A mutational wrench in the HAMP gearbox

HAMP domains communicate between input and output signalling elements in bacterial proteins. In the Tsr chemoreceptor, they convert axial movement of transmembrane helix 2 into changes in packing of the cytoplasmic kinase-control module (KCM). Zhou et al . suggest transmembrane helix 2 'tugs' on HAMP to destabilize x-da packing of the parallel four-helix bundle of the HAMP homodimer. Attractants would inhibit tugging. HAMP stability may be inversely related to stability of the a-d packing of the anti-parallel four-helix bundle of KCM, a relationship possibly facilitated by HAMP/KCM helical mismatch. The beauty of this idea lies in its simplicity and testability.     

Induction of the immune response to cell surface antigens in vitro

Summary Two tissue culture incubation systems are described in which immune responses to cell surface antigens have been demonstrated In the one-way “mixed lymphocyte interaction” system, a specific stimulation of thymidine uptake was induced by a particulate membrane antigen fraction, the microsomal lipoproteins (MLP)when low levels (0.01 to 0.001 μg per ml) were incubated with spleen or lymph node cells from nonsensitized mice. No stimulation was seen when allogeneic MLP was used at high levels, 10 μg per ml, nor at any level with syngeneic MLP. Specific effectors were demonstrated after 72-hr incubation with stimulatory levels of allogeneic MLP in three separate in vitro assays, a plaque-forming cell reduction assay, a tumor target assay, and an antigen-binding cell assay. In the latter assay, [¹²⁵I]MLP was used as the source of antigen. This system has limited potential inasmuch as mouse spleen cells do not survive in it beyond the 4th day of culture. The second tissue culture system, the Marbrook system, has much greater possibilities because at least 25% of the inoculum is recovered 7days later. In this culture system a cell-free sheep erythrocyte membrane preparation can induce, plaque-forming cells in the absence of macrophages. Using a sensitive radioimmunoassay, frees specific antibody was detected in culture supernatant fluids. With the same culture system, allogeneic lymphocytotoxic cells (killer) have been induced with spleen cells from unprimed mice in strains differing at the major histocompatibility locus (H-2). Allogeneic MLP induced very significant “killer” cell activity with spleen cells from primed mice. In a syngeneic tumor systems, significant amounts of killer cell activity were induced with unprimed spleen cell inocula, and much larger amounts induced with spleen cells from immunized mice. Presented in the formal symposium on Carcinogenesis in Vitro, at the 25th Annual Meeting of the Tissue Culture Association, Miami Beach, Florida, June 3–6, 1974. This work was supported by Public Health Service Rescarch grants CA 07973 and CA 10815 from the National Cancer Institute.     

Effects of cytosine arabinoside on in vivo and in vitro mouse limb development

Summary When pregnant mice were exposed to 40 mg per kg of cytosine arabinoside (ara-C) on days 10 to 12 of gestation, adactylous limbs with large, distally located blisters were found when the fetuses were examined on day 18. Embryonic limbs exposed transplacentally under identical conditions and explanted to culture exhibited the same morphological abnormality as did limbs exposed directly in culture to 0.1 to 1 μg per ml of ara-C. Two noncytotoxic analogues of ara-C, uridine arabinoside (ara-U) and hypoxanthine arabinoside (ara-HX), had no influence on morphological differentiation of limbs in vitro. Ara-C alone caused a dose-related decrease in uptake of³H-thymidine and³⁵SO₄ in cultured limb buds. Production of this morphologically distinct malformation in vitro will allow detailed biochemical investigations on the effect of ara-C limb ectodermal-mesenchymal interactions. Supported by NIH Postdoctoral Fellowship No. CM02660. Supported by NIEHS Predoctoral Fellowship No. ES00127. Supported by NIEHS Toxicology Training Grant No. ES00127.