Discovery of new low-molecular-weight p53–Mdmx disruptors and their anti-cancer activities

Although several p53–Mdm2-binding disruptors have been identified to date, few studies have been published on p53–Mdmx-interaction inhibitors. In the present study, we demonstrated that o-aminothiophenol derivatives with molecular weights of 200–300 selectively inhibited the p53–Mdmx interaction. S-2-Isobutyramidophenyl 2-methylpropanethioate (K-178) (1c) activated p53, up-regulated the expression of its downstream genes such as p21 and Mdm2, and preferentially inhibited the growth of cancer cells with wild-type p53 over those with mutant p53. Furthermore, we found that the S-isobutyryl-deprotected forms 1b and 3b of 1c and S-2-benzamidophenyl 2-methylpropanethioate (K-181) (3c) preferentially inhibited the p53–Mdmx interaction over the p53–Mdm2 interaction, respectively, by using a Flag-p53 and glutathione S-transferase (GST)-fused protein complex (Mdm2, Mdmx, DAPK1, or PPID). In addition, the interaction of p53 with Mdmx was lost by replacing a sulfur atom with an oxygen atom in 1b and 1c. These results suggest that sulfides such as 1b, 3b, 4b, and 5b interfere with the binding of p53–Mdmx, resulting in the dissociation of the two proteins. Furthermore, the results of oral administration experiments using xenografts in nude mice indicated that 1c reduced the volume of tumor masses to 49.0% and 36.6% that of the control at 100 mg/kg and 150 mg/kg, respectively, in 40 days.     

ELISA absorbance cut-off method affects malaria sporozoite rate determination in wild Afrotropical Anopheles

ABSTRACT. Malaria sporozoite infection rates in a mixed species group of 244 Anopheles gambiae Giles sensu lato and 115 An. funestus Giles wild female mosquitoes were compared using three methods to determine cutoff absorbance values for positivity of a Plasmodium falciparum Welch enzyme-linked immunosorbent assay (ELISA). Positive controls were based on P. falciparum circumsporozoite protein. As negative controls, four wild male Anopheles were included on each microtitre plate; tests were repeated on four consecutive days for each plate.
Infection rates were estimated at 13.1–22.8% using the mean absorbance value of negative controls plus three standard deviations, 11.7–12.8% using double the mean and 12.5–13.6% using the fixed cut-off value of 0.20 (allowing for 20% variation in negative control absorbance values).
Observed agreement for positivity or negativity among samples tested four times was 98.6% for the 2× mean method, 97.2% for the fixed cut-off 0.20 value, but only 82.7% for the mean +3 SD method. It was concluded that the 2× mean cut-off method is most reliable for field studies. P. falciparum sporozoite rates of 12.2% in An. funestus and 11.9% in An. gambiae s. l . were thus determined on the basis of the 2× mean cut-off method.
This comparative evaluation demonstrates that vector infectivity rates can be seriously over-estimated from sporozoite ELISA tests, by as much as 87% in one case considered here, depending on the absorbance cut-off method applied for negative controls.     

Comparative analysis of antigenic strength and in vivo serum antibodies concentration of tetanus toxoid vaccine adsorbed in Pakistan

Clostridium tetani produce tetanospasmin, a potent exotoxin; that causes tetanus or lockjaw disease. Scientists developed an anti-tetanus toxoid to protect the body from the spasm's neurotoxic effect. In Pakistan recently, 478 cases of neonatal tetanus were reported. The study was carried out at The National Control Laboratory for Biologicals Islamabad, aiming to decipher the effectiveness of the most frequently used tetanus toxoid vaccine adsorbed in Pakistan in comparison to standard reference vaccine having earlier known consistent values. The vaccines included domestic public sector, domestic private sector, imported private sector I, and imported private sector II. The triplicate experiments on purebred Swiss albino mice were performed by immunizing with Tetanus toxoid and then tested parallel with standard reference vaccine. Various analytical tests were performed on the test organism that included flocculation test/identity test, antibody quantification using enzyme-linked immunosorbent assay (ELISA), potency test, abnormal toxicity test, osmolality, pH test, liquid sub-visible particle test, and sterility test. Results of all the vaccines were compared in comparison with the standard reference vaccine. Absorbances of test vaccines were recorded at the lowest dilution by ELISA. The domestic private sector, imported private sector I, imported private sector II and standard reference vaccine were flocculated at mean dilution (Mean: 0.24, 95% CI: 0.1903–0.2897), and the domestic public sector was flocculated at mean dilution (Mean: 0.23, 95% CI: 0.2052–0.2548). All the products were found within the normal ranges where it was concluded that the maximum average titer of 2.81 was observed at dilution 10^?1.6, indicating that these vaccines were adequate/suitable for the prevention of tetanus.     

Detection of coccidioidal antibodies in serum of a small rodent community in Baja California,Mexico

Coccidioidomycosis (Valley Fever) represents a serious threat to inhabitants of endemic areas of North America. Despite successful clinical isolations of the fungal etiological agent, Coccidioides spp., the screening of environmental samples has had low effectiveness, mainly because of the poor characterization of Coccidioides ecological niche. We explored Valle de las Palmas, Baja California, Mexico, a highly endemic area near the U.S.–Mexico border, where we previously detected Coccidioides via culture-independent molecular methods. By testing the serum from 40-trapped rodents with ELISA, we detected antibodies against Coccidioides in two species: Peromyscus maniculatus and Neotoma lepida. This study comprises the first report of wild rodent serum tested for coccidioidal antibodies, and sets the basis to analyze this pathogen in its natural environment and explore its potential ecological niche.     

