ABSTRACT
Peptide libraries displayed by T7 phage, which contain random cDNA fragments insets, were screened for their ability to bind to a biotinylated derivative of clarithromycin. Phage particles bound to an immobilized derivative of the antibiotic were isolated and the inserted cDNA was amplified and sequenced. A common selected peptide sequence, composed of 19 amino acids, was obtained and a synthetic peptide with this sequence was produced. Surface plasmon resonance experiments showed that the synthetic peptide immobilized on a sensor chip bound to clarithromycin and the dissociation constant was determined to be 2.1 x 10(-3) M. The dissociation constants of other macrolide antibiotics, erythromycin, roxithromycin, azithromycin and josamycin were also determined to be 5.4 x 10(-3) M, 6.2 x 10(-5) M, 1.1 M and 3.4 x 10(-2) M, respectively. These results indicated that T7 phage display method might be useful to determine relatively weak interactions between small molecule drugs and the selected peptides which could represent a possible binding site conserved in binding proteins.
Subject(s)
Anti-Bacterial Agents/metabolism , Bacteriophage T7/genetics , Clarithromycin/metabolism , Peptide Library , Peptides/metabolism , Amino Acid Sequence , Biotinylation , Clarithromycin/analogs & derivatives , Gene Library , Ligands , Molecular Sequence Data , Molecular Structure , Peptides/chemistry , Peptides/genetics , Protein Binding , Surface Plasmon ResonanceABSTRACT
The bone metabolic processes of proliferation and differentiation in preterm and term newborns have yet to be fully elucidated. Seventy-four umbilical cord blood samples were collected from preterm and term newborns delivered at 27 to 42 gestational weeks (GWs). Carboxy-terminal propeptide of type I procollagen (PICP), pyridinoline cross-linked telopeptide domain of type I collagen (ICTP), alkaline phosphatase (ALP), and bone-specific alkaline phosphatase (BAP) were measured. Calcitonin (CT), estrogen (E2), intact parathyroid hormone, and insulin-like growth factor-I (IGF-I) were also examined in 20 or 23 randomly selected samples. We conducted cross-sectional regression analyses for bone metabolic markers, fetal growth markers including GWs, birth weight (BW), height (BH) and head circumference (HC), and bone related hormones. PICP and ICTP activities were very high, but decreased significantly with fetal growth based on GWs, BW, BH, and HC changes (GWs, BW, and BH to both PICP and ICTP, P < 0.0001; HC to ICTP, P < 0.0001; HC to PICP, P < 0.05), while BAP and ALP did not change significantly. E2 and CT both showed a significant positive correlation with Ca (P < 0.05), but neither hormone had any apparent correlation with PICP, ALP, BAP, or ICTP. These results suggest very active bone formation and resorption of type I collagen to be dependent on fetal growth and that fetal osteoblasts dominate the proliferation phase of development rather than the maturation phase. However, factors contributing to high bone turnover in the fetus remain to be elucidated.
Subject(s)
Biomarkers/analysis , Bone and Bones/metabolism , Collagen Type I/metabolism , Fetal Development/physiology , Hormones/physiology , Body Height , Cross-Sectional Studies , Hormones/metabolism , Humans , Infant, Newborn , Osteogenesis , Regression AnalysisABSTRACT
We hypothesized that serial changes in platelet (PLT) mitochondrial enzyme (ME) activities might correspond to the effects of medications for mitochondrial encephalomyopathy and stroke-like episodes (MELAS). Cytochrome c and sodium dichloroacetate (DCA) were given to a 7-year-old girl with MELAS who had an A3243G mitochondrial DNA mutation. The effects were evaluated with whole PLT-ME assays, developed by our group, using a microplate-reader. During cytochrome c treatment, complex II+III (II+III), complex IV (IV) and citrate synthase (CS) activities showed gradual but statistically significant decrease. II+III activity dropped below normal. II+III/CS activity was initially below normal, followed by a transient improvement, then decreased again before the appearance of central nervous system symptoms. II+III, IV, II+III/CS and IV/CS activities reached their lowest levels in association with a stroke-like episode, then increased with DCA treatment. Our results suggest that progressive mitochondrial dysfunction may occur before the stroke-like episodes in MELAS and that DCA treatment may increase mitochondrial activities. Our whole PLT-ME assay system may be useful for serially evaluating mitochondrial functions in relation to clinical symptoms.
