Effects of retinoic acid on protein synthesis in cultured melanoma cells

Retinoic acid reduces the growth rate of mouse S91 melanoma cells in culture and increases the proportion of cells in the G₁ phase of the cell cycle. Because of the integral role protein synthesis has been shown to play in growth control we studied the effect of retinoic acid on the protein synthesis machinery with a cell-free system developed from the melanoma cells. This system was capable of translating endogenous mRNA, exogenous globin mRNA, and the synthetic template poly(U). Of the above activities of the protein synthesis system only the translation of endogenous mRNA was reduced significantly in the cell-free system prepared from retinoic acid-treated cells. Analyses of the amount and function of RNA revealed that treatment with retinoic acid leads to reductions in total RNA content, in the proportion of ribosomes in polysomes, in the amount of poly(A)RNA, and in the amount of polysome-associated mRNA. All these effects of retinoic acid contribute to the decrease in protein synthesis activity of treated cells. Two-dimensional electrophoresis anlaysis of L-[³⁵S]methionine-labeled proteins produced by untreated and treated cells revealed only a few quantitative differences. We suggest that retinoic acid-induced suppression of protein synthesis activity may be the cause for growth inhibition.     

Sites of action of D2O in intact and skinned crayfish muscle fibers

Summary The effect on tension development of replacing 90% of the H₂O of the bathing saline with D₂O was studied on intact single fibers, and on skinned fibers before and after the latter were treated so as to eliminate Ca-accumulation by the sarcoplasmic reticulum (SR). Excitation-contraction coupling (ECC) of intact fibers is not abolished, but is depressed by D₂O so that higher depolarizations are required to elicit a given tension. The reduction in tension at a given level of depolarization is not due to inhibition of the contractile system. The latter showed an enhanced Ca sensitivity; that is, skinned fibers respond to Ca concentrations that are 1–2 orders of magnitude smaller in D₂O than in H₂O saline. When bathed in D₂O saline, intact fibers or skinned fibers with functional SR can still accumulate and release Ca in sufficient quantities to allow repeated induction of maximum tensions. Relaxation is slowed in all three types of preparation, perhaps because of an increased affinity of troponin to Ca in D₂O salines.     

The restriction of zoonotic PERV transmission by human APOBEC3G

The human APOBEC3G protein is an innate anti-viral factor that can dominantly inhibit the replication of some endogenous and exogenous retroviruses. The prospects of purposefully harnessing such an anti-viral defense are under investigation. Here, long-term co-culture experiments were used to show that porcine endogenous retrovirus (PERV) transmission from pig to human cells is reduced to nearly undetectable levels by expressing human APOBEC3G in virus-producing pig kidney cells. Inhibition occurred by a deamination-independent mechanism, likely after particle production but before the virus could immortalize by integration into human genomic DNA. PERV inhibition did not require the DNA cytosine deaminase activity of APOBEC3G and, correspondingly, APOBEC3G-attributable hypermutations were not detected. In contrast, over-expression of the sole endogenous APOBEC3 protein of pigs failed to interfere significantly with PERV transmission. Together, these data constitute the first proof-of-principle demonstration that APOBEC3 proteins can be used to fortify the innate anti-viral defenses of cells to prevent the zoonotic transmission of an endogenous retrovirus. These studies suggest that human APOBEC3G-transgenic pigs will provide safer, PERV-less xenotransplantation resources and that analogous cross-species APOBEC3-dependent restriction strategies may be useful for thwarting other endogenous as well as exogenous retrovirus infections.     

Transglucosidic reactions of the Aspergillus niger family 3 beta-glucosidase: qualitative and quantitative analyses and evidence that the transglucosidic rate is independent of pH

The hydrolytic and transglucosidic reactions of the Aspergillus niger Family 3 beta-glucosidase were characterized. Michaelis-Menten plots of the rates of aglycone formation were normal (hyperbolic) at low [substrate]. However, at high [substrate] the rates decreased at pH below approximately 5.5 but increased at pH above approximately 5.5. Each decrease or increase took the form of a second hyperbola adjoining the first. Thin layer chromatography, gas-liquid chromatography, and NMR analyses indicated that the substrates became transglucosidic acceptors when present at high concentrations. When pNPGlc and cellobiose reacted as acceptors, the C6 hydroxyl of the non-reducing substrate component reacted to form beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl-p-nitrophenol and beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl-(1-4)-D-glucopyranose, respectively. The acceptor action accounted for the second adjoining hyperbolas. Rate equations were derived for the production of the aglycone and the transglucosidic intermediate, and these equations described the data very well. Hydrolytic Vmax {Vmax(h)}, hydrolytic Km {Km(h)}, transglucosidic Vmax {Vmax(t)}, and transglucosidic Km {Km(t)} values were obtained by non-linear regression analysis using these equations. Vmax(h) pH profiles were bell shaped with optima between pH 4 and 4.5 but the Vmax(t) values did not change substantially between pH 3 and 7. These differences in the pH profiles explain the decreasing and increasing adjoining hyperbolas since Vmax(t) is lower than Vmax(h) at pH less than approximately 5.5 but higher than Vmax(h) at pH greater than approximately 5.5. The reason for these pH effects is that the value of the hydrolytic rate constant (k3) decreases while the value of the transglucosidic rate constant (k4) does not change between pH 3 and 7. The study also showed that gentiobiose forms by an intermolecular reaction of the C6 hydroxyl of Glc rather than an intramolecular reaction and that an equatorial orientation of the C2 hydroxyl, the presence of a C6 primary hydroxyl and beta-linkages with oligosaccharide acceptors are important for acceptor reactivity.     

