Adsorption of Salmon Calcitonin to PLGA Microspheres

Purpose. The interaction of salmon calcitonin (sCT) and poly (d,l-lactide-co-glycolide) was detected during preparation and evaluation of microspheres. The purpose of this study was to quantitate the extent and nature of the interaction. Methods. Blank microspheres were prepared by an aqueous emulsification solvent extraction technique. Adsorption studies were carried out at six concentrations of sCT and three concentrations of microspheres. Adsorption isotherms were constructed using the Langmuir and Freundlich treatments. Results. Adsorption at 1 mg/ml sCT concentration resulted in almost complete depletion of the peptide from the adsorption medium with the time to reach maximum adsorption decreasing with increasing microsphere concentration. At sCT concentrations below 100 µg/ml, a true equilibrium occurred in 1 hour or less while at higher concentrations (up to 350 µg/ml), a transient equilibrium was reached in 1 to 2 hours, followed by further adsorption of the peptide. The adsorption followed the Langmuir isotherm at concentrations below 200 µg/ml, indicating formation of a monolayer. Multilayer interaction, described by the Freundlich isotherm, occurred at higher concentrations and resulted in complete depletion of sCT from the adsorption medium. The affinity constant during monolayer formation was 0.09 and the plateau surface concentration was 5.1 µg/mg. The multilayer peptide-peptide adsorption showed a lower affinity (0.025) but higher capacity (24 µg/mg) than the monolayer peptide-polymer adsorption. Conclusions. The results show that poly (d,l-lactide-co-glycolide) microspheres have a high adsorption capacity for sCT which must be considered in formulating a controlled delivery product of this peptide.     

Crystal structures of the copper and nickel complexes of RNase A: Metal-induced interprotein interactions and identification of a novel copper binding motif

We report the crystal structures of the copper and nickel complexes of RNase A. The overall topology of these two complexes is similar to that of other RNase A structures. However, there are significant differences in the mode of binding of copper and nickel. There are two copper ions per molecule of the protein, but there is only one nickel ion per molecule of the protein. Significant changes occur in the interprotein interactions as a result of differences in the coordinating groups at the common binding site around His-105. Consequently, the copper- and nickel-ion-bound dimers of RNase A act as nucleation sites for generating different crystal lattices for the two complexes. A second copper ion is present at an active site residue His-119 for which all the ligands are from one molecule of the protein. At this second site, His-119 adopts an inactive conformation (B) induced by the copper. We have identified a novel copper binding motif involving the α-amino group and the N-terminal residues.     

Structural and functional analysis of de‐N‐acetylase PgaB from periodontopathogen Aggregatibacter actinomycetemcomitans

The oral pathogen Aggregatibacter actinomycetemcomitans uses pga gene locus for the production of an exopolysaccharide made up of a linear homopolymer of β‐1,6‐N‐acetyl‐d ‐glucosamine (PGA). An enzyme encoded by the pgaB of the pga operon in A. actinomycetemcomitans is a de‐N‐acetylase, which is used to alter the PGA. The full length enzyme (AaPgaB) and the N‐terminal catalytic domain (residues 25–290, AaPgaBN) from A. actinomycetemcomitans were cloned, expressed and purified. The enzymatic activities of the AaPgaB enzymes were determined using 7‐acetoxycoumarin‐3‐carboxylic acid as the substrate. The AaPgaB enzymes displayed significantly lower de‐N‐acetylase activity compared with the activity of the deacetylase PdaA from Bacillus subtilis, a member of the CE4 family of enzymes. To delineate the differences in the activity and the active site architecture, the structure of AaPgaBN was determined. The AaPgaBN structure has two metal ions in the active site instead of one found in other CE4 enzymes. Based on the crystal structure comparisons among the various CE4 enzymes, two residues, Q51 and R271, were identified in AaPgaB, which could potentially affect the enzyme activity. Of the two mutants generated, Q51E and R271K, the variant Q51E showed enhanced activity compared with AaPgaB, validating the requirement that an activating aspartate residue in the active site is essential for higher activity. In summary, our study provides the first structural evidence for a di‐nuclear metal site at the active site of a member of the CE4 family of enzymes, evidence that AaPgaBN is catalytically active and that mutant Q51E exhibits higher de‐N‐acetylase activity.     

Dose-response relationship of selective serotonin reuptake inhibitors treatment-emergent hypomania in depressive disorders

Objective: The notion that antidepressant treatment‐associated hypomania or mania being pharmacologically induced has been challenged. To determine whether selective serotonin reuptake inhibitors (SSRI) induced hypomania is secondary to medication effects, we examined the dose–response relationship of SSRI‐induced hypomania in two patients with depressive disorder. Method: Case study. Result: Hypomanic symptoms emerged during treatment with sertraline at the dose of 300 mg per day in a 45‐year‐old male with major depression. Paroxetine treatment at the dose of 80 mg per day induced hypomania in a 37‐year‐old female with dysthymia and trichitillomania. These patients have no family or personal history of bipolar disorder. Hypomania resolved when sertraline was decreased to 200 mg per day and paroxetine to 40 mg per day. No hypomanic switch was observed during 18–24 months follow‐up. Conclusion: In the absence of risk factors for manic switch, SSRI‐induced hypomania may be dose‐dependent medication effects.     

