Toc159- and Toc75-independent import of a transit sequence-less precursor into the inner envelope of chloroplasts

Chloroplast envelope quinone oxidoreductase (ceQORH) is an inner plastid envelope protein that is synthesized without cleavable chloroplast transit sequence for import. In the present work, we studied the in vitro-import characteristics of Arabidopsis ceQORH. We demonstrate that ceQORH import requires ATP and is dependent on proteinaceous receptor components exposed at the outer plastid surface. Competition experiments using small subunit precursor of ribulose-bisphosphate carboxylase/oxygenase and precursor of ferredoxin, as well as antibody blocking experiments, revealed that ceQORH import does not involve the main receptor and translocation channel proteins Toc159 and Toc75, respectively, which operate in import of proteins into the chloroplast. Molecular dissection of the ceQORH amino acid sequence by site-directed mutagenesis and subsequent import experiments in planta and in vitro highlighted that ceQORH consists of different domains that act concertedly in regulating import. Collectively, our results provide unprecedented evidence for the existence of a specific import pathway for transit sequence-less inner plastid envelope membrane proteins into chloroplasts.     

Recovery of RNA from flow-sorted fixed cells.

We describe a method for the preparation of RNA from ethanol-fixed cells, allowing analysis of the RNA from cells "frozen" in a given physiological state. This technique may have important applications in experiments which require prolonged cell manipulations before RNA preparation, such as investigations of cell-cycle-regulated gene expression, which require the preparation of cells for cell-cycle flow analysis, and even for long-term cell sorting. It eliminates all the inconveniences associated with the use of fresh cells, and allows cell-cycle biologists to couple flow cytometry methodology with the advancing techniques of molecular biology.     

Acylated Steryl Glycoside Synthesis in Seedlings of Nicotiana tabacum L

In tobacco seedlings (Nicotiana tabacum L.), glucose from supplied uridine diphosphate-[U-¹⁴C]glucose was first incorporated into steryl glycosides and later into acylated steryl glycosides. However, when [¹⁴C]cholesterol was used as substrate, the acylated steryl glycosides became labeled earlier than the steryl glycosides. With [¹⁴C]cholesteryl glucoside as substrate, most of the radioactive label was recovered as free sterol, and the acylated steryl glycosides were not readily labeled; however, palmitoyl [¹⁴C]cholesteryl glucoside was rapidly converted to steryl glycoside. In feeding experiments with free sterol, an unknown, highly radioactive steroid component was isolated. Incorporation of radioactivity into the unknown occurred before the acylated steryl glycosides were labeled.     

Bayesian profiling for cost with zeros to decompose total cost into probability of cost and mean nonzero cost

Cost of health care can vary substantially across hospitals, centers, or providers. Data from electronic health records provide information for studying patterns of cost variation and identifying high or low cost centers. Cost data often include zero values when patients receive no care, and joint two-part models have been developed for clustered cost data with zeros. Standard methods for center comparisons, sometimes called profiling, can use these methods to incorporate zero values into total cost. However, zero costs also provide opportunities to further examine sources of cost variation and outliers. For example, a hospital may have high (or low) cost due to frequency of nonzero cost, amount of nonzero cost, or a combination of those. We give methods for decomposing hospital differences in total cost with zeros into components for probability of use (i.e., of nonzero cost) and for cost of use (mean of nonzero cost). The components multiply to total cost and quantify components on the same easily interpreted multiplicative scales. The methods are based on Bayesian hierarchical models and counterfactual arguments, with Markov chain Monte Carlo estimation. We used simulated data to illustrate use, interpretation, and visualization of the methods in diverse situations, and applied the methods to 30,024 patients at 57 US Veterans Administration hospitals to characterize outlier hospitals in one year cost of inpatient care following a cardiac procedure. Twenty eight percent of patients had zero cost. These methods are useful in providing insight into cost variation and outliers for planning future studies or interventions.     

