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Dnm1p belongs to a family of dynamin-related GTPases required to remodel different cellular membranes. In budding yeast, Dnm1p-containing complexes assemble on the cytoplasmic surface of the outer mitochondrial membrane at sites where mitochondrial tubules divide. Our previous genetic studies suggested that Dnm1p's GTPase activity was required for mitochondrial fission and that Dnm1p interacted with itself. In this study, we show that bacterially expressed Dnm1p can bind and hydrolyze GTP in vitro. Coimmunoprecipitation studies and yeast two-hybrid analysis suggest that Dnm1p oligomerizes in vivo. With the use of the yeast two-hybrid system, we show that this Dnm1p oligomerization is mediated, in part, by a C-terminal sequence related to the GTPase effector domain (GED) in dynamin. The Dnm1p interactions characterized here are similar to those reported for dynamin and dynamin-related proteins that form higher order structures in vivo, suggesting that Dnm1p assembles to form rings or collars that surround mitochondrial tubules. Based on previous findings, a K705A mutation in the Dnm1p GED is predicted to interfere with GTP hydrolysis, stabilize active Dnm1p-GTP, and stimulate a ratelimiting step in fission. Here we show that expression of the Dnm1 K705A protein in yeast enhances mitochondrial fission. Our results provide evidence that the GED region of a dynaminrelated protein modulates a rate-limiting step in membrane fission.

Pulmonary deposition of alpha-particle-emitting radon daughters is estimated to account for 10% of all lung cancer deaths in the USA. However, the nature and timing of early (preneoplastic) genetic alterations in radon-associated lung cancer are still relatively uncertain. The purpose of this investigation was to determine whether genomic instability occurs after exposure of cultured normal human bronchial epithelial cells to six equal, fractionated doses of alpha-particles (total doses 2-4 Gy). Two weeks after the final exposure, foci of phenotypically altered cells (PACs) were detected in 0, 63 and 77% of control, low and high dose cultures respectively. Of these, 18% exhibited extended life spans relative to unexposed controls. Elevated frequencies of binucleated cells (BNCs), a marker of genomic instability, were observed in 60 and 38% of the PAC cultures from the low and high dose groups respectively. The micronucleus assay also showed evidence of genomic instability in 40 and 38% of PAC cultures from the low dose and high dose groups respectively. No changes in microsatellite length, another marker of genomic instability, were detected in any of the PAC samples with the 28 markers used for this assay. However, one PAC (L2) showed a hemizygous deletion at 9p13.3. Another PAC (H9), which exhibited the highest frequency of cells containing micronuclei (MN), exhibited a hemizygous deletion at 7q31.3. Each loss may represent a stable mutation that resulted either directly from irradiation or later in progeny of exposed cells because of alpha-particle-induced genomic instability. The fact that elevated levels of BNCs and MN were present in the progeny many generations after irradiation indicates that the genetic alterations detected with these two markers were not a direct consequence of radiation exposure, but of resulting genomic instability, which may be an early change after exposure to alpha-particles.

The minimum region in salmon calcitonin (sCT) which induces antigenicity and gastrointestinal disturbances has been identified by examining the cross-reactivity of several sCT fragments and CT analogs with antisera from sCT-treated patients, and by examining inhibition of gastrointestinal motility of these sCT fragments and CT analogs in conscious dogs. Sixteen residues at the N-terminus of sCT comprised the minimum fragment capable of inducing both activities. Human CT (hCT) showed no antigenicity and a four-order weaker inhibition of gastrointestinal motility than sCT. Based on these data, we synthesized the human and salmon chimeric CT, ACT-15, in which the 16 N-terminal residues were those of hCT and the 16 C-terminal residues were those of sCT. ACT-15 had no cross-reactivity with the antisera and had almost the same weak gastrointestinal inhibition effect as hCT in dog and rat models. Nevertheless, it retained a hypocalcemic activity and an analgesic activity comparable to sCT. These results suggest that the amino acid residues in the N-terminal half of CT are responsible for the formation of antibodies and the induction of gastrointestinal disturbances, but may not influence calcium metabolism or analgesia. Clinical studies of ACT-15 will be needed to confirm this hypothesis.

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