CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28–1 mutant and to a lesser extent in a cdc7–1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.     

The imbalanced supertree of flowering-plant phylogeny

Two contrasting approaches have been used to construct the overall tree of life from molecular data: one involves the analysis of single large datasets, while the other involves joining many independent smaller analyses into a supertree. A recent study uses the latter approach to produce the most complete phylogeny yet of flowering plant families.     

Water status and growth of loblolly pine (Pinus taeda L.) callus

Water status of Pinus taeda L. callus supported on Murashige and Skoog (MS) liquid medium was characterized over an 8 week period using thermocouple psychrometry. Medium with 30 gl⁻¹ sucrose was used to produce a high water potential (Ψ_w) of −0.4 MPa (H), and the same medium was used to create a moderate Ψ_w of −0.7 MPa (M) by the addition of 10% polyethylene glycol (PEG, w/v, MW=8000). Calli were produced from cotyledon explants on H medium for 2 weeks and then transferred to either M or H medium. Callus absorption of PEG accounted for 40% of the callus dry weight and less than 7% of the callus fresh weight. Callus dry weight (without the PEG fraction) on M medium was 40% of that observed on H medium. Fresh weight on M medium was only 15% of that observed on H medium. The Ψ_w of both H and M media remained constant throughout the culture period. On H medium, callus Ψ_w and osmotic potential (Ψ_s) both increased 0.05 MPa/week with the callus Ψ_w approaching that of the external medium. On M medium, callus Ψ_w and Ψ_s both decreased more than 0.1 MPa/week with the callus Ψ_w decreasing greatly below that of the external medium. The latter was attributed to a rapidly produced osmotic shock induced upon callus transfer and/or PEG which caused less callus hydration and resulted in reduced growth. Callus turgor potential (Ψ_p) was estimated to be +0.02 to +0.09 MPa and turgor was maintained as callus Ψ_w increased or decreased. After 8 weeks, cell volumes from callus on M medium were 50 to 60% less than on H medium, suggesting that reduced cell volumes were related to turgor maintenance.     

Persistent reovirus infections of L cells select mutations in viral attachment protein sigma1 that alter oligomer stability.

During maintenance of L-cell cultures persistently infected with reovirus, mutations are selected in viruses and cells. Cells cured of persistent infection support growth of viruses isolated from persistently infected cultures (PI viruses) significantly better than that of wild-type (wt) viruses. In a previous study, the capacity of PI virus strain L/C to grow better than wt strain type 1 Lang (T1L) in cured cells was mapped genetically to the S1 gene (R. S. Kauffman, R. Ahmed, and B. N. Fields, Virology 131:79-87, 1983), which encodes viral attachment protein sigma1. To investigate mechanisms by which mutations in S1 confer growth of PI viruses in cured cells, we determined the S1 gene nucleotide sequences of L/C virus and six additional PI viruses isolated from independent persistently infected L-cell cultures. The S1 sequences of these viruses contained from one to three mutations, and with the exception of PI 2A1 mutations in each S1 gene resulted in changes in the deduced amino acid sequence of sigma1 protein. Using electrophoresis conditions that favor migration of sigma1 oligomers, we found that sigma1 proteins of L/C, PI 1A1, PI 3-1, and PI 5-1 migrated as monomers, whereas sigma1 proteins of wt reovirus and PI 2A1 migrated as oligomers. These findings suggest that mutations in sigma1 protein affecting stability of sigma1 oligomers are important for the capacity of PI viruses to infect mutant cells selected during persistent infection. Since no mutation was found in the deduced amino acid sequence of PI 2A1 sigma1 protein, we used T1L X PI 2A1 reassortant viruses to identify viral genes associated with the capacity of this PI virus to grow better than wt in cured cells. The capacity of PI 2A1 to grow better than T1L in cured cells was mapped to the S4 gene, which encodes outer-capsid protein sigma3. This finding suggests that in some cases, mutations in sigma3 protein in the absence of sigma1 mutations confer growth of PI viruses in mutant cells. To confirm the importance of the S1 gene in PI virus growth in cured cells, we used T1L X PI 3-1 reassortant viruses to genetically map the capacity of this PI virus to grow better than wt in cured cells. In contrast to our results using PI 2A1, we found that growth of PI 3-1 in cured cells was determined by the sigma1-encoding S1 gene. Given that the sigma1 and sigma3 proteins play important roles in reovirus disassembly, findings made in this study suggest that stability of the viral outer capsid is an important determinant of the capacity of reoviruses to adapt to host cells during persistent infection.     

