB10.BR)F₁], the GVHR mortality seen in the presence of an additional incompatibility for DBA/2 non-H-2 antigens [(DBA/2 X B10.BR)F₁recipients] is significantly delayed but only in female mice. An analysis of individual BC donors indicated that this protective effect of DBA/2 non-H-2 antigens correlates with incompatibility for gene(s) linked to the Pgm-1 chromosome marker. In contrast, incompatibility for gene(s) linked to Mod-1 and Es-3 markers accelerates GVHR mortality, but only in male mice. Finally, the results obtained with (DBA/2 x B10.D2)F₁ and (DBA/2 x B10.BR)F₁ recipients were compared; they showed that the intensity of the GVHR developed by cells from individual BC donors against a given set of DBA/2 non-H-2 antigens correlates well with that developed by the same BC donor against the same set of non-H-2 antigens plus H-2^k. We conclude that certain non-H-2 genes (and antigens) can modulate the intensity of the GVHR developed across an H-2 barrier. The number of such genes is probably great; their effects are strong and complex, and can be sex-dependent.     

Mechanisms of genetic control of immune responses

The mechanisms underlying Ir gene control of CMI were addressed by examining the DTH and Tprlf responses specific for the synthetic polymers GT, GAT, and GA. We show that BALB/c mice (GAT/GA responders, GT nonresponders) primed with GT fail to develop DTH and Tprlf responses specific for GT, GAT, or GA. GAT immunization resulted in DTH responses that could be elicited not only with GAT and GA but also with GT, demonstrating that GT-specific T_DH are present in nonresponder mice. GT-specific DTH was transferred with Thy-1⁺ Lyt-1⁺2^–, H-2 Irestricted, nylon wool nonadherent cells. GA-primed BALB/c mice developed GAT- and GA-, but not GT-apecific DTH responses, indicating that GA and GT do not cross-react at the T-cell level. The ability of GAT [but not a mixture of GA plus GT, or GT electrostatically complexed to the immunogenic carrier MBSA (GT-MBSA)] to induce GT-specific DTH suggested a requirement for covalent linkage of stimulatory GA and nonstimulatory GT determinants present on the GAT molecule. Similarly, GT-specific in vitro Tprlf responses could be demonstrated in GAT-primed mice exhibiting significant levels of GT-specific DTH but not in GT- or GT-MBSA-primed mice. Tolerization experiments also suggested that GT-specific Th were involved in the development of GT-specific DTH in GAT-primed mice. The GT nonresponsiveness of BALB/c mice for DTH and Tprlf responses could not be reversed by treatments designed to abrogate Ts activity (priming with GT-MBSA and CY injection), nor could GT-primed cells be shown to inhibit the development or elicitation of GT-specific CMI in GAT-primed mice during the afferent and/or efferent stages of DTH. Our results suggest that GT nonresponsiveness does not result from the absence of GT-specific T cells or preferential induction of Ts. The results are discussed in the context of hole-in-the-repertoire and antigen presentation (determinant selection) models of Ir gene control.Abbreviations used in this paper APC antigen-presenting cells - BSA bovine serum albumin - BSS Mishell-Dutton balanced salt solution - CFA complete Freund's adjuvant - CMI cell-mediated immunity - CY cyclophosphamide - DTH delayed-type hypersensitivity - GA poly(Glu⁶⁰Ala⁴⁰) - GAT poly (Glu⁶⁰Ala³⁰Tyr¹⁰) - GT poly(Glu⁵⁰Tyr⁵⁰) - GT-MBSA GT complexed to methylated bovine serum albumin - It immune response - LN lymph node - PPD purified protein derivative of tuberculin - TDH DTH T cells - Th helper T cells - Tprlf T-cell proliferation - Ts suppressor T cells - TsF T-cell suppressor factor(s)