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Transcription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitum beta-lactamase genes cloned from a natural isolate and a high-level beta-lactamase producer.

The gene encoding a class A β‐lactamase was cloned from a natural isolate of Mycobacterium fortuitum (blaF) and from a high‐level amoxicillin‐resistant mutant that produces large amounts of β‐lactamase (blaF). The nucleotide sequences of the two genes differ at 11 positions, including two in the region upstream from the coding sequence. Gene fusions to Escherichia coli lacZ and transcription and expression analysis of the cloned genes in Mycobacterium smegmatis indicated that high‐level production of the β‐lactamase in the mutant is mainly or wholly due to a single base pair difference in the promoter. These analyses also showed that transcription and translation start at the same position. A comparison of the amino acid sequence of BlaF, as predicted from the nucleotide sequence, with the determined N‐terminal amino acid sequence indicated the presence of a typical signal peptide. The fusion of blaF (or biaF) to the E. coli gene phoA resulted in the production of BlaF‐PhoA hybrid proteins that had alkaline phosphatase activity. These results demonstrate that phoA can be used as a reporter gene for studying protein export in mycobacteria.

Cloning and nucleotide sequencing of the gene encoding the β-lactamase from Citrobacter diversus

The gene coding for the class A beta-lactamase of Citrobacter diversus has been cloned and sequenced. It contains the information for a 294-amino-acid precursor protein, including a 27-residue N-terminal signal peptide. The deduced sequence of the N-terminal portion of the mature protein is in excellent agreement with that determined by microsequencing of the protein and readily explains the pI differences observed between the naturally occurring forms I and II of the enzyme. The sequence of the mature protein exhibits a very high degree of similarity with that of the Klebsiella oxytoca class A beta-lactamase.

Use of the chromosomal class A beta-lactamase of Mycobacterium fortuitum D316 to study potentially poor substrates and inhibitory beta-lactam compounds.

Sixteen different compounds usually considered beta-lactamase stable or representing potential beta-lactam inhibitors and inactivators were tested against the beta-lactamase produced by Mycobacterium fortuitum. The compounds exhibiting the most interesting properties were BRL42715, which was by far the best inactivator, and CGP31608 and ceftazidime, which were not recognized by the enzyme. These compounds thus exhibited adequate properties for fighting mycobacterial infections. Although cloxacillin, dicloxacillin, cefoxitin, and CP65207-2 exhibited poor inhibitory efficiency against the enzyme, they were also rather poor substrates and might be considered potential antimycobacterial agents. By contrast, CGP31523A and ceftamet were good substrates.

Lactoferrin Derived Peptides: Mechanisms of Action and their Perspectives as Antimicrobial and Antitumoral Agents

Antimicrobial peptides, AMPs, exert their function acting mainly at the membrane level. In the wide AMPs panorama a particular position is occupied by lactoferrin derived peptides. They also possess antiviral, antifungal and antitumor activities and their parent molecules are available in several mammalian fluids and in dairy industries waste.

Immunohistochemical localization of ascorbate oxidase in cucurbita pepo medullosa

Abstract Antibodies raised against homogeneous ascorbate oxidase (AAO) were used for the immunohistochemical localization of the enzyme in Cucurbita pepo medullosa. Ascorbate oxidase is present in all the specimens examined (vegetative and reproductive organs). At the cellular level the enzyme is associated with the cell wall and cytoplasm. The ubiquitous distribution of ascorbate oxidase suggest a role of general relevance for plant cells.

Biochemistry: Can Non-steroidal Anti-inflammatory Drugs Act as Metalloproteinase Modulators? An In-vitro Study of Inhibition of Collagenase Activity

The in‐vitro effects of some non‐steroidal anti‐inflammatory drugs and some analgesic drugs on collagenase activity were studied by use of a self‐quenched fluorogenic esapeptide as substrate. The increased fluorescence signal arising as a result of peptide cleavage by collagenase was recorded and related to the inhibitory potency of the drugs. The effective concentrations in collagenase modulation were also correlated with the levels of the drugs in the plasma and synovial fluids of patients receiving therapeutic doses.

Purification of a 76-kDa iron-binding protein from human seminal plasma by affinity chromatography specific for ribonuclease: structural and functional identity with milk lactoferrin.

