The conserved axial ligand methionine 121 from Pseudomonas aeruginosa azurin (Az) has been replaced by isostructural unnatural amino acid analogues, oxomethionine (OxM), difluoromethionine (DFM), ...trifluoromethionine (TFM), selenomethionine (SeM), and norleucine (Nle) using expressed protein ligation. The replacements resulted in <6 nm shifts in the S(Cys)−Cu charge transfer (CT) band in the electronic absorption spectra and <8 gauss changes in the copper hyperfine coupling constants (A ∥) in the X-band electron paramagnetic resonance spectra, suggesting that isostructural replacement of Met resulted in minimal structural perturbation of the copper center. The slight blue shifts of the CT band follow the trend of stronger electronegativity of the ligands. This trend is supported by 19F NMR studies of the fluorinated methionine analogues. However, the order of A ∥ differs, suggesting additional factors influencing A ∥. In contrast to the small changes in the UV−vis and EPR spectra, a large variation of >227 mV in reduction potential was observed for the series of variants reported here. Additionally, a linear correlation was established between the reduction potentials and hydrophobicity of the variants. Extension of this analysis to other type 1 copper-containing proteins reveals a linear correlation between change in hydrophobicity and change in reduction potential, independent of the protein scaffold, experimental conditions, measurement techniques, and steric modifications. This analysis has also revealed for the first time high and low potential states for type 1 centers, and the difference may be attributable to destabilization of the protein fold by disruption of hydrophobic or hydrogen bonding interactions that stabilize the type 1 center.
Targeting specific neuronal cell types is a major challenge for unraveling their function and utilizing specific cells for gene therapy strategies. Viral vector tools are widely used to target ...specific cells or circuits for these purposes. Here, we use viral vectors with short promoters of neuropeptide genes to target distinct neuronal populations in the hypothalamus of rats and mice. We show that lowering the amount of genomic copies is effective in increasing specificity of a melanin-concentrating hormone promoter. However, since too low titers reduce transduction efficacy, there is an optimal titer for achieving high specificity and sufficient efficacy. Other previously identified neuropeptide promoters as those for oxytocin and orexin require further sequence optimization to increase target specificity. We conclude that promoter-driven viral vectors should be used with caution in order to target cells specifically.
The proximal tubule contains the highest expression of angiotensinogen mRNA and protein within the kidney and plays a vital role in the renal renin-angiotensin system. To study the regulation of ...angiotensinogen expression in the kidney in more detail, the proximal tubule needs to be accurately isolated from the rest of the nephron and separated into its three segments. The purpose of this study was to design a novel protocol using specific markers for the separation of proximal tubule cells into the three proximal tubule segments and to determine angiotensinogen expression in each segment. Kidneys were removed from C57BL/6J mice. The proximal tubules were aspirated from region of a Percoll gradient solution of the appropriate density. The proximal tubule was then separated into its three segments using segment-specific membrane proteins, after which each segment was characterized by a different specific marker (sodium-glucose transporter 2 for Segment 1; carbonic anhydrase IV for Segment 2; ecto-adenosine triphosphatase for Segment 3). The isolation of proximal tubules into three segments was successful, and angiotensinogen mRNA in Segment 2 and 3 and angiotensinogen protein in all three segments were confirmed. This protocol will be helpful for future studies of the detailed mechanisms of the intrarenal renin-angiotensin system.
Two questions important to the success in metalloenzyme design are how to attach or anchor metal cofactors inside protein scaffolds and in what way such positioning affects enzymatic properties. We ...have previously reported a dual anchoring method to position a nonnative cofactor, MnSalen (1), inside the heme cavity of apo sperm whale myoglobin (Mb) and showed that the dual anchoring can increase both the activity and enantioselectivity over single anchoring methods, making this artificial enzyme an ideal system to address the above questions. Here, we report systematic investigations of the effect of different covalent attachment or anchoring positions on reactivity and selectivity of sulfoxidation by the MnSalen-containing Mb enzymes. We have found that changing the left anchor from Y103C to T39C has an almost identical effect of increasing rate by 1.8-fold and increasing selectivity by +15% for S, whether the right anchor is L72C or S108C. At the same time, regardless of the identity of the left anchor, changing the right anchor from S108C to L72C increases the rate by 4-fold and selectivity by +66%. The right anchor site was observed to have a greater influence than the left anchor site on the reactivity and selectivity in sulfoxidation of a wide scope of other ortho-, meta- and para-substituted substrates. The 1·Mb(T39C/L72C) showed the highest reactivity (TON up to 2.32 min–1) and selectivity (ee % up to 83%) among the different anchoring positions examined. Molecular dynamic simulations indicate that these changes in reactivity and selectivity may be due to the steric effects of the linker arms inside the protein cavity. These results indicate that small differences in the anchor positions can result in significant changes in reactivity and enantioselectivity, probably through steric interactions with substrates when they enter the substrate-binding pocket, and that the effects of right and left anchor positions are independent and additive in nature. The finding that the anchoring arms can influence both the positioning of the cofactor and steric control of substrate entrance will help design better functional metalloenzymes with predicted catalytic activity and selectivity.
Rats that have restricted access to food at a fixed time point of the circadian phase display high levels of food anticipatory activity (FAA). The orexigenic hormone ghrelin has been implicated in ...the regulation of FAA. However, it is not known via which brain area ghrelin exerts this effect. Growth hormone secretagogue receptor 1a (GHS-R1a) is highly expressed in the hypothalamus, including the dorsomedial hypothalamus (DMH) and the ventromedial hypothalamus (VMH). These two hypothalamic areas have been reported to play a role in FAA.
