Measurement of sarcoplasmic reticulum Ca2+ content in intact amphibian skeletal muscle fibres with 4-chloro-m-cresol.

Single skeletal muscle fibres were isolated from the toad (Bufo marinus) and isometric force and myoplasmic free calcium concentration ([Ca2+]i) were measured. Brief applications of 4-chloro- m-cresol (4-CmC, 0.2-5 mM) elevated [Ca2+]i reversibly in a dose-dependent manner. The lowest concentration of 4-CmC which reliably gave maximal [Ca2+]i was 2 mM and it was, therefore, used for measurement of sarcoplasmic reticulum (SR) Ca2+ content. Tetanic stimulations (100 Hz) increased [Ca2+]i from a resting level of 105 +/- 47 nM (n = 10) to 1370 +/- 220 nM (n = 6). Application of 2 mM 4-CmC produced a contracture that was 54 +/- 16% (n = 6) of the tetanic force and elevated [Ca2+]i to a peak of 3520 +/- 540 nM (n = 8). Both force and [Ca2+]i levels (resting and tetanic) were restored after 10 min of washout of 4-CmC. In skinned muscle fibres, the myofibrillar Ca(2+)-sensitivity was not changed by 4-CmC, but maximal force was reduced to 74 +/- 10% (n = 4). The magnitude of the peak of the 4-CmC-induced Ca2+ transient was not significantly changed by removal of extracellular Ca2+ nor by inhibiting the SR Ca2+ pump with 2,5-di-tert-butylhydroquinone. Treatment of intact fibres with 30 mM caffeine produced a peak Ca2+ level that was indistinguishable from 2 mM 4-CmC. These results indicate that it is possible to measure the SR Ca2+ content in the same fibre with 4-CmC without loss of normal muscle function.     

OBPC Symposium: Maize 2004 &; beyond—Plant regeneration,gene discovery,and genetic engineering of plants for crop improvement

Summary The development of robust plant regeneration technology in cereals, dicots and ornamentals that is in turn coupled to a high-frequency DNA transfer technology is reported. Transgenic cereals that include maize, Tripsacum, sorghum, Festuca and Lolium, in addition to dicots that include soybean, cotton and various ornamentals such as petunia, begonia, and geranium have been produced following either somatic embryogenesis or direct organogenesis independent of genotype. Coupled with these regeneration protocols, we have also identified several interesting genes and promoters for incorporation into various crops and ornamentals. In addition, the phenomenon of direct in vitro flowering from cotyledonary nodes in soybean is described. In in vitro flowering, the formation of a plant body is suppressed and the cells of the cotyledonary node produce complete flowers from which fertile seed is recovered. This in vitro flowering technology serves as a complementary tool to chloroplast transformation for developing a new transgenic pollen containment strategy for crop species. Recently, the center has undertaken to screen the expression response of the 24 000 Arabidopsis genes to nitric oxide. This signaling molecule upregulated 342 genes and downregulated 80 genes. The object here was to identify a population of promoters that can be manipulated by using a signaling molecule. In addition, in keeping with the mission of enhancing greenhouse profitability for North West Ohio growers, we cloned a number of genes responsive for disease resistance from ornamentals that play an important role in disease management and abiotic stress. We have constructed a plant transformation vector with CBF3 gene under the rd29A promoter for engineering cold and freezing tolerance in petunia. Leaf dises of Petunia×hybrida v26 were used for Agrobacterium-mediated transformation, and 44 hygromycin-resistant T0 plants were obtained. The presence of CBF3 gene was confirmed in all the transgenic plants by PCR and Southern analyses.     

Pre-steady-state kinetic studies of the fidelity and mechanism of polymerization catalyzed by truncated human DNA polymerase lambda

DNA polymerase lambda (Pollambda), a member of the X-family DNA polymerases, possesses an N-terminal BRCT domain, a proline-rich domain, and a C-terminal polymerase beta-like domain (tPollambda). In this paper, we determined a minimal kinetic mechanism and the fidelity of tPollambda using pre-steady-state kinetic analysis of the incorporation of a single nucleotide into a one-nucleotide gapped DNA substrate, 21-19/41-mer (primer-primer/template). Our kinetic studies revealed an incoming nucleotide bound to the enzyme.DNA binary complex at a rate constant of 1.55 x 10(8) M(-1) s(-1) to form a ground-state ternary complex while the nucleotide dissociated from this complex at a rate constant of 300 s(-1). Since DNA dissociation from tPollambda (0.8 s(-1)) was less than 3-fold slower than polymerization, we measured saturation kinetics for all 16 possible nucleotide incorporations under single turnover conditions to eliminate the complication resulting from multiple turnovers. The fidelity of tPollambda was estimated to be in the range of 10(-2)-10(-4) and was sequence-dependent. Surprisingly, the ground-state binding affinity of correct (1.1-2.4 microM) and incorrect nucleotides (1.4-8.4 microM) was very similar while correct nucleotides (3-6 s(-1)) were incorporated much faster than incorrect nucleotides (0.001-0.2 s(-1)). Interestingly, the misincorporation of dGTP opposite a template base thymine (0.2 s(-1)) was more rapid than all other misincorporations, leading to the lowest fidelity (3.2 x 10(-2)) among all mismatched base pairs. Additionally, tPollambda was found to possess weak strand-displacement activity during polymerization. These biochemical properties suggest that Pollambda likely fills short-patched DNA gaps in base excision repair pathways and participates in mammalian nonhomologous end-joining pathways to repair double-stranded DNA breaks.     

Roles of Atox1 and p53 in the trafficking of copper-64 to tumor cell nuclei: implications for cancer therapy

Owing to its cytotoxicity, free copper is chelated by protein side chains and does not exist in vivo. Several chaperones transport copper to various cell compartments, but none have been identified that traffic copper to the nucleus. Copper-64 decays by β ⁺ and β ^? emission, allowing positron emission tomography and targeted radionuclide therapy for cancer. Because the delivery of ⁶⁴Cu to the cell nucleus may enhance the therapeutic effect of copper radiopharmaceuticals, elucidation of the pathway(s) involved in transporting copper to the tumor cell nucleus is important for optimizing treatment. We identified Atox1 as one of the proteins that binds copper in the nucleus. Mouse embryonic fibroblast cells, positive and negative for Atox1, were used to determine the role of Atox1 in ⁶⁴Cu transport to the nucleus. Mouse embryonic fibroblast Atox1^+/+ cells accumulated more ⁶⁴Cu in the nucleus than did Atox1^?/? cells. HCT 116 colorectal cancer cells expressing p53 (+/+) and not expressing p53 (?/?) were used to evaluate the role of this tumor suppressor protein in ⁶⁴Cu transport. In cells treated with cisplatin, the uptake of ⁶⁴Cu in the nucleus of HCT 116 p53^+/+ cells was greater than that in HCT 116 p53^?/? cells. Atox1 expression increased in HCT 116 p53^+/+ and p53^?/? cells treated with cisplatin; however, Atox1 localized to the nuclei of p53^+/+ cells more than in the p53^?/? cells. The data presented here indicate that Atox1 is involved in copper transport to the nucleus, and cisplatin affects nuclear transport of ⁶⁴Cu in HCT 116 cells by upregulating the expression and the nuclear localization of Atox1.