A simple three-step method for design and affinity testing of new antisense peptides: an example of erythropoietin.

Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide-receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope) as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense-antisense (epitope-paratope) peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s) could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.

One hundred kidney transplantations in Tuzla.

INTRODUCTION: Kidney transplantation assures considerably better quality of life than the treatment of end-stage renal disease patients with dialysis. GOAL: Authors intended to present results of kidney transplantations that were performed for over 13 years in UCC Tuzla. EXAMINEES AND METHODS: Total of 100 transplantations have been done over 13 years. The gender and age structure have been presented, as well as number of transplantations per year, type of transplantation (living related donor, living unrelated donor, deceased donor), number and percentage of donors and results of transplantations expressed as survival of both the patient and transplanted kidney/ renal graft. We also wanted to presented other important events such as dates of introduction of certain drugs, dates of first cadaver transplantation, transplantation with desensitization protocols and dates of first living unrelated (spousal/emotional) transplantation. RESULTS: The survival of patients and renal grafts were demonstrated by Kaplan-Meier curve, and obtained results were fully in range of results recommended in other literature and by other authors. One-year survival of graft is 94%, with five-year survival being 75%. One-year survival of patients is 95%, and five-year survival of patients was 84%. DISCUSSION: Our results have been compared to those from other studies, gaining suggestions for transplantation improvement. CONCLUSION: Among all modifications of renal replacement therapy transplantation is by far the method of choice because, its well known advantages aside, it also has an economical advantage over chronic treatment with dialysis and it should therefore become interesting to healthcare systems.

Genetic coding algorithm for sense and antisense peptide interactions.

Sense and antisense peptides, i.e. peptides specified by complementary DNA and RNA sequences, interact with increased probability. Biro, Blalock, Mekler, Root-Bernstein and Siemion investigated the recognition rules of peptide-peptide interaction based on the complementary coding of DNA and RNA sequences in 3'→5' and 5'→3' directions. After more than three decades of theoretical and experimental investigations, the efficiency of this approach to predict peptide-peptide binding has been experimentally verified for more than 50 ligand-receptor systems, and represents a promising field of research. The natural genetic coding algorithm for sense and antisense peptide interactions combines following elements: of amino acid physico-chemical properties, stereochemical interaction, and bidirectional transcription. The interplay of these factors influences the specificity of sense-antisense peptide interactions, and affects the selection and evolution of peptide ligand-receptor systems. Complementary mRNA codon-tRNA anticodon complexes, and recently discovered Carter-Wolfenden tRNA acceptor-stem code, provide the basis for the rational modeling of peptide interactions based on their hydrophobic and lipophilic amino acid physico-chemical properties. It is shown that the interactions of complementary amino acid pairs according to the hydrophobic and lipophilic properties strongly depend on the central (second) purine base of the mRNA codon and its pyrimidine complement of the tRNA anticodon. This enables the development of new algorithms for the analysis of structure, function and evolution of protein and nucleotide sequences that take into account the residue's tendency to leave water and enter a nonpolar condensed phase considering its mass, size and accessible surface area. The practical applications of the sense-antisense peptide modeling are illustrated using different interaction assay types based on: microscale thermophoresis (MST), tryptophan fluorescence spectroscopy (TFS), nuclear magnetic resonance spectroscopy (NMR), and magnetic particles enzyme immunoassay (MPEIA). Various binding events and circumstances were considered, e.g., in situations with-short antisense peptide ligand (MST), L- and D-enantiomer acceptors (TFS), in low affinity conditions (NMR), and with more than one antisense peptide targeting hormone (MPEIA).

Modulation of neutrophil and inflammation markers in chronic obstructive pulmonary disease by short-term azithromycin treatment.

The anti-inflammatory potential of azithromycin in chronic obstructive pulmonary disease (COPD) patients was explored following a standard oral dosing regimen. Patients with moderate and severe COPD were treated with azithromycin (500 mg, n=16) or placebo (n=8) once daily for 3 days in a randomized, double blind design, to compare effects on inflammation markers with those seen in a previous study in healthy volunteers. A battery of tests was made on serum, blood neutrophils and sputum on days 1 (baseline), 3, 4, 11, 18 and 32. In comparison to placebo, azithromycin resulted in an early transient increase in serum nitrites plus nitrates (day 3), associated with a tendency towards an increase in the blood neutrophil oxidative burst to phorbol myristic acetate. Subsequently, prolonged decreases in blood leukocyte and platelet counts, serum acute phase protein (including C reactive protein) and soluble E-selectin and blood neutrophil lactoferrin concentrations and a transient decrease in serum interleukin-8 were observed. Blood neutrophil glutathione peroxidase activity showed a prolonged increase after azithromycin treatment. The biphasic facilitatory-then-inhibitory response to azithromycin seen in healthy volunteers is not so clearly detectable in COPD patients, only potential anti-inflammatory effects. Treatment for longer periods may give therapeutic anti-inflammatory benefit in these patients.

Azithromycin modulates neutrophil function and circulating inflammatory mediators in healthy human subjects.

Effects on human neutrophils and circulating inflammatory mediators were studied in 12 volunteers who received azithromycin (500 mg/day, p.o.) for 3 days. Blood was taken 1 h before treatment, 2.5, 24 h and 28 days after the last dose. An initial neutrophil degranulating effect of azithromycin was reflected in rapid decreases in azurophilic granule enzyme activities in cells and corresponding increases in serum. The oxidative response to a particulate stimulus was also acutely enhanced. These actions were associated with high plasma and neutrophil drug concentrations. A continuous fall in chemokine and interleukin-6 serum concentrations, within the non-pathological range, accompanied a delayed down-regulation of the oxidative burst and an increase in apoptosis of neutrophils up to 28 days after the last azithromycin dose. Neutrophils isolated from blood at this time point still contained detectable drug concentrations. Acute neutrophil stimulation could facilitate antibacterial effects of azithromycin, while delayed, potentially anti-inflammatory activity may curtail deleterious inflammation.