Enhanced expression and purification of nucleotide-specific ribonucleases MC1 and Cusativin.

Combinations of ribonucleases (RNases) are commonly used to digest RNA into oligoribonucleotide fragments prior to liquid chromatography-mass spectrometry (LC-MS) analysis. The distribution of the RNase target sequences or nucleobase sites within an RNA molecule is critical for achieving a high mapping coverage. Cusativin and MC1 are nucleotide-specific endoribonucleases encoded in the cucumber and bitter melon genomes, respectively. Their high specificity for cytidine (Cusativin) and uridine (MC1) make them ideal molecular biology tools for RNA modification mapping. However, heterogenous recombinant expression of either enzyme has been challenging because of their high toxicity to expression hosts and the requirement of posttranslational modifications. Here, we present two highly efficient and time-saving protocols that overcome these hurdles and enhance the expression and purification of these RNases. We first purified MC1 and Cusativin from bacteria by expressing and shuttling both enzymes to the periplasm as MBP-fusion proteins in T7 Express lysY/IqE. coli strain at low temperature. The RNases were enriched using amylose affinity chromatography, followed by a subsequent purification via a C-terminal 6xHIS tag. This fast, two-step purification allows for the purification of highly active recombinant RNases significantly surpassing yields reported in previous studies. In addition, we expressed and purified a Cusativin-CBD fusion enzyme in P. pastoris using chitin magnetic beads. Both Cusativin variants exhibited a similar sequence preference, suggesting that neither posttranslational modifications nor the epitope-tags have a substantial effect on the sequence specificity of the enzyme.

Molecular thin films on solid surfaces: mechanisms of melting.

We use molecular dynamics simulations to study the melting of pentane and hexane monolayers adsorbed on the basal plane of graphite. For both of these systems, the temperature-dependent structures and the melting temperatures agree well with experiment. A detailed analysis reveals that a mechanism involving the promotion of molecules to the second layer underlies melting in these systems. In the second-layer promotion mechanism, a small fraction of molecules transition into the second layer around the melting temperature, leaving vacant space in the first layer to facilitate disordering. The second-layer promotion mechanism arises because of the weaker molecule-surface interaction in our study than that in previous studies. The weaker molecule-surface interaction is consistent with experimental temperature-programmed desorption studies.

Accelerated molecular dynamics of temperature-programed desorption.

We use accelerated molecular dynamics to simulate temperature-programed desorption (TPD) of n-pentane from the basal plane of graphite in the first atomistic simulations to probe TPD over laboratory time scales. Although the simulated TPD spectra agree with experiment, a detailed analysis reveals underlying kinetic phenomena that contrast the standard experimental interpretation and opens new possibilities for understanding molecular kinetics at solid surfaces.

Accelerated molecular dynamics simulation of the thermal desorption of n-alkanes from the basal plane of graphite.

We utilize accelerated molecular dynamics to simulate alkane desorption from the basal plane of graphite. Eight different molecules, ranging from n-pentane to n-hexadecane, are studied in the low coverage limit. Acceleration of the molecular dynamics simulations is achieved using two different methods: temperature acceleration and a compensating potential scheme. We find that the activation energy for desorption increases with increasing chain length. The desorption prefactor increases with chain length for molecules ranging from pentane to decane. This increase subsides and the value of the preexponential factor fluctuates about an apparently constant value for decane, dodecane, tetradecane, and hexadecane. These trends are consistent with data obtained in experimental temperature-programed desorption (TPD) studies. We explain the dependence of the preexponential factor on alkane chain length by examining conformational changes within the alkane molecules. For the shorter molecules, torsional motion is not activated over experimental temperature ranges. These molecules can be treated as rigid rods and their partial loss in translational and rotational entropies upon adsorption increases as chain length increases, leading to an increasing preexponential factor. At their typical TPD peak temperatures, torsions are activated in the longer adsorbed chain molecules to a significant extent which increases with increasing chain length, increasing the entropy of the adsorbed molecule. This increase counteracts the decrease in entropy due to a loss of translation and rotation, leading to a virtually constant prefactor.

Effects of oral dehydroepiandrosterone on bone density in young women with anorexia nervosa: a randomized trial.

