Reciprocal Fusions of Two Genes in the Formaldehyde Detoxification Pathway in Ciliates and Diatoms

doi:10.1093/molbev/msi151 Mol. Biol. Evol.      

Inhibition of 5,10-methenyltetrahydrofolate synthetase

The interaction of 5-formyltetrahydrofolate analogs with murine methenyltetrahydrofolate synthetase (MTHFS) was investigated using steady-state kinetics, molecular modeling, and site-directed mutagenesis. MTHFS catalyzes the irreversible cyclization of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Folate analogs that cannot undergo the rate-limiting step in catalysis were inhibitors of murine MTHFS. 5-Formyltetrahydrohomofolate was an effective inhibitor of murine MTHFS (K(i)=0.7 microM), whereas 5-formyl,10-methyltetrahydrofolate was a weak inhibitor (K(i)=10 microM). The former, but not the latter, was slowly phosphorylated by MTHFS. 5-Formyltetrahydrohomofolate was not a substrate for murine MTHFS, but was metabolized when the MTHFS active site Y151 was mutated to Ala. MTHFS active site residues do not directly facilitate N10 attack on the on the N5-iminium phosphate intermediate, but rather restrict N10 motion around N5. Inhibitors specifically designed to block N10 attack appear to be less effective than the natural 10-formyltetrahydrofolate polyglutamate inhibitors.     

Mechanisms and kinetics of procathepsin D activation.

In vitro, procathepsin D is activated to pseudocathepsin D by incubation at low pH. To investigate the mechanism of this activation, recombinant human procathepsin D and two mutants were generated in a baculovirus expression system. One mutant carried a point mutation within the catalytic domain, which resulted in a catalytically inactive enzyme form (D77A). The other carried a point mutation within the propeptide, which prevented activation by processing at the 'autoproteolysis-site' (L26P). Neither mutant is capable of processing itself to form pseudocathepsin D, and L26P is not able to process D77A. Despite the inability of L26P to cleave either its own or a wild-type prosequence, it did exhibit activity against a synthetic peptide substrate. The ability of intact precursor (zymogen) to cleave a peptide, but not a protein substrate, offers new insights into the mechanism of inhibition by the propeptide. Mature cathepsin D can process the inactive D77A mutant to the pseudoform, demonstrating that processed species are capable of cleaving zymogen molecules in an intermolecular interaction. In addition, kinetic studies provide evidence for a two-phase mechanism for the conversion of procathepsin D to pseudocathepsin D, one phase where the first molecules of pseudocathepsin D are formed at a low rate and a second phase where the process is autocatalytically accelerated by newly formed pseudocathepsin D molecules. Finally, with the help of the mutants L26P and D77A it was observed that at least two additional proteinase activities, found in conditioned media from insect cell culture, are capable of activating procathepsin D by cleaving it within the proregion. This observation suggests that there are likely to be multiple proteinases in the extracellular matrix that are capable of activating procathepsin D, thereby triggering the second autocatalytic phase. This may also be important for solid tumors, where the presence of cathepsin D has been correlated with tumor growth and invasion.     

The Safety of Ultrasound Guided Tenotomy and Debridement for Upper and Lower Extremity Tendinopathies: A Retrospective Study

BackgroundUltrasound guided tenotomy (USGT) is a minimally invasive treatment option for patients with chronic tendinopathy. There are conflicting findings in the literature with some studies reporting severe complications and others reporting none. This variability is likely due to the small sample sizes of previous studies. We aimed to evaluate the risks associated with USGT and outcomes across multiple tendinopathy/fasciopathy sites in a large clinical sample.MethodsPatients who had USGT were identified by retrospective review of charts. Complications, satisfaction, and outcomes (pain, quality of life) were assessed at baseline prior to the procedure (outcomes only), short-term follow up, and long term follow up.ResultsA total of 262 patients with 289 procedures were identified through chart review. There was a low complication rate of 0.7% including one superficial wound infection and one case of wound hypersensitivity. The majority of patients reported improvement in pain by short-term and long-term follow-up and improvement in function by long-term follow-up. The majority of responders reported being either ‘very satisfied’ or ‘somewhat satisfied’ with the procedure at short-term follow-up.ConclusionThis study found that USGT is a safe procedure with a low complication rate in a heterogeneous sample. Study findings provide preliminary evidence on the utility of USGT to reduce pain and improve function with a high rate of patient satisfaction.Level of Evidence: IV     

Evidence for panmixia despite barriers to gene flow in the southern African endemic, <Emphasis Type="Italic">Caffrogobius caffer</Emphasis> (Teleostei: Gobiidae)

Background  

Oceanography and life-history characteristics are known to influence the genetic structure of marine species, however the relative role that these factors play in shaping phylogeographic patterns remains unresolved. The population genetic structure of the endemic, rocky shore dwelling Caffrogobius caffer was investigated across a known major oceanographic barrier, Cape Agulhas, which has previously been shown to strongly influence genetic structuring of South African rocky shore and intertidal marine organisms. Given the variable and dynamic oceanographical features of the region, we further sought to test how the pattern of gene flow between C. caffer populations is affected by the dominant Agulhas and Benguela current systems of the southern oceans.     

Fine root biomass estimates from minirhizotron imagery in a shrub ecosystem exposed to elevated CO2

Fine root biomass and C content are critical components in ecosystem C models, but they cannot be directly determined by minirhizotron techniques, and indirect methods involve estimating 3-dimensional values (biomass/ soil volume) from 2-dimensional measurements. To estimate biomass from minirhizotron data, a conversion factor for length to biomass must be developed, and assumptions regarding depth of view must be made. In a scrub-oak ecosystem in central Florida, USA, root length density (RLD) was monitored for 10 years in a CO₂ manipulation experiment using minirhizotron tubes. In the seventh year of the study, soil cores were removed from both ambient and elevated CO₂ chambers. Roots from those cores were used to determine specific root length values (m/g) that were applied to the long-term RLD data for an estimation of root biomass over 10 years of CO₂ manipulation. Root length and biomass estimated from minirhizotron data were comparable to determinations from soil cores, suggesting that the minirhizotron biomass model is valid. Biomass estimates from minirhizotrons indicate the <0.25 mm diameter roots accounted for nearly 95% of the total root length in 2002. The long-term trends for this smallest size class (<0.25 mm diameter) mirrored the RLD trends closely, particularly in relation to suspected root closure in this system. Elevated CO₂ did not significantly affect specific root length as determined by the soil cores. A significant treatment effect indicated smallest diameter fine roots (<0.25 mm) were greater under elevated CO₂ during the early years of the study and the largest (2–10 mm) had greater biomass under elevated CO₂ during the later years of the study. Overall, this method permits long-term analysis of the effects of elevated CO₂ on fine root biomass accumulation and provides essential information for carbon models.