RNA-directed construction of structurally complex and active ligase ribozymes through recombination

RNA-directed recombination can be used to catalyze a disproportionation reaction among small RNA substrates to create new combinations of sequences. But the accommodation of secondary and tertiary structural constraints in the substrates by recombinase ribozymes has not been explored. Here, we show that the Azoarcus group I intron can recombine oligoribonucleotides to construct class I ligase ribozymes, which are catalytically active upon synthesis. The substrate oligonucleotides, ranging in size from 58 to 104 nucleotides (nt), along with the 152-nt ligase ribozymes they reconstitute, can contain significant amounts of secondary structure. However, substrate recognition by the Azoarcus ribozyme depends on the existence of a single accessible CAU triplet for effective recombination. A biphasic temperature reaction profile was designed such that the sequential recombination/ligation events could take place in a thermocycler without human intervention. A temperature-dependent pH shift of the reaction buffer contributes to the success of the net reaction. When the substrate for the ligase ribozyme is introduced into the reaction mixture, as much as 11% can be observed being converted to product by the recombined ligase in the same reaction vessel. Recombination followed by ligation can also occur under isothermal conditions at 37 degrees C. Tertiary structure formation of the ligase upon construction can provide some protection from cleavage by the Azoarcus ribozyme when compared to the constituent substrates. These data suggest that RNA-directed recombination can, in fact, articulate complex ribozymes, and that there are logical rules that can guide the optimal placement of the CAU recognition sequence.     

Ecogeographic size variations in Sifakas: a test of the resource seasonality and resource quality hypotheses

Ecogeographic size variations have been documented in some but not all sifakas. Few morphometric or body weight data have been available for two critically endangered subspecies of diademed sifakas: Perrier's sifakas (Propithecus diadema perrieri) and silky sifakas (Propithecus diadema candidus). The objectives of our study were to determine size variations in sifakas and if these variations are related to resource quality and/or resource seasonality. P. d. perrieri and P. d. candidus were captured, weighed, and measured in northern Madagascar. Body weights and morphometrics were compared with other subspecies of diademed sifakas and indris (Indri indri). Differences in body weights and morphometrics between taxa are particularly pronounced for P. d. perrieri compared to P. d. diadema, P. d. edwardsi, and I. indri. Most morphometrics varied in comparisons between P. d. candidus and the other Indriidae (P. d. diadema, P. d. edwardsi, and I. indri). Average body size in sifakas is positively correlated with annual rainfall and negatively correlated with length of dry season. Sifaka body size is not correlated with protein-to-fiber ratios. Thus, size variations in sifakas are related to resource seasonality rather than resource quality. The relationships between the temporal availability of food resources and sifaka body size reflect complex and regionally varying causalities. Detailed, longitudinal information on the ecological factors underlying food selection and nutrient requirements in sifakas are needed to determine the relationship between ecogeographic variables and body size in sifakas.     

Drosophila muscle regulation characterized by electron microscopy and three-dimensional reconstruction of thin filament mutants

Wild-type and mutant thin filaments were isolated directly from "myosinless" Drosophila indirect flight muscles to study the structural basis of muscle regulation genetically. Negatively stained filaments showed tropomyosin with periodically arranged troponin complexes in electron micrographs. Three-dimensional helical reconstruction of wild-type filaments indicated that the positions of tropomyosin on actin in the presence and absence of Ca(2+) were indistinguishable from those in vertebrate striated muscle and consistent with a steric mechanism of regulation by troponin-tropomyosin in Drosophila muscles. Thus, the Drosophila model can be used to study steric regulation. Thin filaments from the Drosophila mutant heldup(2), which possesses a single amino acid conversion in troponin I, were similarly analyzed to assess the Drosophila model genetically. The positions of tropomyosin in the mutant filaments, in both the Ca(2+)-free and the Ca(2+)-induced states, were the same, and identical to that of wild-type filaments in the presence of Ca(2+). Thus, cross-bridge cycling would be expected to proceed uninhibited in these fibers, even in relaxing conditions, and this would account for the dramatic hypercontraction characteristic of these mutant muscles. The interaction of mutant troponin I with Drosophila troponin C is discussed, along with functional differences between troponin C from Drosophila and vertebrates.     