Dietary fat increases energy intake across the range of typical consumption in the United States

Objective: The fat content of a diet has been shown to affect total energy intake, but controlled feeding trials have only compared very high (40% of total calories) fat diets with very low (20% of total calories) fat diets. This study was designed to measure accurately the voluntary food and energy intake over a range of typical intake for dietary fat. Methods and Procedures: Twenty‐two non‐obese subjects were studied for 4 days on each of three diets, which included core foods designed to contain 26, 34, and 40% fat, respectively of total calories and ad lib buffet foods of similar fat content. All diets were matched for determinants of energy density except dietary fat. Subjects consumed two meals/day in an inpatient unit and were provided the third meal and snack foods while on each diet. All food provided and not eaten was measured by research staff. Results: Voluntary energy intake increased significantly as dietary fat content increased (P = 0.008). On the 26% dietary fat treatment, subjects consumed 23.8% dietary fat (core and ad lib foods combined) and 2,748 ± 741 kcal/day (mean ± s.d.); at 34% dietary fat, subjects consumed 32.7% fat and 2,983 ± 886 kcal/day; and at 40% dietary fat subjects consumed 38.1% fat and 3,018 ± 963 kcal/day. Discussion: These results show that energy intake increases as dietary fat content increases across the usual range of dietary fat consumed in the United States. Even small reductions in dietary fat could help in lowering total energy intake and reducing weight gain in the population.     

The highly conserved nuclear lamin Ig-fold binds to PCNA: its role in DNA replication

This study provides insights into the role of nuclear lamins in DNA replication. Our data demonstrate that the Ig-fold motif located in the lamin C terminus binds directly to proliferating cell nuclear antigen (PCNA), the processivity factor necessary for the chain elongation phase of DNA replication. We find that the introduction of a mutation in the Ig-fold, which alters its structure and causes human muscular dystrophy, inhibits PCNA binding. Studies of nuclear assembly and DNA replication show that lamins, PCNA, and chromatin are closely associated in situ. Exposure of replicating nuclei to an excess of the lamin domain containing the Ig-fold inhibits DNA replication in a concentration-dependent fashion. This inhibitory effect is significantly diminished in nuclei exposed to the same domain bearing the Ig-fold mutation. Using the crystal structures of the lamin Ig-fold and PCNA, molecular docking simulations suggest probable interaction sites. These findings also provide insights into the mechanisms underlying the numerous disease-causing mutations located within the lamin Ig-fold.     

Transduction of the scorpion toxin maurocalcine into cells. Evidence that the toxin crosses the plasma membrane

Maurocalcine (MCa) is a 33-amino-acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. External application of MCa to cultured myotubes is known to produce Ca2+ release from intracellular stores. MCa binds directly to the skeletal muscle isoform of the ryanodine receptor, an intracellular channel target of the endoplasmic reticulum, and induces long lasting channel openings in a mode of smaller conductance. Here we investigated the way MCa proceeds to cross biological membranes to reach its target. A biotinylated derivative of MCa was produced (MCa(b)) and complexed with a fluorescent indicator (streptavidine-cyanine 3) to follow the cell penetration of the toxin. The toxin complex efficiently penetrated into various cell types without requiring metabolic energy (low temperature) or implicating an endocytosis mechanism. MCa appeared to share the same features as the so-called cell-penetrating peptides. Our results provide evidence that MCa has the ability to act as a molecular carrier and to cross cell membranes in a rapid manner (1-2 min), making this toxin the first demonstrated example of a scorpion toxin that translocates into cells.     

Reduced mobilization of Rev-responsive element-deficient lentiviral vectors

Infection of cells transduced with a lentiviral vector by human immunodeficiency virus (HIV) could lead to packaging of the lentiviral vector RNA into HIV particles and unintended transfer of the vector. To prevent this, the Rev-responsive element (RRE) of an HIV-1 vector was functionally replaced by a heterologous RNA element (MS2). Providing Rev fused to an MS2 binding protein allowed efficient vector production. Mobilization of the vector from infected target cells was below the level of detection and at least 10(3)- to 10(4)-fold lower than for the RRE-containing vector. Thus, RRE-deficient lentiviral vectors provide a novel approach to reduce the risk of vector mobilization.     

Effect of sterols on the permeability of alcohol-treated red beet tissue

Alcohols and hydrogen peroxide altered the permeability of membranes of Beta vulgaris root cells. Generally alcohols increased the permeability of membranes without going through an induction period except methanol which required a 10- to 15-hour induction period. The membrane effect of methanol could be inhibited with CaCl₂, cholesterol, β-sitosterol, and stigmasterol. Cholesterol was the most effective inhibitor, followed by β-sitosterol and stigmasterol; and at the same concentration, the sterols were more effective than CaCl₂, the classic membrane stabilizer.