Salamander diversity alters stream macroinvertebrate community structure

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Expanding ART for treatment and prevention of HIV in South Africa: estimated cost and cost-effectiveness 2011-2050

Background

Antiretroviral Treatment (ART) significantly reduces HIV transmission. We conducted a cost-effectiveness analysis of the impact of expanded ART in South Africa.

Methods

We model a best case scenario of 90% annual HIV testing coverage in adults 15–49 years old and four ART eligibility scenarios: CD4 count <200 cells/mm³ (current practice), CD4 count <350, CD4 count <500, all CD4 levels. 2011–2050 outcomes include deaths, disability adjusted life years (DALYs), HIV infections, cost, and cost per DALY averted. Service and ART costs reflect South African data and international generic prices. ART reduces transmission by 92%. We conducted sensitivity analyses.

Results

Expanding ART to CD4 count <350 cells/mm³ prevents an estimated 265,000 (17%) and 1.3 million (15%) new HIV infections over 5 and 40 years, respectively. Cumulative deaths decline 15%, from 12.5 to 10.6 million; DALYs by 14% from 109 to 93 million over 40 years. Costs drop $504 million over 5 years and $3.9 billion over 40 years with breakeven by 2013. Compared with the current scenario, expanding to <500 prevents an additional 585,000 and 3 million new HIV infections over 5 and 40 years, respectively. Expanding to all CD4 levels decreases HIV infections by 3.3 million (45%) and costs by $10 billion over 40 years, with breakeven by 2023. By 2050, using higher ART and monitoring costs, all CD4 levels saves $0.6 billion versus current; other ART scenarios cost $9–194 per DALY averted. If ART reduces transmission by 99%, savings from all CD4 levels reach $17.5 billion. Sensitivity analyses suggest that poor retention and predominant acute phase transmission reduce DALYs averted by 26% and savings by 7%.

Conclusion

Increasing the provision of ART to <350 cells/mm3 may significantly reduce costs while reducing the HIV burden. Feasibility including HIV testing and ART uptake, retention, and adherence should be evaluated.     

Pressure dissociation of integration host factor-DNA complexes reveals flexibility-dependent structural variation at the protein-DNA interface

E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236–39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences with differing flexibility have interactions with IHF that compensate for unfavorable deformation energy. Time-resolved Förster resonance energy transfer (FRET) shows that global topologies are indistinguishable for three complexes with oligonucleotides of different flexibility. However, pressure perturbation shows that the volume change upon binding is smaller with increasing flexibility. We interpret these results in the context of Record and coworker's model for IHF binding (J. Mol. Biol. 310, 379–401, 2001). We propose that the volume changes reflect differences in hydration that arise from structural variation at IHF–DNA interfaces while the resulting energetic compensation maintains the same net binding energy.     

Mesenchymal Stem Cells Promote Mammosphere Formation and Decrease E-Cadherin in Normal and Malignant Breast Cells

Introduction

Normal and malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew, referred to as stem cells, or tumor initiating cells (TIC). These cells can be enriched by growth as “mammospheres” in three-dimensional cultures.

Objective

We tested the hypothesis that human bone-marrow derived mesenchymal stem cells (MSC), which are known to support tumor growth and metastasis, increase mammosphere formation.

Results

We found that MSC increased human mammary epithelial cell (HMEC) mammosphere formation in a dose-dependent manner. A similar increase in sphere formation was seen in human inflammatory (SUM149) and non-inflammatory breast cancer cell lines (MCF-7) but not in primary inflammatory breast cancer cells (MDA-IBC-3). We determined that increased mammosphere formation can be mediated by secreted factors as MSC conditioned media from MSC spheroids significantly increased HMEC, MCF-7 and SUM149 mammosphere formation by 6.4 to 21-fold. Mammospheres grown in MSC conditioned media had lower levels of the cell adhesion protein, E-cadherin, and increased expression of N-cadherin in SUM149 and HMEC cells, characteristic of a pro-invasive mesenchymal phenotype. Co-injection with MSC in vivo resulted in a reduced latency time to develop detectable MCF-7 and MDA-IBC-3 tumors and increased the growth of MDA-IBC-3 tumors. Furthermore, E-cadherin expression was decreased in MDA-IBC-3 xenografts with co-injection of MSC.

Conclusions

MSC increase the efficiency of primary mammosphere formation in normal and malignant breast cells and decrease E-cadherin expression, a biologic event associated with breast cancer progression and resistance to therapy.     

Modified inoculum for radiometric susceptibility testing ofMycobacterium tuberculosis

Two-hundred and fifteen isolates ofMycobacterium tuberculosis were evaluated with the BACTEC 460 radiometric method for susceptibility to isoniazid, rifampin, ethambutol, and streptomycin (SM); a revised protocol for inoculum preparation was used. Fresh clinical isolates were subcultured into 7H9 broth and then photometrically adjusted to the equivalent of a 0.5 McFarland standard, one-half the recommended inoculum density. This method produced an overall 98.3% correlation with a conventional agar method. The sensitivity of this procedure was good for all drugs tested except for the lowest concentration of SM (2 g/ml). Specificity was excellent for all drugs tested. After repeat testing, only four discrepancies were found, yielding a 99.8% correlation between the two systems. The time required for susceptibility tests averaged 4.6 days. This method for inoculum preparation effectively minimized the number of susceptibility tests exceeding the threshold value before the fourth day of incubation. This allowed for definite trends of the growth index values to become established before interpretation of results.