Young women with anorexia nervosa (AN) have subnormal levels of dehydroepiandrosterone (DHEA) and estrogen that may be mechanistically linked to the bone loss seen in this disease. The purpose of this study was to compare the effects of a 1-yr course of oral DHEA treatment vs. conventional hormonal replacement therapy (HRT) in young women with AN. Sixty-one young women were randomly assigned to receive oral DHEA (50 mg/d) or HRT (20 micro g ethinyl estradiol/0.1 mg levonorgestrel). Anthropometric, nutrition, and exercise data were acquired every 3 months, and bone mineral density (BMD) and body composition were measured by dual energy x-ray absorptiometry (DXA) every 6 months over 1 yr. Serum samples were obtained for measurements of hormones, proresorptive cytokines, and bone formation markers, and urine was collected for determinations of bone resorption markers at each visit. In initial analyses, total hip BMD increased significantly and similarly (+1.7%) in both groups. Hip BMD increases were positively correlated with increases in IGF-I (r = 0.44; P = 0.030) and the bone formation marker, bone-specific alkaline phosphatase increased significantly only in the DHEA treatment group (P = 0.003). However, both groups gained significant amounts of weight over the year of therapy, and after controlling for weight gain, no treatment effect was detectable. There was no significant change in lumbar BMD in either group. Both bone formation markers, bone-specific alkaline phosphatase and osteocalcin, increased transiently at 6-9 months in those subjects receiving DHEA compared with the estrogen-treated group (P < 0.05). Both DHEA and HRT significantly reduced levels of the bone resorption markers, urinary N-telopeptides (P < 0.05). There was a positive correlation between changes in IGF-I and changes in weight, body fat determined by DXA, and estradiol for both groups. In addition, patients receiving DHEA exhibited improvement on three validated psychological instruments (Eating Attitudes Test, Anorexia Nervosa Subtest, and Spielberger Anxiety Inventory). Both DHEA and HRT had similar effects on hip and spinal BMD. Over the year of treatment, maintenance of both hip and spinal BMD was seen, but there was no significant increase after accounting for weight gain. Compared with HRT, DHEA appeared to have anabolic effects, evidenced by the positive correlation between increases in hip DXA measurements and IGF-I and significant increases in bone formation markers. Both therapies significantly decreased bone resorption. Replicating results from studies of the elderly, DHEA resulted in improvements in specific psychological parameters in these young women.

Physiologic regulators of bone turnover in young women with anorexia nervosa.

OBJECTIVE: To clarify the role of physiologic regulators of bone turnover in patients with anorexia nervosa (AN). STUDY DESIGN: Adolescent girls with AN (n = 61) had anthropometric, nutrition, and exercise data acquired, and bone mineral density (BMD) and body composition measured by dual energy x-ray absorptiometry. Serum samples were obtained for hormones, proresorptive cytokines, and bone formation markers, and urine for bone resorption markers. RESULTS: In bivariate correlation analyses, significant (P <.05) predictors of lumbar BMD included height, weight, and exercise. In multiple regression models, these significant relationships held, even after controlling for the duration of amenorrhea and AN. For total body BMD, the same positive predictors were found and percentage of body fat was a negative correlate. For hip BMD, exercise and weight were found to be positive predictors. Dehydroepiandrosterone sulfate (DHEAS) was inversely correlated with N-telopeptides (NTx), and insulin-like growth factor I (IGF-I) was directly correlated with osteocalcin. Proresorptive cytokine levels were low or undetectable. CONCLUSIONS: Exercise and weight were positive predictors of BMD. These data are the first to suggest a relationship between DHEAS and increased bone resorption in AN. IGF-I was correlated with bone formation indices. Low cytokine levels suggest that these factors do not mediate the increased bone resorption of AN.

Reduced primary antibody responses in a genetic animal model of depression.

OBJECTIVE: Clinical depression is associated with multiple abnormalities of immune function, including reduced virus-specific responses. This study tested the hypothesis that the Flinders Sensitive Line (FSL) rat, a promising genetic animal model of depression, would exhibit reductions in antigen-specific primary antibody responses to immunization. METHODS: FSL (N = 13) and control Flinders Resistant Line (FRL; N = 14) rats were immunized with the protein antigen keyhole limpet hemocyanin (KLH; 300 microg/kg), and KLH-specific immunoglobulin (Ig)M, IgG, IgG1, and IgG2a responses were measured before and 3, 5, 7, 11, and 14 days after immunization. In separate experiments, production of interferon-gamma (IFN-gamma) by cells from naive and KLH-immunized animals was measured in vitro to determine whether strain differences in antibody production might be associated with differential production of this regulatory cytokine. RESULTS: KLH-specific production of IgM (p <.01) and IgG2a (p <.05) was significantly reduced in the FSL rats compared with the FRL controls. There were no strain differences in IgG or IgG1 production. Although IFN-gamma production between the two strains was similar in naive animals, cells from KLH-immunized FSL rats produced significantly less IFN-gamma when stimulated with KLH in vitro than cells from KLH-immunized FRL controls (p =.01). CONCLUSIONS: This study extends previous reports of altered immune function in the FSL rats to include reduced in vivo antigen-specific antibody responses. Moreover, diminished production of IFN-gamma by KLH-primed lymphocytes may contribute to lower antibody production in these animals. Collectively, these data suggest deficiencies in type 1 T-helper cell-mediated immunity in the FSL rats.