To examine the role of GHS-R1a signaling in the DMH and VMH in FAA.
Adeno-associated virus expressing a shRNA directed against GHS-R1a was used to establish local knockdown of GHS-R1a in the DMH and VMH in rats. Rats were subsequently subjected to a restricted feeding schedule (RFS).
Under ad libitum conditions, knockdown of GHS-R1a in the VMH increased food intake and body weight gain. In addition, GHS-R1a knockdown in VMH and DMH reduced body temperature and running wheel activity (RWA). When rats were subjected to a RFS, the main effect of GHS-R1a knockdown in both DMH and VMH was a decrease in RWA and an attenuation of body weight loss. Rats with knockdown of GHS-R1a in DMH and VMH showed a delay in onset of FAA. In addition, GHS-R1a knockdown in DMH resulted in a reduction of FAA amplitude.
This is the first study to investigate the effect of local hypothalamic knockdown of GHS-R1a on FAA. Our results implicate hypothalamic GHS-R1a signaling in the regulation of FAA. Nevertheless, some FAA remained, suggesting that a distributed network of brain areas and signaling pathways is involved in the development of FAA.
The outdoor lifetime and performance of organic photovoltaics (OPVs) using boron subphthalocyanine (BsubPc) derivatives as electron-accepting materials is presented. The protocols followed are based ...on the most advanced level of outdoor testing established by the International Summit on OPV Stability (ISOS). The stability of each BsubPc is compared using three different sets of encapsulated planar heterojunction OPVs, with each set containing a different BsubPc as the electron-accepting layer. The performance and stability of each set is tested outdoors using an epoxy glue and a glass coverslip as protection from the ambient environment. Outdoor testing continued until the OPVs reached 80 or 50% of their original power conversion efficiency, as determined by frequent indoor characterization. OPVs utilizing chloro-BsubPc are shown to exhibit the highest stability and performance, while the stability of the other two BsubPc derivatives is reduced presumably as a result of their phenoxy or phenyl functionalization in the molecular axial positions. The established structure–property relationship and guidance for the design of future compounds for application in planar heterojunction OPVs are contrary to, and could not have been anticipated from, time zero laboratory testing.
The study of patients with bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets. We have identified a patient with a chronic ...bleeding disorder expressing a homozygous P2RY(12) mutation, predicting an arginine to cysteine (R122C) substitution in the G-protein-coupled P2Y(12) receptor. This mutation is found within the DRY motif, which is a highly conserved region in G-protein-coupled receptors (GPCRs) that is speculated to play a critical role in regulating receptor conformational states.
To determine the functional consequences of the R122C substitution for P2Y(12) function.
We performed a detailed phenotypic analysis of an index case and affected family members. An analysis of the variant R122C P2Y(12) stably expressed in cells was also performed.
ADP-stimulated platelet aggregation was reduced as a result of a significant impairment of P2Y(12) activity in the patient and family members. Cell surface R122C P2Y(12) expression was reduced both in cell lines and in platelets; in cell lines, this was as a consequence of agonist-independent internalization followed by subsequent receptor trafficking to lysosomes. Strikingly, members of this family also showed reduced thrombin-induced platelet activation, owing to an intronic polymorphism in the F2R gene, which encodes protease-activated receptor 1 (PAR-1), that has been shown to be associated with reduced PAR-1 receptor activity.
Our study is the first to demonstrate a patient with deficits in two stimulatory GPCR pathways that regulate platelet activity, further indicating that bleeding disorders constitute a complex trait.
Objectives. To determine the effects on balance and gait of a Wii-Fit program compared to a walking program in subjects with mild Alzheimer’s dementia (AD). Methods. A prospective randomized (1 : 1) ...pilot study with two intervention arms was conducted in an assisted living facility with twenty-two mild AD subjects. In both groups the intervention occurred under supervision for 30 minutes daily, five times a week for eight weeks. Repeated measures ANOVA and paired t-tests were used to analyze changes. Results. Both groups showed improvement in Berg Balance Scale (BBS), Tinetti Test (TT) and Timed Up and Go (TUG) over 8 weeks. However, there was no statistically significant difference between the groups over time. Intragroup analysis in the Wii-Fit group showed significant improvement on BBS (P=0.003), and TT (P=0.013). The walking group showed a trend towards improvement on BBS (P=0.06) and TUG (P=0.07) and significant improvement in TT (P=0.006). Conclusion. This pilot study demonstrates the safety and efficacy of Wii-Fit in an assisted living facility in subjects with mild AD. Use of Wii-Fit resulted in significant improvements in balance and gait comparable to those in the robust monitored walking program. These results need to be confirmed in a larger, methodologically sound study.
Placental health is critical to fetal growth and maternal health during gestation. However, investigating placental flow in an
isolated system where inflow is independently controlled has yet to be ...developed in the rat. Here, we describe a novel technique, isolated perfused placenta technique that allows for analysis of placental pressure outflow pressure, placental flow in rats at gestational day 20. Using this method, we successfully perfused placentas from dams and were able to observe increases in outflow pressure and flow as the inflow pressure to the placenta was increased in a step wise fashion. This method will help to advance the functional analysis of placental flow and therefore placental resistance and efficiency.
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