[Abstract unavailable

The influence of the nucleotide at position ‐3 relative to the AUG initiation codon on the initiation of protein synthesis was studied in two different in vitro translation systems using synthetic mRNAs. The four mRNAs, transcribed from cDNAs directed by an SP6 promoter, were identical except for mutations at nucleotide ‐3. In each case, translation of mRNAs produced a single protein of Mr = 12,600. Relative translational efficiencies showed a hierarchy in the reticulocyte lysate system (100, 85, 61 and 38% for A, G, U and C in position ‐3, respectively) but no differences in the wheat germ system. Differential mRNA degradation or polypeptide chain elongation were excluded as causes of the differences observed in translation in the reticulocyte lysate. mRNA competition increased the differences observed in translational efficiencies in reticulocyte lysate but showed no effect in wheat germ. Analysis of 61 plant and 209 animal mRNA sequences revealed qualitative and quantitative differences between the consensus sequences surrounding AUG initiation codons. Whereas the consensus sequence for animals was CACCAUG that for plants was AACAAUGGC. Both the structural and functional findings suggest that the factors which select AUG initiation codons in plants and animals differ significantly.

SummaryAAA domain-containing 3A (ATAD3A) is a member of the AAA-ATPase family. Three forms of ATAD3 have been identified: ATAD3A, ATAD3B and ATAD3C. In this study, we examined the type and expression of ATAD3 in lung adenocarcinoma (LADC). Expression of ATAD3A was detected by reverse transcription-polymerase chain reaction, immunoblotting, immunohistochemistry and confocal immunofluorescent microscopy. Our results show that ATAD3A is the major form expressed in LADC. Silencing of ATAD3A expression increased mitochondrial fragmentation and cisplatin sensitivity. Serum deprivation increased ATAD3A expression and drug resistance. These results suggest that ATAD3A could be an anti-apoptotic marker in LADC.

Dynamin-related protein 1 (DRP1), an 80 kDa GTPase, is involved in mitochondrial fission and anticancer drugmediated cytotoxicity, which implicate an association with disease progression of cancer. In this study we investigated the prognostic value of DRP1 in lung adenocarcinomas. Using immunohistochemistry, we measured the expression of DRP1 in 227 patients with lung adenocarcinomas. Expression of DRP1 was confirmed by immunoblotting. The correlation between DRP1 expression and clinicopathological parameters was analyzed by statistical analysis. Difference of survivals between different groups was compared by a logrank test. The results showed that DRP1 expression was detected in 202 patients with lung adenocarcinomas. Among these, nuclear DRP1 (DRP1 nuc ) was detected in 184 patients. A significant difference was found in cumulative survival between patients with high DRP1 nuc levels and those with DRP1 cyt levels (Po0.001). In vitro, hypoxia increased DRP1 nuc levels and cisplatin resistance. Antibodies specific to DRP1 co-precipitated a human homologue of yeast Rad23 protein A (hHR23A) and silencing of hHR23A decreased the nuclear DRP1 level and cisplatin resistance. In conclusion, DRP1 nuc is highly expressed in lung adenocarcinomas, and correlates with poor prognosis. Nuclear DRP1 may increase drug resistance during hypoxia, and hHR23A is essential for nuclear transportation of DRP1. Our results suggest that other than the protein level alone, intracellular distribution of the protein is critical for determining the protein function in cells.

