The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure

Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79). In Saccharomyces cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA damage response. The biological function of H3K79 methylation in mammals, however, remains poorly understood. Using gene targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES) cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres. These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.     

Hdm2 nuclear export, regulated by insulin-like growth factor-I/MAPK/p90Rsk signaling, mediates the transformation of human cells

Insulin-like growth factor (IGF)-I receptor activation leads to enhanced proliferation and cell survival via the MAP kinase and phosphatidylinositol 3-kinase-signaling pathways. Upon stimulation by IGF-I, the Hdm2 oncoprotein is phosphorylated by AKT, leading to its rapid nuclear translocation and subsequent inhibition of p53. We now show that IGF-I stimulation regulates the nuclear export of Hdm2 and p53 via the MAP kinase pathway. Inhibition of p38 MAPK or MEK via pharmacological means or expression of dominant negative proteins inhibited the cytoplasmic accumulation of Hdm2 and increased Hdm2 and p53 protein levels, whereas constitutively active p90Rsk promoted the nuclear export of Hdm2. Expression of constitutively active p90Rsk with E1A, oncogenic H-Ras, and hTERT resulted in the anchorage-independent growth of normal human fibroblasts. Our findings link p90Rsk-mediated modulation of Hdm2 nuclear to cytoplasmic shuttling with the diminished ability of p53 to regulate cell cycle checkpoints that ultimately leads to transformation.     

Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production

Inhibition of mitochondrial respiratory chain complex I by rotenone had been found to induce cell death in a variety of cells. However, the mechanism is still elusive. Because reactive oxygen species (ROS) play an important role in apoptosis and inhibition of mitochondrial respiratory chain complex I by rotenone was thought to be able to elevate mitochondrial ROS production, we investigated the relationship between rotenone-induced apoptosis and mitochondrial reactive oxygen species. Rotenone was able to induce mitochondrial complex I substrate-supported mitochondrial ROS production both in isolated mitochondria from HL-60 cells as well as in cultured cells. Rotenone-induced apoptosis was confirmed by DNA fragmentation, cytochrome c release, and caspase 3 activity. A quantitative correlation between rotenone-induced apoptosis and rotenone-induced mitochondrial ROS production was identified. Rotenone-induced apoptosis was inhibited by treatment with antioxidants (glutathione, N-acetylcysteine, and vitamin C). The role of rotenone-induced mitochondrial ROS in apoptosis was also confirmed by the finding that HT1080 cells overexpressing magnesium superoxide dismutase were more resistant to rotenone-induced apoptosis than control cells. These results suggest that rotenone is able to induce apoptosis via enhancing the amount of mitochondrial reactive oxygen species production.     

REPRODUCTIVE RATES OF HUMPBACK WHALES OFF CALIFORNIA

From 1986 to 1996 we examined the reproductive rates, calving rates, and reproductive histories of mature females as part of photo-identification studies of humpback whales that feed off California, Oregon, and Washington during summer and fall. Annual reproductive rates were measured by two methods: proportion of all whales that were calves based on sightings (0.6%-5.9% per year, mean = 3.6%, SD = 1.6) and based on individually identified animals (1.1%-8.0% per year, mean = 4.1%, SD = 1.8). The reproductive rate based on sightings varied significantly by year ( G test, P < 0.001), region ( G test, P < 0.001), and by month ( G test, P < 0.05). Seventy-nine sexually mature females were identified with 97 calves out of a total of 844 known individuals over the 11-yr study. Mother-calf separation on the feeding grounds was recorded in several instances. The apparent reproductive rates of this population are considerably lower than rates of 4%–15% reported from other feeding areas for this species. Our estimates are likely biased downward because this population has been increasing at about 5% per year. Calves may have been missed due to early weaning and because of our sampling from small boats late in the season. We also found evidence of geographic segregation of mother-calf pairs within our large study area. Despite these factors, we conclude the reproductive rate of this population appears to be lower than has been reported in other areas.     