A pink-colored iron-binding protein has been found in large amount in human seminal plasma and identified as a lactoferrin isoform. Its purification, by a modification of a three-step chromatography procedure developed in an attempt to purify a ribonuclease from the same fluid, provided about 15-18 mg of pure protein from 100 ml of seminal plasma. Despite its ability to bind a ribonuclease ligand during the affinity step, the iron-binding protein did not display any detectable RNase activity in a standard assay with yeast RNA as substrate. It showed an apparent molecular weight of 76 kDa and resulted to be quite similar, if not identical, to human milk lactoferrin in many respects. Its N-terminal sequence (31 amino acid residues) starting with Arg-3 was identical to that of one of the N-terminally truncated lactoferrin variants isolated from human milk. Moreover, the amino acid sequence of a number of peptides, which represented about 23% of the entire sequence, has been also shown to be identical to that of the corresponding peptides of human milk lactoferrin. Double diffusion analysis revealed full recognition by antibodies anti-human milk lactoferrin of the human seminal plasma protein. Using immunoblotting analysis, both human milk lactoferrin and human seminal protein were recognized by antibodies anti-milk lactoferrin. When tested for its iron binding capacity, with Fe-NTA as iron donor, the protein purified was able to bind iron up to 100% saturation, as judged by absorbance at 465 nm.

Human erythrocyte damage at the initial stages of oxidative stress.

Specific fluorescent probes have been used to monitor changes in erythrocyte membranes in the first stages of the hemolytic process induced by irradiation with visible light in the presence of protoporphyrin IX.Although no change, or even a slight increase of fluorescence anisotropy, occurred with two probes having a preferential binding to membrane proteins, such as fluorescamine and 3-pyrene maleimide, the fluorescence anisotropy of two lipophilic probes, namely diphenyl-hexatriene and anilino-naphtalene sulfonate, underwent a substantial decrease upon irradiation.Concomitantly, a dramatic decrease of ATPase activity and an increase of thiobarbituric-reacting substances were observed in erythrocyte membranes. Instead, there was no effect on the activities of the intracellular enzymes glucose-6-phosphate dehydrogenase and pyruvate kinase.These findings are consistent with the hypothesis that protoporphyrin-sensitized irradiation induces, primarily in the erythrocyte membrane, the peroxidation of the lipid component, which results in an increase of the fluidity of the bilayer. Hemolysis eventually occurs because of an osmotic imbalance resulting from the combination of increased passive diffusion and decreased active ion transport.

Interactions of biapenem with active-site serine and metallo-beta-lactamases.

Biapenem, formerly LJC 10,627 or L-627, a carbapenem antibiotic, was studied in its interactions with 12 beta-lactamases belonging to the four molecular classes proposed by R. P. Ambler (Philos. Trans. R. Soc. Lond. Biol. Sci. 289:321-331, 1980). Kinetic parameters were determined. Biapenem was readily inactivated by metallo-beta-lactamases but behaved as a transient inhibitor of the active-site serine enzymes tested, although with different acylation efficiency values. Class A and class D beta-lactamases were unable to confer in vitro resistance toward this carbapenem antibiotic. Surprisingly, the same situation was found in the case of class B enzymes from Aeromonas hydrophila AE036 and Bacillus cereus 5/B/6 when expressed in Escherichia coli strains.

Encapsulation of ampicillin in reverse-phase evaporation liposomes: a direct evaluation by derivative spectrophotometry

Abstract Ampicillin has been entrapped in phospholipid vesicles by reverse-phase evaporation technique. The relationship between lipid composition and the encapsulation efficiency or the release of ampicillin-loaded liposomes was studied in vitro using a derivative spectrophotometry assay. The encapsulation degree was closely dependent on the lipid mixture used in liposome preparation: in particular, phosphatidylcholine (DPPC) vesicles containing cholesterol (CHO) or lipopolysaccharide (LPS) had a lower entrapment efficiency than liposomes prepared with DPPC alone, whereas the presence of cardiolipin (CD conferred an opposite trend. From the release kinetics it appeared that vesicles leaked the carried drug by a biphasic feature both dialyzing against buffer or in bulk solution. These observations indicate that for the in vivo use of ampicillin-loaded liposomes as chemotherapeutic agents one must pay attention to the lipid composition of the vesicle, in order to modulate the dose-response effect.