Tissue culture of Chrysosplenium americanum and its potential for flavonoid production

Chrysosplenium americanum (Saxifragaceae) accumulates a variety of partially O-methylated flavonol glucosides. Because of the semi-aquatic nature of this plant and its extensive contamination with endogenous organisms, the initiation of shoot and callus cultures could only be achieved after (a) using a special surface sterilization procedure, (b) production of new shoots from initial explants, and (c) selective elimination of organogenic structures during several subcultures. HPLC analysis of the cultured tissues established the presence of a number of flavonoids characteristic of the intact plant, though in lower and variable concentrations. However, shoot and callus cultures exhibited flavonoid profiles similar to those of the intact leaves and roots, respectively.Abbreviations BAP benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA 3-indoleacetic acid - NAA 1-naphtaleneacetic acid - HPLC high pressure liquid chromatography     

Positive influence of tetracycline on human fetal kidney in serum-free organ culture

Summary Human fetal kidney explants can be maintained during 5 days in Leibovitz’s L15, a basic serum-free medium. Because culture conditions are minimal for growth and differentiation, DNA synthesis drastically decreases during the first 48 h, but stabilizes thereafter. The addition of insulin plus transferrin significantly restores this important cellular function in kidneys of fetuses younger than 16 wk. However, renal explants from older fetuses are more difficult to culture: they respond less to growth factors and are more prone to necrosis. The objective of this study was to verify the influence of tetracycline, an antibiotic with anti-collagenase potential, on cultured kidney explants aged 17 to 20 wk. The addition of 20μg/ml tetracycline did not influence DNA synthesis nor the effectiveness of insulin plus transferrin on cell proliferation. Nor did it change the activities of alkaline phosphatase and γ-glutamyltransferase, two enzymic markers of brush border differentiation. After 5 days in L15 alone, explants often showed necrosis and an important reduction in both weight and volume. Insulin plus transferrin significantly restored these parameters to control values observed at Day 0, but evidence of necrosis was still present. Tetracycline alone markedly reduced explant necrosis resulting in a significant increase in weight and volume. The effectiveness of insulin plus transferrin on explant morphometry was not improved when tetracycline was added as third factor. These results indicate that insulin plus transferrin restores explant mass through cell proliferation, whereas tetracycline does so possibly through a reduction in extracellular matrix degradation. The two effects are not additive in cultured mid-term fetal kidneys.     

Streptomycin-induced conformational changes in the 70-S bacterial ribosome.

Conformational alterations induced by streptomycin in the bacterial ribosome have been investigated using as probes, ethidium bromide, N-[14C]ethylmaleimide and a spin label nitroxide analog of N-ethylmaleimide. 1. The binding of the antibiotic to the ribosome does not affect the reactivity of sulfhydryl groups towards N-ethylmaleimide. 2. The motional freedom of spin labels bound to ribosomal proteins S1 and S18 is increased but it is hardly affected at other labeled sites. This observation suggests that the binding of streptomycin causes a local loosening of the ribosomal structure. 3. Ribosomes are found to bind less ethidium bromide in the presence of streptomycin, which suggests that the binding of streptomycin decreases the degree of organization of ribosomal RNA.     

Modification of RBC properties by an autoantibody. Binding to RBC senescent antigen.

It has been reported that natural antibodies alter aging RBCs, so increasing their vulnerability for removal from the blood stream. Results presented indicate that binding of anti-Gal, a natural antibody, to erythrocytes increases RBC rigidity in terms of microviscosity and deformability. Removal of RBCs from circulation could, at least in part, depend on these alterations.     

A Bayesian Two‐Part Latent Class Model for Longitudinal Medical Expenditure Data: Assessing the Impact of Mental Health and Substance Abuse Parity

Summary In 2001, the U.S. Office of Personnel Management required all health plans participating in the Federal Employees Health Benefits Program to offer mental health and substance abuse benefits on par with general medical benefits. The initial evaluation found that, on average, parity did not result in either large spending increases or increased service use over the four‐year observational period. However, some groups of enrollees may have benefited from parity more than others. To address this question, we propose a Bayesian two‐part latent class model to characterize the effect of parity on mental health use and expenditures. Within each class, we fit a two‐part random effects model to separately model the probability of mental health or substance abuse use and mean spending trajectories among those having used services. The regression coefficients and random effect covariances vary across classes, thus permitting class‐varying correlation structures between the two components of the model. Our analysis identified three classes of subjects: a group of low spenders that tended to be male, had relatively rare use of services, and decreased their spending pattern over time; a group of moderate spenders, primarily female, that had an increase in both use and mean spending after the introduction of parity; and a group of high spenders that tended to have chronic service use and constant spending patterns. By examining the joint 95% highest probability density regions of expected changes in use and spending for each class, we confirmed that parity had an impact only on the moderate spender class.     

Probing the Huntingtin 1-17 Membrane Anchor on a Phospholipid Bilayer by Using All-Atom Simulations

Mislocalization and aggregation of the huntingtin protein are related to Huntington’s disease. Its first exon—more specifically the first 17 amino acids (Htt17)—is crucial for the physiological and pathological functions of huntingtin. It regulates huntingtin’s activity through posttranslational modifications and serves as an anchor to membrane-containing organelles of the cell. Recently, structure and orientation of the Htt17 membrane anchor were determined using a combined solution and solid-state NMR approach. This prompted us to refine this model by investigating the dynamics and thermodynamics of this membrane anchor on a POPC bilayer using all-atom, explicit solvent molecular dynamics and Hamiltonian replica exchange. Our simulations are combined with various experimental measurements to generate a high-resolution atomistic model for the huntingtin Htt17 membrane anchor on a POPC bilayer. More precisely, we observe that the single α-helix structure is more stable in the phospholipid membrane than the NMR model obtained in the presence of dodecylphosphocholine detergent micelles. The resulting Htt17 monomer has its hydrophobic plane oriented parallel to the bilayer surface. Our results further unveil the key residues interacting with the membrane in terms of hydrogen bonds, salt-bridges, and nonpolar contributions. We also observe that Htt17 equilibrates at a well-defined insertion depth and that it perturbs the physical properties—order parameter, thickness, and area per lipid—of the bilayer in a manner that could favor its dimerization. Overall, our observations reinforce and refine the NMR measurements on the Htt17 membrane anchor segment of huntingtin that is of fundamental importance to its biological functions.