Molecular cloning of cDNA for human prostatic acid phosphatase

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.     

Detection of ionizing radiations with the SOS Chromotest, a bacterial short-term test for genotoxic agents

The effects of 3 types of ionizing radiation, gamma-rays, neutrons and accelerated alpha-particles, were examined using the SOS Chromotest, a bacterial colorimetric assay for genotoxic agents based on the measurement of the SOS response in Escherichia coli. The SOS Chromotest appeared to be a sensitive and simple assay to detect quantitatively these radiations as well as their biological effects. The range of adsorbed doses for which induction was observed was similar for the 3 types of radiation, the minimum inducing doses being in the order of 2.5-5 Gy. We discuss the possible use of these observations to study the molecular action of radiations and to compare their genotoxic effects with those of chemicals.     

Thrombolysis using liposomal-encapsulated streptokinase: an in vitro study

The clot-lysing ability of streptokinase (SK) was examined using membrane-bound thrombi. Encapsulation of SK in large unilamellar phospholipid vesicles (liposomes) resulted in entrapping approximately 30% of its original activity. Measurements of streptokinase activity for liposomal-encapsulated streptokinase (LESK) indicated little loss of activity or leakage in Tris-buffered saline over a 24-hr period at temperatures of 4 and 23 degrees C. However, incubation of free SK and LESK in platelet-poor plasma (PPP) at 37 degrees C resulted in a decrease of SK activity. The retention of SK activity in LESK was considerably higher than that of unentrapped SK. Clot-dissolving time (CDT) was measured by monitoring the pressure drop during slow filtration in plasma through membrane-bound thrombi. The results indicated that both LESK and free SK were able to activate the fibrinolytic system. Without prior incubation in PPP at 37 degrees C, the CDT of a SK and PPP mixture (SK/PPP) was 10.7 +/- 1.9 min (n = 12), while that of a LESK and PPP mixture (LESK/PPP) was 12.4 +/- 1.7 min (n = 12). The CDT-detected clot-lysing abilities of both SK and LESK were diminished by incubation in PPP, but to different extents. After 15- and 30-min incubations, the CDT of SK/PPP increased significantly to 15.5 +/- 1.5 and 24.1 +/- 2.4 min (n = 5, P less than 0.05), respectively. In contrast, the CDT of LESK/PPP increased to 13.3 +/- 0.8 min (n = 5) after 15 min of incubation and to 16.0 +/- 1.1 min (n = 5, P less than 0.05) after a 30-min incubation. These results suggest that entrapment of SK in liposomes preserves the thrombolytic potential of the plasminogen activator by limiting its exposure to the components of the plasma.     

Cloning of a lymphocyte homing receptor reveals a lectin domain

Lymphocytes express cell surface molecules, termed homing receptors, that mediate their selective attachment to specialized high endothelial venules found within secondary lymphoid organs. Previous work has demonstrated that the adhesive interaction between lymphocytes and the endothelium of peripheral lymph nodes appears to involve a lectin-like activity. Moreover, MEL-14, a monoclonal antibody that blocks lymphocyte-peripheral lymph node binding and presumably recognizes the homing receptor mediating this adhesive interaction, appeared to detect the lectin-like receptor. In this paper we describe the cloning of a murine cDNA that encodes the antigen recognized by the MEL-14 antibody. Characterization of the cDNA encoding the putative mouse peripheral lymph node-specific homing receptor shows that it contains a lectin domain that appears to be involved in the binding of lymphocytes to peripheral lymph node endothelium, thus defining a new type of cellular adhesion molecule. This result supports a novel mechanism for the distribution of lymphocyte populations to various lymphoid organs.     

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2

An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40 degrees C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (V(max)) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-beta-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.     

Limited chloroplast gene transfer via recombination overcomes plastomegenome incompatibility between Nicotiana tabacum and Solanum tuberosum

Green cybrids with a new nucleus-chloroplast combination cannot be selected after protoplast fusion in the intersubfamilial Nicotiana-Solanum combination. As an approach to overcome the supposed plastomegenome incompatibility, a partial plastome transfer by genetic recombination has been considered. After fusions of protoplasts of a light-sensitive Nicotiana tabacum (tobacco) plastome mutant and lethally irradiated protoplasts of wild-type Solanum tuberosum (potato), a single green colony was recovered among 2.5×10⁴ colonies. The regenerated plants had tobacco-like (although abnormal) morphology, but were normally green, and sensitive to tentoxin, demonstrating chloroplast markers of the potato parent. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed recombinant, nonparental patterns. A comparison with physical maps of the parental cpDNA demonstrated the presence of a considerable part of the potato plastome flanked by tobacco-specific regions. This potacco plastome proved to be stable in backcross and backfusion experiments, and normally functional in the presence solely of N. tabacum nucleus.     

Fluorescence-based viability assay for studies of reactive drug intermediates

Studies of drug toxicity, toxicologic structure-function relationships, screening of idiosyncratic drug reactions, and a variety of cytotoxic events and cellular functions in immunology and cell biology require the sensitive and rapid processing of often large numbers of cell samples. This report describes the development of a high-sensitivity, high-throughput viability assay based on (a) the carboxyfluorescein derivative 2'-7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) as a vital dye, (b) instrumentation capable of processing multiple small (less than 100 cells) samples, and (c) a 96-well unidirectional vacuum filtration plate. Double staining of cultured peripheral blood mononuclear cells with BCECF and propidium iodide (PI) showed no overlap between PI+ (nonviable) and BCECF+ (viable) cells by flow cytometric analysis. Optimal conditions were developed for dye loading and minimizing physical cell damage and fluorescence quench during the assay procedure. The ratio of BCECF fluorescence to internal standard fluorescent particles was linear from 40 to greater than 20,000 cells with a signal:noise ratio of approximately 3 at 40 cells/well. Sulfamethoxazole hydroxylamine (SMX-HA) was used as a model toxic drug metabolite to explore the validity of the BCECF procedure. SMX-HA, but not its parent compound sulfamethoxazole, resulted in a dose dependent loss of cellular fluorescence and the parallel accumulation of PI+ nonviable cells. When compared to the currently used tetrazolium dye reduction viability assay, the BCECF method was 3-fold more sensitive, greater than 10-fold faster, and required 1/10-1/100 the cell numbers.     

Lipid peroxidation in Ehrlich ascites cell mitochondria is not determined by the polyunsaturated fatty acid content of the membrane

Lipid peroxidation intensity is compared in Ehrlich Ascites Cell and in liver mitochondria, prepared from tumor bearing mice. Malondialdehyde formation is negligible in intact ascites tumour mitochondria, but it is significantly increased in permeabilised mitochondria and in isolated mitochondrial membranes. We suggest that the resistance against oxidative stress is a consequence of efficient protective mechanisms operating in the intact tumour mitochondria and the low level of polyunsaturated fatty acids under these circumstances cannot be the rate limiting factor in lipid peroxidation. Succinate, an effective inhibitor of mitochondrial lipid peroxidation in liver, cannot determine malondialdehyde formation in ascites tumour mitochondria.