Limulus amebocyte clotting cascade: Roles of endotoxin and adenylate cyclase

Endotoxin, the lipopolysaccharide from the cell wall of Gram-negative bacteria, causes blood clotting in the horseshoe crab,Limulus polyphemus. Minute amounts of endotoxin stimulate the amebocytes in the blood to undergo exocytosis, which release the contents of their secretory granules to form a clot. An endotoxin-binding protein that possesses calmodulin-like activity has been isolated from the amebocyte plasma membrane. This endotoxin-binding protein can activate adenylate cyclase fromBordetella pertussis to the same extent as rat testes calmodulin. The effect of endotoxin and the endotoxin-binding protein on cyclic AMP synthesis inLimulus amebocytes was examined. Amebocytes exposed to endotoxin have increased levels of intracellular cyclic AMP. Amebocyte membranes contain an adenylate cyclase which is stimulated by NaF, guanosine (β,r-imido)triphosphate, and for skolin. This adenylate cyclase is also stimulated by the endotoxin-binding protein and calcium. Exposure of amebocytes to forskolin or dibutyryl cyclic AMP are stimulated to secrete clot components. Activation of adenylate cyclasein vivo by endotoxin via the endotoxin-binding protein may be one of the ways in which endotoxin stimulates secretion. It is suggested that endotoxin may have two actions in theLimulus system: (1) binding of endotoxin to the endotoxin-binding protein activates adenylate cyclase, promoting secretion by the amebocytes; and (2) endotoxin catalyzes a reaction on the secreted material to form a blood clot. This latter reaction is not elicited by forskolin or dibutyryl cyclic AMP.     

Exotic shrub invasion in an undisturbed wetland has little community-level effect over a 15-year period

In this long-term study, we examined the invasion by the exotic shrub glossy buckthorn (Rhamnus frangula L.) and the response of co-occurring plants in a large, undisturbed wetland. We first sampled the vegetation in 1991 and repeated the sample 15 years later using the same, permanently located sample units (n = 165). Despite dramatic increases in the abundance of buckthorn, the invasion elicited little apparent response by the resident plant community. Species richness and cover in the herbaceous plant stratum had no apparent relationship with change in buckthorn cover. The number of shrub species other than buckthorn showed no relationship with change in buckthorn cover, but the cover of other shrubs decreased as buckthorn cover increased. Species composition changed independently of changes in buckthorn cover. These results show that dramatic increases in the abundance of an invasive species do not necessarily cause large changes in the native plant community and suggest disturbance history influences community response to invasion.     

Asymmetric assembly of Merkel cell polyomavirus large T-antigen origin binding domains at the viral origin

The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects immunosuppressed and elderly persons. As in other polyomaviruses, the large T-antigen of MCV recognizes the viral origin of replication by binding repeating G(A/G)GGC pentamers. The spacing, number, orientation, and necessity of repeats for viral replication differ, however, from other family members such as SV40 and murine polyomavirus. We report here the 2.9 Å crystal structure of the MCV large T-antigen origin binding domain (OBD) in complex with a DNA fragment from the MCV origin of replication. Consistent with replication data showing that three of the G(A/G)GGC-like binding sites near the center of the origin are required for replication, the crystal structure contains three copies of the OBD. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing double-stranded DNA was found to be ∼ 740 nM, approximately 8-fold weaker than the equivalent domain in SV40 for the analogous region of the SV40 origin. The difference in affinity is partially attributable to DNA-binding residue Lys331 (Arg154 in SV40). In contrast to SV40, a small protein-protein interface is observed between MCV OBDs when bound to the central region of the origin. This protein-protein interface is reminiscent of that seen in bovine papilloma virus E1 protein. Mutational analysis indicates, however, that this interface contributes little to DNA binding energy.     

Progress in melanoma treatment: Patient's perspectives

Upon the 20th Anniversary of the Society for Melanoma Research, we highlight the perspectives of patients aiming to help improve future experiences, outcomes, and their quality of life over the next 20 years. Five melanoma patients generously shared their inspiring and enlightening stories of diagnosis, treatment, and outcomes. Many patients had excellent medical teams that synergistically worked together to provide an accurate diagnosis, effective treatment options, and supportive care. However, it is clear that health inequities persist in communities where people of color are predominant, affecting early detection, patient experience, and outcomes. These stories shed light on the unique challenges faced by patients and how the lack of melanoma awareness and adequate resources, especially in communities of color or low socioeconomic status, can contribute to disparate outcomes in melanoma care. We expect that these stories will raise awareness about the progress in melanoma treatment but also the existent disparities in melanoma diagnosis and treatment and the importance of early detection and prevention.