Should symptomatic menopausal women be offered hormone therapy?

Many physicians remain uncertain about prescribing hormone therapy for symptomatic women at the onset of menopause. The American Society for Reproductive Medicine (ASRM) convened a multidisciplinary group of healthcare providers to discuss the efficacy and risks of hormone therapy for symptomatic women, and to determine whether it would be appropriate to treat women at the onset of menopause who were complaining of menopausal symptoms. MAJOR FINDINGS: Numerous controlled clinical trials consistently demonstrate that hormone therapy, administered via oral, transdermal, or vaginal routes, is the most effective treatment for vasomotor symptoms. Topical vaginal formulations of hormone therapy should be preferred when prescribing solely for the treatment of symptoms of vulvar and vaginal atrophy. Data from the Women's Health Initiative indicate that the overall attributable risk of invasive breast cancer in women receiving estrogen plus progestin was 8 more cases per 10,000 women-years. No increased risk for invasive breast cancer was detected for women who never used hormone therapy in the past or for those receiving estrogen only. Hormone therapy is not effective for the treatment of cardiovascular disease and that the risk of cardiovascular disease with hormone therapy is principally in older women who are considerably postmenopause. CONCLUSIONS: Healthy symptomatic women should be offered the option of hormone therapy for menopausal symptoms. Symptom relief with hormone therapy for many younger women (at the onset of menopause) with menopausal symptoms outweighs the risks and may provide an overall improvement in quality of life. Hormone therapy should be individualized for symptomatic women. This involves tailoring the regimen and dose to individual needs.     

Detection of high levels of recombination generated during PCR amplification of RNA templates

Recombination during the PCR amplification of DNA templates can be a serious problem for those seeking to genotype heterogeneous populations, yet a boon to those seeking to enhance variation during in vitro evolution. Here, the extent to which PCR generates chimeric full-length products was estimated using a powerful restriction fragment-length polymorphism (RFLP) assay involving the use of fluorescently labeled PCR primers. Three different RNA-encoding DNA templates were assayed: (i) one for a group I ribozyme, (ii) one for a 16S ribosomal RNA (rRNA), and (iii) one for a messenger RNA (mRNA). In all cases, the observed frequency of chimeric PCR products exceeded 20%, and longer templates appear to produce more chimeric products. Although two of these templates have the potential to form secondary structures during the PCR, this tendency does not seem to heighten recombination frequency. These results corroborate previous studies that show that the production of chimeras can be best attenuated to a certain extent by varying the extension times in PCR.     

Kinesiological research: The use of surface electromyography for assessing the effects of spinal manipulation

Decreasing an elevated muscle tone is an often cited benefit of spinal manipulation. Spinal manipulation is theorized to disrupt an assumed pain-spasm-pain cycle that sufferers of low back pain may be experiencing. The current research has mostly investigated the short term influence of a single spinal manipulation on paraspinal muscle activity either at rest (e.g. standing or prone) or during simple movements (e.g. forward bend). The higher quality experiments to date have typically reported both reductions in muscle activity during lying prone or during the fully flexed position of forward bend. The only study measuring the long term influence of spinal manipulation has failed to document any change in muscle activity as measured with surface electromyography. Both manually delivered manipulations and manipulations delivered via a mechanical adjusting device have been associated with changes in muscle activation. Changes in muscle activity at muscles distant from the spinal joints manipulated (e.g. muscles in the upper limbs) have been documented following a single spinal manipulation however rather than the typical reduction in muscle activity an increase in resting activation has been reported. The state of muscle dysfunction (e.g. palpably tender or subjectively taut) may be a factor in achieving a myoelectric response to spinal manipulation. Currently, the clinical significance of short term changes in electromyographic amplitude following manipulation is unknown.     

Phenotypic Characterization of Prostate Cancer LNCaP Cells Cultured within a Bioengineered Microenvironment

Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment.