Studies of the interaction between demeclocycline and human serum albumin by multi-spectroscopic and molecular docking methods.

This study was designed to examine the interaction of demeclocycline (DMCTC) with human serum albumin (HSA) by multi-spectroscopic and molecular docking methods. The inner filter effect was corrected before we calculated the binding parameters. Fluorescence and UV-vis spectroscopy revealed that DMCTC induced the fluorescence quenching of HSA though a static quenching procedure. Thermodynamic analysis by Van Hoff equation found enthalpy change (ΔH) and entropy change (ΔS) were -53.01 kJ mol(-1) and -65.13 J mol(-1)K(-1), respectively, which indicated hydrogen bond and van der Waals force were the predominant force in the binding process. According to fluorescence resonance energy transfer (FRET), the specific binding distances between Trp-214 (donor) and DMCTC (acceptor) were 3.18 nm. Through site marker competitive experiments, subdomain IIA of HSA has been assigned to possess the high-affinity binding site of DMCTC. The three dimensional fluorescence showed that the conformation of HSA was changed after its complexation with DMCTC, and the alternations of protein secondary structure were quantitatively calculated from FT-IR with reduction of α-helices content about 4.8%, ß-sheet from 30.3% to 21.6% and with increases of ß-turn from 15.6% to 22.2%. Furthermore, the binding details between DMCTC and HSA were further confirmed by molecular docking studies, which revealed that DMCTC was bound at subdomain IIA through multiple interactions, such as hydrophobic effect, polar forces and π-π interactions. Moreover, the coexist metal ions such as Al(3+), Fe(3+), Cu(2+), Cr(3+) and Cd(2+) can decrease the binding constants of DMCTC-HSA.

Conserved organization of genes for biosynthesis of chlortetracycline in Streptomyces strains.

By genomic Southern blot analysis, the DNA sequences homologous to the gene cluster responsible for biosynthesis of 6-demethyl-chlortetracycline in Streptomyces aureofaciens NRRL3203 were shown to be highly conserved in independent chlortetracycline- or tetracycline producing Streptomyces strains. By contrast, oxytetracycline-producing Streptomyces strains had no hybridization with the cluster DNA.

Cloning and nucleotide sequence of the gene responsible for chlorination of tetracycline.

Two cosmid clones containing distinct types of self-defense gene of a 6-demethylchlortetracycline producer, Streptomyces aureofaciens NRRL3203, were isolated. The gene responsible for chlorination of tetracycline (chl gene) was subcloned from one of the cosmid clones by complementation of a chlorination-deficient mutant, using a gene cloning system for strain NRRL3203 developed in this study. The nucleotide sequence analysis of a 4.4-kb SacI-BamHI fragment containing the chl gene showed that the predicted product of the chl gene is a polypeptide of 452 amino acids, and that the chl gene was preceded by two open reading frames, which could endode polypeptides of 50 kDa and 32 kDa, respectively. A search for sequences homologous to these ORFs found that the former product strongly resembles that of the 6-hydroxylation enzyme for oxytetracycline biosynthesis, and that the latter product has a weak but significant similarity to the hydroxyindole O-methyltransferase of bovine pineal gland. By Northern blot analysis, these three genes were suggested to be polycistronically transcribed.

Competition between tetracycline and tRNA at both P and A sites of the ribosome of Escherichia coli.

Fluorescence anisotropy studies performed on 6-demethylchlortetracycline, binding to the ribosome of E. coli in competition with tRNA at the P site or at both P and A sites, have provided a quantitative assessment in situ of the interaction of this antibiotic with the A site and have demonstrated that there is also an interaction between tetracycline and the P site.

The label escape error: comparison of measured and theoretical fraction of total bone-trabecular surface covered by single label in normals and patients with osteoporosis.

Thirty-four normal Caucasian women and 69 patients with osteoporosis were given two bone-labeling agents. Transilial biopsies were obtained and embedded undecalcified. Fractional surfaces covered by double label and single label were determined, along with total surface and double label width by fluorescent microscopy. Mean wall thickness was measured on toluidine blue-stained sections. The mathematical model for predicting the amount of single-labeled surface was compared to the actual amount of surface covered by single label. We found an excess of single labels compared to the model in both groups, more so in the normals, and suggest the explanation is that bone-formation pauses at formation sites in both groups but tends not to resume in the patients. This results in a shortened functional life span of the osteoblast and bone loss. Further, the data suggest that for accurate expression of bone formation rates in trabecular bone using a volume referent, one should use the whole bone tissue including bone and marrow and should express the bone-forming surface as the double-labeled surface plus one-half the measured single-labeled surface.

Demethylchlortetracycline-binding proteins in uninvolved gastric mucosa of gastric carcinoma and gastric ulcer patients. Demonstration of a difference between the uninvolved mucosa of ulcer and cancer patients.

The uninvolved gastric mucosa of gastric ulcer and gastric carcinoma patients has been compared in in vitro studies as regards their capacity to bind demethylchlortetracycline (DMCT). Dialysis experiments demonstrated excessive binding of DMCT in gastric cancer. Several electrophoretic fractions were observed that bound DMCT; it was demonstrated that these fractions differed in the uninvolved mucosa of gastric ulcer and gastric cancer patients.