Peroxo heteroligand vanadates(V): Synthesis,spectra-structure relationships,and stability toward decomposition

Effect of heteroligands (L) on the properties of vanadium peroxides was investigated by preparing a number of peroxovanadium complexes, which were characterized by analysis, IR, UV/V and NMR spectra. X-ray structures for some were obtained. The vanadates(V) contain the cation M(I)=Na, K, NH₄, Rb or Cs. Diperoxo complexes include M(I)[VO(O₂)₂L], where L=dipyridyl, o-phenanthroline; M(I)₃[VO(O₂)₂(C₂O₄)]; K₂[(nicotinic acid) {VO(O₂)₂}₂]H₂O;M(I)₄[O{VO(O₂)₂}₂ cystine]2H₂O; H₄[O{VO(O₂)₂(adenine)₂)₂]2H₂O; and K₂H₂[O{VO(O₂)₂(adenosine)}₂]2H₂O. Monoperoxo vanadates(V) correspond to the formula M(I)₂[VO(O₂)L]₂ for L=citrate and malate; M(I)₂[VO(O₂)L] for L=nitrilotriacetate; M(I)[VO(O₂)L] for L=iminodiacetate, tartrate and EDTA; and [HVO₂(O₂)(adenosine)]2H₂O. Syntheses of these heteroligand peroxovanadium compounds are sensitive to pH, temperature and the concentration of the components. The stability towards decomposition in solid state, mother-liquid and pure water solutions depends upon the heteroligand. Characteristic (V=O) and (O-O) stretching frequency bands in IR can be correlated with the corresponding bond lengths and the [peroxoV(V)] charge transfer bands in UV/V spectra. Intramolecular one-electron transfer in peroxo vanadates(V) can trigger the generation of radicals, and its dependency upon the nature of the heteroligand is discussed.     

Physiological Changes in Cultured Sorghum Cells in Response to Induced Water Stress : II. Soluble Carbohydrates and Organic Acids

Eight cultivars Sorghum bicolor (L.) Moench were grown as callus cultures under induced, prolonged water stress (8 weeks), with polyethylene glycol in the medium. Concentrations of soluble carbohydrates and organic acids in callus were measured at the end of the growth period to determine differences in response to prolonged water stress. Sucrose, glucose, fructose, and malate were the predominant solutes detected in all callus at all water potentials. All cultivars had high levels of solutes in the absence of water stress and low levels in the presence of prolonged water stress. However, at low water potentials, low levels of solutes were observed in drought-tolerant cultivar callus and high solute levels were observed in drought-susceptible cultivar callus. Estimated sucrose concentrations were significantly higher in water-stressed, susceptible cultivar callus. Large solute concentrations in susceptible cultivar callus were attributed to osmotic adjustment and/or reduced growth during water stress.     

PPARγ and LXR Signaling Inhibit Dendritic Cell-Mediated HIV-1 Capture and trans-Infection

Dendritic cells (DCs) contribute to human immunodeficiency virus type 1 (HIV-1) transmission and dissemination by capturing and transporting infectious virus from the mucosa to draining lymph nodes, and transferring these virus particles to CD4+ T cells with high efficiency. Toll-like receptor (TLR)-induced maturation of DCs enhances their ability to mediate trans-infection of T cells and their ability to migrate from the site of infection. Because TLR-induced maturation can be inhibited by nuclear receptor (NR) signaling, we hypothesized that ligand-activated NRs could repress DC-mediated HIV-1 transmission and dissemination. Here, we show that ligands for peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor (LXR) prevented proinflammatory cytokine production by DCs and inhibited DC migration in response to the chemokine CCL21 by preventing the TLR-induced upregulation of CCR7. Importantly, PPARγ and LXR signaling inhibited both immature and mature DC-mediated trans-infection by preventing the capture of HIV-1 by DCs independent of the viral envelope glycoprotein. PPARγ and LXR signaling induced cholesterol efflux from DCs and led to a decrease in DC-associated cholesterol, which has previously been shown to be required for DC capture of HIV-1. Finally, both cholesterol repletion and the targeted knockdown of the cholesterol transport protein ATP-binding cassette A1 (ABCA1) restored the ability of NR ligand treated cells to capture HIV-1 and transfer it to T cells. Our results suggest that PPARγ and LXR signaling up-regulate ABCA1-mediated cholesterol efflux from DCs and that this accounts for the decreased ability of DCs to capture HIV-1. The ability of NR ligands to repress DC mediated trans-infection, inflammation, and DC migration underscores their potential therapeutic value in inhibiting HIV-1 mucosal transmission.     

Genetic diversity from pre-bottleneck to recovery in two sympatric pinniped species in the Northwest Atlantic

Conservation successes of the past several decades provide natural settings to study post-bottleneck evolutionary processes in species undergoing recovery. Here, we study the impact of demographic change on genetic diversity in parallel natural experiments of historical decline and subsequent recovery in two sympatric pinniped species in the Northwest Atlantic, the gray seal (Halichoerus grypus atlantica) and harbor seal (Phoca vitulina concolor). We compare genetic diversity at the mitochondrial control region today to diversity in archaeological specimens, which represent the populations prior to the regional bounties of the late 1800s to mid-1900s that drastically reduced population sizes and led to local extirpations. We further assess genetic diversity throughout recovery, using biological collections from ongoing long-term studies of both species. Overall, the genetic data are consistent with the historical presence of large, genetically diverse populations of pinnipeds prior to human exploitation, and suggest that gray seals were more dramatically impacted by historical bottlenecks than harbor seals in the Northwest Atlantic. Current mitochondrial diversity in both species is relatively high, and we observe little change over the past several decades during a period of roughly parallel rapid population increases. However, there remain large differences in haplotype composition between pinniped populations of pre-exploitation and today, a lasting genetic signature of historical exploitation that is likely to persist into the future.