Diagnosis value of some acute-phase proteins in purulent-inflammatory diseases of uterine appendages

We investigated concentrations of acute-phase proteins a-2-macroglobulin (MG) and lactoferrin (LF) in blood serum of 78 women with various types of uterine appendages inflammatory processes. Coefficient MG/LF was used as an additional diagnostic criterum of purulent-necrotic destruction of organs and tissues and allowed us to choose proper treatment options. MG values were assessed by method of rocket immune electroforesis using monospecific antiserum to the given protein, LF level was assessed by enzyme linked immunoassay based method (ELISA). Standard was performed when coefficient MG/LF was greater than 1, and if value of coefficient MG/LF was less than 1, we performed surgical treatment. Using coefficient MG/LF as a diagnostic criterion of existence of organic destruction in uterine appendages allowed us to optimize the selection of treatment program.

Cloning and Characterization of Arylamine N -Acetyltransferase Genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: Increased Expression Results in Isoniazid Resistance

ABSTRACT Arylamine N-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote Salmonella typhimurium. NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it. nat genes were cloned fromMycobacterium smegmatis and Mycobacterium tuberculosis, and expressed in Escherichia coli andM. smegmatis. The induced M. smegmatis NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from S. typhimurium recognizes NAT fromM. smegmatis and cross-reacts with recombinant NAT fromM. tuberculosis. Overexpression of mycobacterialnat genes in E. coli results in predominantly insoluble recombinant protein; however, with M. smegmatisas the host using the vector pACE-1, NAT proteins from M. tuberculosis and M. smegmatis are soluble. M. smegmatis transformants induced to express the M. tuberculosis nat gene in culture demonstrated a threefold higher resistance to isoniazid. We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.

Sensitive RIA for the Specific Determination of Insulin Lispro

Insulin lispro is an insulin analog in which the primary sequence has been altered by the inversion of amino acids B28 and B29. To date, it has not been possible to specifically measure insulin lispro in the presence of endogenous insulin because of the high degree of homology between these peptides. However, the specific determination of insulin lispro offers advantages over quantifying total concentrations of immunoreactive insulin. We therefore immunized guinea pigs and screened for antibodies with increased affinity and selectivity for insulin lispro. We prepared a monospecific antiserum by a novel immunoadsorption strategy using despentapeptide insulin. The antiserum was used to develop a competitive RIA for insulin lispro. The RIA has a low limit of quantification (17.2 pmol/L); has no interference from insulin, proinsulin, or C-peptide; and has interassay CVs of 2.6–13.4%. The new RIA is useful for measuring serum concentrations of insulin lispro.

Molecular characterization of antigen 24, a specific immunodominant antigen family from Leishmania infantum

Leishmania infantum immunoelectrophoretic antigen 24 (AG 24), a visceral leishmaniasis associated immunodominant antigen, has been characterized with a monospecific antiserum by combining SDS–PAGE, immunoblotting, metabolic labelling, radio-immunoprecipitation and in vitro poly A+ mRNA translation. AG 24 appeared to correspond to a multi-antigen family of 6–9 members ranging from 20 to 31 kDa and proteinic by nature with no post-translational modifications. A similar banding pattern was recognized by infection sera. AG 24 was not found exposed on the cell surface.

Subcellular Localization of Quinate: Oxidoreductase from Phaseolus mungo L. Sprouts

Quinate:oxidoreductase (QORase, EC 1.1.1.24) was isolated and purified from etiolated mung bean (Phaseolus mungo L.) sprouts and a monospecific antiserum was raised in rabbit to the homogeneous protein. Highly intact etioplasts were isolated from the same plant material. The stroma of the purified etioplasts was enzymatically characterized. Contamination by cytosol, mitochondria and vacuole was estimated from activities of marker en­zymes. QORase activity was localized in the stroma (about 91% for both NAD+ and NADP+ as a cofactor). Western blotting and immunoprinting of the stroma proteins revealed a single band that migrated identically with the purified QORase. The results suggest that the QOR-ase is localized predominantly, if not exclusively, in the etioplast stroma. The physiological role of the enzyme is discussed

Using the CL-770 Spectrophotometer to Quantify Serum Immunoglobulins

Serum immunoglobulins (A, G, M) are usually quantified by radial gel immunodiffusion (RID). It is based on measuring the diameter of the precipitation ring formed when the serum is added to the wells cut out in the agar layer in which the monospecific antiserum is preliminarily dispersed. The gel plates are placed in a humid chamber for 24-48 hours. At the end of the incubation, the diameter of the precipitation ring is measured. Under standard experimental conditions, the diameter of the precipitation ring is directly proportional to the concentration of the studied immunoglobulin. The amount of immunoglobulins is determined by constructing a calibration graph. The results of determining the concentration of serum immunoglobulins by the RID method are issued only on the 3rd day of the study. Clinicians are not always satisfied with the timing of the results and the quality of the study, since the latter depends on many factors - thoroughness (conducting the experiment, the quality and thickness of the gel, the scale of the schedule, etc.)

Cell-free and cell-bound hemolytic activities of Proteus penneri determined by different Hly determinants

A collection of 45 Proteus penneri strains was characterized with respect to their hemolytic activity and representative cell-free or only cell-bound hemolysin possessing strains were chosen for further study. Extracellular Proteus penneri hemolysin, which was investigated earlier by hybridization, reacted with monospecific antiserum against α-hemolysin of Escherichia coli. In this paper we also show, using the colony hybridization technique, that the α-hemolysin-like determinant is widely distributed among Proteus penneri strains. Because one of the strains tested, which expressed a high activity of cell-bound hemolytic factor, did not carry such a Hly determinant, the presence of a second hemolysin is postulated. We cannot demonstrate any difference in hybridization patterns of α- and β-hemolytic Proteus penneri strains and accumulation of the toxin molecule inside the cells was also not observed. The existence of another control mechanism, external to the hly operon, for hemolysin gene is suggested. Key words: Proteus penneri, hemolytic activity.