A concise methodology for the stereoselective synthesis of O-glycosylated amino acid building blocks: complete 1HNMR assignments and their application in solid-phase glycopeptide synthesis

A facile strategy for the stereoselective synthesis of suitably protected O-glycosylated amino acid building blocks, namely, N^α-Fmoc-Ser-[Ac₄-β-d -Gal-(1-3)-Ac₂α or β-d -GalN₃]-OPfp and N^α-Fmoc-Thr-[Ac₄-β-d -Gal-(1-3)-Ac₂-α or β-d -GalN₃]-OPfp is described. What is new and novel in this report is that Koenigs-Knorr type glycosylation of an aglycon serine/threonine derivative (i.e. N^α-Fmoc-Ser-OPfp or N^α-Fmoc-Thr-OPfp) with protected β-d -Gal(1-3)-d -GalN₃ synthon mediated by silver salts resulted in only α-and/or β-isomers in excellent yields under two different reaction conditions. The subtle differences in stereoselectivity were demonstrated clearly when glycosylation was carried out using only AgClO₄ at -40°C which afforded α-isomer in a quantitative yield (α:β= 5:1). On the other hand, the β-isomer was formed exclusively when the reaction was performed in the presence of Ag₂CO₃AgClO₄ at room temperature. A complete assignment of ¹H resonances to individual sugar ring protons and the characteristic anomeric α-1H and β-1H in Ac₄Galβ(1-3)Ac₂GalN₃α and/or β linked to Ser/Thr building blocks was accomplished unequivocally by two-dimensional double-quantum filtered correlated spectroscopy and nuclear Overhauser enhancement and exchange spectroscopy NMR experiments. An unambiguous structural characterization and documentation of chemical shifts, including the coupling constants for all the protons of the aforementioned a- and p-isomers of the O-glycosylated amino acid building blocks carrying protected β-d -Gal(1-3)-d -GalN₃, could serve as a template in elucidating the three-dimensional structure of glycoproteins. The synthetic utility of the building blocks and versatility of the strategy was exemplified in the construction of human salivary mucin (MUC7)-derived, O-linked glycopeptides with varied degrees of glycosylation by solid-phase Fmoc chemistry. Fmoc/tert-butyl-based protecting groups were used for the peptidic     

Economic,clinical, and quality-of-life outcomes associated with olanzapine treatment in mania. Results from a randomized controlled trial

INTRODUCTION: The objectives of this study were to determine the economic, clinical, and quality-of-life outcomes associated with olanzapine treatment in patients diagnosed with mania. METHODS: Patients with a diagnosis of bipolar I disorder with manic or mixed episodes were enrolled in a randomized controlled trial. The study design comprised a 3-week acute phase followed by a 49-week open label extension. In the open label extension, the use of lithium and fluoxetine was permitted for patients who experienced breakthrough symptoms. Clinical, economic, and quality-of-life outcomes of treatment were assessed. RESULTS: During the acute phase, olanzapine patients experienced a statistically significant greater mean improvement from baseline on the Y-MRS total score compared to the placebo patients. In the open label extension, patients experienced a statistically significant mean change of 11.8 units on the Y-MRS from the end of the acute phase. When compared to costs incurred in the previous 12 months of therapy, patients experienced savings of almost $900 per month during the 49 weeks of olanzapine therapy. These cost savings were largely driven by reductions in in-patient costs during the open label extension. Health-related quality of life improvements measured by the SF-36 were seen on several dimensions both in the 3-week acute phase as well as in the 49-week open label extension. CONCLUSION: From a clinical, economic, and quality-of-life outcomes standpoint, olanzapine had a significant impact in the treatment of mania, and could be considered a cost-effective treatment option for use in this population if these findings are extrapolated to non-clinical trial populations.     

Extended evaluation of serotonin transporter gene functional polymorphisms in subjects with post-stroke depression.

OBJECTIVE: As an extension of our previous observation, relating a serotonin transporter gene-linked promoter region (5-HTTLPR) diallelic functional polymorphism (short [S] and long [L] alleles) to the risk of post-stroke major depression (PSD), this study investigated the role of 2 other functional polymorphisms of the serotonin transporter gene (5-HTT) in the same sample of subjects with PSD. METHOD: In a clinical sample of 26 patients with PSD and 25 unrelated nondepressed stroke patients of Caucasian descent, we examined the frequencies of a functional single nucleotide variant (A/G) within the promoter region (rs25531) and located in L (16-repeat) and S (14-repeat) alleles of 5-HTTLPR, and a variable number tandem repeat (VNTR) polymorphism in intron 2. RESULTS: There were significant intergroup differences in the allelic frequencies of 5-HTTLPR/rs25531 (SA, LA, and LG) (P < 0.05) and in the combined frequencies of lower-expressing alleles (SA and LG) and higher-expressing alleles (LA) (P < 0.025) between subjects with PSD and nondepressed stroke. However, the differences in the combined frequencies of lower-expressing (SA/SA, SA/LG, and LG/LG), intermediate-expressing (SA/LA and LA/LG), and higher-expressing (LA/LA) genotypes of 5-HTTLPR were not significant. Further, no significant intergroup differences were found in the allelic and genotypic frequencies of the intron 2 VNTR. CONCLUSIONS: These findings strengthen the support for an association between PSD and lower-expressing alleles of 5-HTTLPR.     

Anti-angiotensin therapy: new perspectives

Activation of the renin-angiotensin system (RAS) plays an important role in the pathogenesis of heart failure. Thus, strategies for the treatment of heart failure have focused on agents that block the RAS. More recently, the role of angiotensin receptor blockers (ARBs) in heart failure therapy has been better defined. This article examines the rationale and role of ARBs in the treatment of patients with heart failure on the basis of evidence from clinical trials.