Independent intramolecular mediators of folding, activity, and inhibition for the Plasmodium falciparum cysteine protease falcipain-2

The Plasmodium falciparum cysteine protease falcipain-2 is a trophozoite hemoglobinase and potential antimalarial drug target. Unlike other studied papain family proteases, falcipain-2 does not require its prodomain for folding to active enzyme. Rather, folding is mediated by an amino-terminal extension of the mature protease. As in related enzymes, the prodomain is a potent inhibitor of falcipain-2. We now report further functional evaluation of the domains of falcipain-2 and related plasmodial proteases. The minimum requirement for folding of falcipain-2 and four related plasmodial cysteine proteases was inclusion of a 14-15-residue amino-terminal folding domain, beginning with a conserved Tyr. Chimeras of the falcipain-2 catalytic domain with extensions from six other plasmodial proteases folded normally and had kinetic parameters (k(cat)/K(m) 124,000-195,000 M(-1) s(-1)) similar to those of recombinant falcipain-2 (k(cat)/K(m) 120,000 M(-1) s(-1)), indicating that the folding domain is functionally conserved across the falcipain-2 subfamily. Correct folding also occurred when the catalytic domain was refolded with a separate prodomain-folding domain construct but not with an isolated folding domain peptide. Thus, the prodomain mediated interaction between the other two domains when they were not covalently bound. The prodomain-catalytic domain interaction was independent of the active site, because it was blocked by free inactive catalytic domain but not by the active site-binding peptide leupeptin. The folded catalytic domain retained activity after purification from the prodomain-folding domain construct (k(cat)/K(m) 168,000 M(-1) s(-1)), indicating that the folding domain is not required for activity once folding has been achieved. Activity was lost after nonreducing gelatin SDS-PAGE but not native gelatin PAGE, indicating that correct disulfide bonds are insufficient to direct appropriate folding. Our results identify unique features of the falcipain-2 subfamily with independent mediation of activity, folding, and inhibition.     

Data on the push–out bond strength of three different root canal treatment sealers

It is of interest to document data on the push – out bond strength of three different root canal treatment sealers such as MTA Fillapex (MTA based), AH plus (Epoxy Resin based) and Apexit plus (Calcium hydroxide based). Forty-five freshly extracted human maxillary central incisors with closed apices were selected randomly. All the teeth were sectioned at cement-enamel junction using a diamond disc before starting the root canal preparation to obtain root length of 12 mm. All teeth were instrumented using ProTaper rotary instruments. 5.25% sodium hypochlorite was used for irrigation between instrumentation followed by 17% EDTA, and final rinse by saline. Obturation procedures were done using the gutta-percha single cone technique. 45 roots were randomly assigned to 3 groups of 15 for obturation with gutta-percha cones and 1 of the 3 sealers (n=15). Group 1 = MTA Fillapex sealer + gutta-percha: Group 2 = AH plus sealer + gutta-percha:Group 3 = Apexit plus sealer + gutta-percha. The roots were sectioned horizontally to its canal into 3 sections: Coronal, Mid-root and Apical-thirds using a precision cutting machine, with a thickness of 3 mm. The specimens were subjected to push-out test using a universal testing machine that carried a plunger. The loading speed was 1mm/min until the dislodgment of the material occurred. The independent t- test was used to compare the mean scores among the study groups. The level of significance was set at 5% for all tests. After the push-out bond strength test, each sample was evaluated under stereomicroscope (40x) to determine the mode of failure and recorded as one of the following categories: adhesive, cohesive or mixed. The observations thus obtained were subjected to statistical analysis using Student - t test. AH Plus showed significantly higher values than MTA Fillapex and Apexit plus (p < 0.05). Amongst the push-out bond strength AH Plus sealer showed significant difference from MTA Fillapex and Apexit plus groups. There was no significant difference between MTA Fillapex and Apexit plus however (p>0.05). Microscopic analysis displayed that the majority of the modes were cohesive failures for AH Plus, adhesive failures for MTA Fillapex and mixed failures for Apexit Plus. . Thus, AH Plus had the highest bond strength and MTA Fillapex had the lowest bond strength to root dentin. Mean push-out bond strength values were ranked as follows; AH Plus >Apexit Plus > MTA Fillapex. Microscopic analysis displayed that the majority of the modes were cohesive failures of AH Plus, adhesive failures for MTA Fillapex and mixed failures for Apexit Plus.     

Dendrimer versus linear conjugate: Influence of polymeric architecture on the delivery and anticancer effect of paclitaxel

The relative difference in polymeric architectures of dendrimer and linear bis(poly(ethylene glycol)) (PEG) polymer in conjugation with paclitaxel has been described. Paclitaxel, a poorly soluble anticancer drug, was covalently conjugated with PAMAM G4 hydroxyl-terminated dendrimer and bis(PEG) polymer for the potential enhancement of drug solubility and cytotoxicity. Both conjugates were characterized by 1NMR, HPLC, and MALDI/TOF. In addition, molecular conformations of dendrimer, bis(PEG), paclitaxel, and its polymeric conjugates were studied by molecular modeling. Hydrolysis of the ester bond in the conjugate was analyzed by HPLC using esterase hydrolyzing enzyme. In vitro cytotoxicity of dendrimer, bis(PEG), paclitaxel, and polymeric conjugates containing paclitaxel was evaluated using A2780 human ovarian carcinoma cells. Cytotoxicity increased by 10-fold with PAMAM dendrimer-succinic acid-paclitaxel conjugate when compared with free nonconjugated drug. Data obtained indicate that the nanosized dendritic polymer conjugates can be used with good success as anticancer drug carriers.     

