ABSTRACT
We determined the nucleotide sequences of blaCARB-4 encoding CARB-4 and deduced a polypeptide of 288 amino acids. The gene was characterized as a variant of group 2c carbenicillin-hydrolyzing beta-lactamases such as PSE-4, PSE-1, and CARB-3. The level of DNA homology between the bla genes for these beta-lactamases varied from 98.7 to 99.9%, while that between these genes and blaCARB-4 encoding CARB-4 was 86.3%. The blaCARB-4 gene was acquired from some other source because it has a G+C content of 39.1%, compared to a G+C content of 67% for typical Pseudomonas aeruginosa genes. DNA sequencing revealed that blaAER-1 shared 60.8% DNA identity with blaPSE-3 encoding PSE-3. The deduced AER-1 beta-lactamase peptide was compared to class A, B, C, and D enzymes and had 57.6% identity with PSE-3, including an STHK tetrad at the active site. For CARB-4 and AER-1, conserved canonical amino acid boxes typical of class A beta-lactamases were identified in a multiple alignment. Analysis of the DNA sequences flanking blaCARB-4 and blaAER-1 confirmed the importance of gene cassettes acquired via integrons in bla gene distribution.
Subject(s)
Carbenicillin/metabolism , Penicillins/metabolism , beta-Lactamases/chemistry , Amino Acid Sequence , Base Sequence , Molecular Sequence Data , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Bombesin synthesis has previously been achieved by the Merrifield method. Severe conditions and repetitive acidic treatments have led to the selection of a milder method for improving effectiveness. This study describes a new technique for the synthesis of bombesin and its analogs based on the use of fluorenylmethoxycarbonyl (Fmoc) protected amino acids and continuous flow solid phase procedure. Peptide elongation is realized by the intermediaries of derived pentafluorophenyl ester amino acids. Peptides of different lengths were synthesized in order to determine the minimal length of peptide chain needed to obtain immunoreactivity comparable to that of natural bombesin. Purification and analysis of the products by FPLC and amino acid determination were performed. Immunoreactivity study of these peptides was measured by a competition radioimmunoassay with an antiserum directed against the C-terminal portion. Results suggest that a decapeptide analog is sufficient for bombesin to be recognized by the antibody or as a carrier in targeting therapy.
Subject(s)
Bombesin/chemical synthesis , Amino Acid Sequence , Bombesin/analogs & derivatives , Bombesin/chemistry , Chemistry, Pharmaceutical , Molecular Sequence DataABSTRACT
We have evaluated the effectiveness of daunorubicin against an SCCL cell line (NCI-H69) after conjugation to transferrin. Our results show that the conjugate is at least 10 times more active than the free drug in this in vitro system.
Subject(s)
Carcinoma, Small Cell/drug therapy , Daunorubicin/pharmacology , Lung Neoplasms/drug therapy , Transferrin/pharmacology , Carcinoma, Small Cell/chemistry , Drug Combinations , Drug Screening Assays, Antitumor , Humans , Lung Neoplasms/chemistry , Receptors, Transferrin/analysis , Tumor Cells, CulturedABSTRACT
We report some modifications of the semiautomated tetrazolium-based assay for the measurement of anchorage-dependent and -independent mammalian cells. The various factors affecting color production, such as the concentration of tetrazolium, incubation period, the type and volume of solvent, were optimized. Using KCN and daunorubicin as cytotoxic agents, the influence of dead cells was studied on the measurement. The assay was tested with mouse leukemia P388 cells, H69 small cell carcinoma cells growing in suspension and anchorage dependent colon adenocarcinoma cells (LoVo). Centrifugation of the microtitration plate was eliminated by the use of a Skatron supernatant collection system. Although the use of the MTT assay is rapid and precise, we found that care should be taken when using this assay for short-term cytotoxicity assays since non-viable cells also reduce the tetrazolium.