Human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) has been shown to induce host cell death by increasing the permeability of mitochondrial outer membrane (MOM). The mechanism underlying the damage to the mitochondria by Vpr, however, is not clearly illustrated. In this study, Vpr that is introduced, via transient transfection or lentivirus infection, into the human embryonic kidney cell line HEK293, human CD4 + T lymphoblast cell line SupT1, or human primary CD4 + T cells serves as the model system to study the molecular mechanism of Vpr-mediated HIV-1 pathogenesis. The results show that Vpr injures MOM and causes a loss in membrane potential (MMP) by posttranscriptionally reducing the expression of mitofusin 2 (Mfn2) via VprBP-DDB1-CUL4A ubiquitin ligase complex, gradually weakening MOM, and increasing mitochondrial deformation. Vpr also markedly decreases cytoplasmic levels of dynamin-related protein 1 (DRP1) and increases bulging in mitochondria-associated membranes (MAM), the specific regions of endoplasmic reticulum (ER) which form physical contacts with the mitochondria. Overexpression of Mfn2 and DRP1 significantly decreased the loss of MMP and apoptotic cell death caused by Vpr. Furthermore, by employing time-lapse confocal fluorescence microscopy, we identify the transport of Vpr protein from the ER, via MAM to the mitochondria. Taken together, our results suggest that Vpr-mediated cellular damage may occur on an alternative protein transport pathway from the ER, via MAM to the mitochondria, which are modulated by Mfn2 and DRP1.

. It also mediates the DNA cleavage activity and cytotoxicity of clinically important anticancer agents such as etoposide. We have examined the activity of topoisomerase H during the first cell cycle of quiescent BALB/c 3T3 cells following serum stimulation. Etoposide-mediated DNA break frequency in vivo was used as a parameter of topoisomerase II activity, and enzyme content was assayed by immunoblotting. Density-arrested A31 cells exhibited a much lower sensitivity to the effects of etoposide than did actively proliferating cells. Upon serum stimulation of the quiescent cells, however, there was a marked increase in drug sensitivity which began during S phase and reached its peak just before mitosis. Maximal drug sensitivity during this period was 2.5 times greater than that of log-phase cells. This increase in drug sensitivity was associated with an increase in intracellular topoisomerase II content as determined by immunoblotting. The induction of topoisomerase 11-mediated drug sensitivity was aborted within 1 h of exposure of cells to the protein synthesis inhibitor cycloheximide, but the DNA synthesis inhibitor aphidicolin had no effect. In contrast to the sensitivity of cells to drug-induced DNA cleavage, maximal cytotoxicity occurred during S phase. A 3-h exposure to cycloheximide before etoposide treatment resulted in nearly complete loss of cytotoxicity. Our findings indicate that topoisomerase II activity fluctuates with cell cycle progression, with peak activity occurring during the G2 phase. This increase in topoisomerase II is protein synthesis dependent and may reflect a high rate of enzyme turnover. The dissociation between maximal drug-induced DNA cleavage and cytotoxicity indicates that the topoisomerase-mediated DNA breaks may be necessary but are not sufficient for cytotoxicity and that other factors which are particularly expressed during S phase may be important as well.Type II DNA topoisomerases are ATP-dependent enzymes which catalyze the breakage and reunion of duplex DNA, allowing a second segment of DNA to pass through the break site (1, 23). They are required for the segregation of DNA following the conclusion of replication (4) and appear to be required at mitosis (12). During the breakagereunion cycle a covalent phosphotyrosyl bond between enzyme and DNA is formed at the 5' terminus of each strand. A number of commonly used anticancer agents such as intercalating agents and epipodophyllotoxins can stabilize this DNA protein intermediate, and convincing evidence exists that this interaction does mediate their cytotoxicity (15,16,25). A better understanding of the enzyme would be useful in improving the efficacy of these drugs. In addition, these topoisomerase-specific drugs have become useful probes in understanding the fundamental functions and control mechanisms of the enzyme.Topoisomerase II activity and content varies as a function of the proliferative status of the cell (5,19,29,30 Cell culture. BALB/c 3T3 (A31) were grown at 37°C in a monolayer in alpha minimal essential medium...

GM-CSF can significantly reduce the severity and duration of chemotherapy-induced oral mucositis after PFL chemotherapy.

These results indicate that FUMHD is associated with dermal CMV manifestation. Nonetheless, the induction mechanism of FUMHD with CMV infection has yet to be determined.