Keeping single minded expression on the straight and narrow

Recent Developmental Cell papers by De Renzis et al. (2006) and Bardin and Schweisguth (2006) show that members of the Bearded family interact with Neuralized to regulate trafficking of the Notch ligand Delta. This allows precise control of Notch signaling and restriction of single minded expression to a single row of cells.     

The essential mosquito-stage P25 and P28 proteins from Plasmodium form tile-like triangular prisms

P25 and P28 proteins are essential for Plasmodium parasites to infect mosquitoes and are leading candidates for a transmission-blocking malaria vaccine. The Plasmodium vivax P25 is a triangular prism that could tile the parasite surface. The residues forming the triangle are conserved in P25 and P28 from all Plasmodium species. A cocrystal structure shows that a transmission-blocking antibody uses only its heavy chain to bind Pvs25 at a vertex of the triangle.     

Muscle contributions to support and progression during single-limb stance in crouch gait

Pathological movement patterns like crouch gait are characterized by abnormal kinematics and muscle activations that alter how muscles support the body weight during walking. Individual muscles are often the target of interventions to improve crouch gait, yet the roles of individual muscles during crouch gait remain unknown. The goal of this study was to examine how muscles contribute to mass center accelerations and joint angular accelerations during single-limb stance in crouch gait, and compare these contributions to unimpaired gait. Subject-specific dynamic simulations were created for ten children who walked in a mild crouch gait and had no previous surgeries. The simulations were analyzed to determine the acceleration of the mass center and angular accelerations of the hip, knee, and ankle generated by individual muscles. The results of this analysis indicate that children walking in crouch gait have less passive skeletal support of body weight and utilize substantially higher muscle forces to walk than unimpaired individuals. Crouch gait relies on the same muscles as unimpaired gait to accelerate the mass center upward, including the soleus, vasti, gastrocnemius, gluteus medius, rectus femoris, and gluteus maximus. However, during crouch gait, these muscles are active throughout single-limb stance, in contrast to the modulation of muscle forces seen during single-limb stance in an unimpaired gait. Subjects walking in crouch gait rely more on proximal muscles, including the gluteus medius and hamstrings, to accelerate the mass center forward during single-limb stance than subjects with an unimpaired gait.     

Combinatorial effect of fish oil (Maxepa) and 1α,25-dihydroxyvitamin D3 in the chemoprevention of DMBA-induced mammary carcinogenesis in rats

The present study demonstrates the anti-tumor effects of combined supplementations of dietary fish oil (Maxepa) and 1α,25-dihydroxyvitamin D₃ (vitamin D₃) on 7,12-dimethylbenz(α)anthracene (DMBA)-induced rat mammary carcinogenesis. Female Sprague–Dawley rats at 50 days of age were treated with 7,12-dimethylbenz(α)anthracene (DMBA; 0.5 mg/100 g body weight) by a single tail vein injection in an oil emulsion. Both fish oil (rich in EPA and DHA) and vitamin D₃ were administered orally at a dose of 0.5 ml/day/rat and 0.3 μg/100 μL propylene glycol twice a week respectively and continued to 35 weeks after DMBA administration. Fish oil in combination with vitamin D₃ resulted in a significant reduction in incidence, multiplicity and volume of mammary tumors. These supplementation also inhibited DMBA-induced mammary 7-methylguanine DNA adducts formation, which was measured by HPLC-fluorescence assay (at four sequential time points; ANOVA, F = 42.56, P < 0.0001). Immunohistochemical analysis revealed that the effect of fish oil and vitamin D₃ occurred through suppression of cell proliferation (BrdU-LI: P < 0.0001). Fish oil and vitamin D₃ together also reduced the mRNA expression of iNOS (84%, P < 0.05). In view of their natural availability, non-toxicity and acceptability; combined supplementation of fish oil and vitamin D₃ might be effective for chemoprevention of mammary carcinogenesis.     

Light-regulated overexpression of an Arabidopsis phytochrome A gene in rice alters plant architecture and increases grain yield

The phytochromes are a family of red/far-red light absorbing photoreceptors that control plant developmental and metabolic processes in response to changes in the light environment. We report here the overexpression of Arabidopsis thaliana PHYTOCHROME A (PHYA) gene in a commercially important indica rice variety (Oryza sativa L. Pusa Basmati-1). The expression of the transgene was driven by the light-regulated and tissue-specific rice rbcS promoter. Several independent homozygous sixth generation (T₅) transgenic lines were characterized and shown to accumulate relatively high levels of PHYA protein in the light. Under both far-red and red light, PHYA-overexpressing lines showed inhibition of the coleoptile extension in comparison to non-transgenic seedlings. Furthermore, compared with non-transgenic rice plants, mature transgenic plants showed significant reduction in plant height, internode length and internode diameter (including differences in cell size and number), and produced an increased number of panicles per plant. Under greenhouse conditions, rice grain yield was 6–21% higher in three PHYA-overexpressing lines than in non-transgenic plants. These results demonstrate the potential of manipulating light signal-transduction pathways to minimize the problems of lodging in basmati/aromatic rice and to enhance grain productivity.