Accessibility of an epitope common to all histone H3 variants in folded and unfolded chromatin as studied by a monoclonal antibody

In a recent publication the isolation and some characteristics of an anti-histone 3 monoclonal antibody, 1GB3 were described (Muller et al. FEBS Lett. 182: 459–464, 1985). We now report that the epitope recognized is phylogenetically conserved and located in the N-terminal part of H3, most likely between residues 40 and 50. Using the ELISA technique we found this region to be accessible in chromatin to the monoclonal antibody. The effect of non-ionic detergents on the adsorbtion of chromatin on microtiter plates was studied in this context.Immunological analysis of the reaction of the monoclonal antibody with chromatin by immunoinhibition and immunosedimentation shows that the H3 epitope is accessible in both folded and unfolded chromatin fibre as well as in high- and low-molecular weight oligonucleosomes.Abbreviations BSA Bovine srum albumin - mab Monoclonal antibody - PBS Phosphate buffered saline - PMSF Phenylmethyl sulfonyl fluoride     

An order-specific monoclonal antibody to Diptera reveals the impact of alternative prey on spider feeding behavior in a complex food web

Generalist predators have the capacity to restrict pest population growth, especially early in the season before densities increase. However, their polyphagous feeding habits sometimes translate into reduced pest consumption when they target alternative prey. An order-specific monoclonal antibody was developed to examine the strength of trophic connections between Diptera, a major category of non-pest prey, and linyphiid spiders in alfalfa. We report the development and characterization of a monoclonal antibody with order-level specificity to Diptera. This antibody elicited strong absorbance to 22 Diptera from 13 families, no false-positive reactivity to non-dipteran invertebrates, and antigen detection periods following prey consumption that were comparable between spiders. Over 900 field-collected females of the linyphiid spiders Erigone autumnalis and Bathyphantes pallidus were screened for Diptera antigen. Significantly more B. pallidus screened positive for Diptera (40%) compared to E. autumnalis (16%), indicating differential reliance on these prey. In parallel with the collection of spiders for gut-content analysis, prey availability was estimated at web sites. The two spiders exhibited different feeding responses to prey availability. Consumption of Diptera by B. pallidus was strongly correlated with Diptera abundance whilst the availability of other potential prey did not influence predation rates. Conversely, E. autumnalis did not prey upon Diptera in proportion to availability, but increased Collembola activity-density reduced dipteran consumption. Integration of molecular gut-content analysis with precise sampling of prey demonstrated how two closely related linyphiid spiders exhibit different feeding responses to the availability of prey under natural field conditions. Elucidating the feeding preferences of natural enemies is critical to effective incorporation of biological control by generalist predators in the management of agricultural pests.     

Determination of human factor VIII (AHG-associated protein)-antigen in endothelial cells from Xenopus laevis (XTH cells)

Summary AHG-associated protein (AHG-a.p.), the antigen of the blood-clotting factor VIII complex, is a specific endothelial cell marker. Primary (p-XTH) and established (XTH-2) endothelial cells from the hearts of Xenopus laevis tadpoles were assayed for the presence of this marker by means of immunological cross-reaction (recognition of common antigenic sites) with antiserum against human AHG-a.p. Radial imtnunodiffusion and rocket immunoelectrophoresis proved to be insufficiently sensitive, whereas immunofluorescence and a newly evaluated ELISA technique gave positive results. The very high sensitivity of the ELISA (less than 1/240000 of the AHG-a.p. in 0.1 ml human standard plasma can be detected) and the removal of interfering proteins by gel filtration also revealed the presence of AHG-a.p. in the fetal calf serum used in the culture medium; earlier investigations into this subject by a one-step radioimmunoassay had reported negative results. Specially adapted XTH-2 cells were grown in a proteinand serum-free hydrolysate medium in order to demonstrate the presence of a Xenopus-derived antigen that was immunoreactive with the anti-human AHG-a.p.     

Haptoglobin biosynthesis in rats Immunological identification of polysomes synthesizing haptoglobin and quantitation of haptoglobin in the cytoplasm of liver cells

A quantitative enzyme-linked immunosorbent assay was developed and utilized to study the stimulation of haptoglobin biosynthesis during an acute inflammatory challenge. A 10-fold increase in intracellular haptoglobin was measured at the peak of the inflammatory response. The increase in serum haptoglobin levels was concomitant with the intracellular levels, demonstrating the secretory output is also elevated during the inflammatory period. A monospecific antihaptoglobin was produced and used to detect the specific polysomes involved in haptoglobin synthesis. The amount of radioactively labeled antibody bound to the nascent haptoglobin chain was increased approx. 3-fold during the inflammatory response, indicating that new haptoglobin was being synthesized and suggesting an increase in functional haptoglobin mRNA resulting from the inflammatory signal.     

ELISA analyses of malate dehydrogenases from nonsulfur purple phototrophic bacteria

Abstract The accumulation of ppGpp in three streptococci starved for isoleucine was studied via HPLC analysis of cell extracts prepared from mechanically disrupted bacteria. Starvation was achieved either by reduction of isoleucine in the growth medium or the addition of pseudomonic acid. The results indicate that while both treatments produced a physiological response similar to that described for stringent strains of other bacteria, in the streptococci, stringency was not necessarily coupled with ppGpp.     

Immunochemical identification of mung bean actin like protein and its cellular involvement during germination

Actin like protein, extracted and purified fromVigna radiata (mung bean) seedling, has been found to give positive enzyme-linked immunosorbent assay with mouse monoclonal antiactin antibody. In vivo studies show that cytochalasin B at sublethal dose inhibits the chromosomal movement at metaphase stage during germination. Fromin vitro studies it is found that the actin like protein isolated from mung bean seedling has a cytochalasin B binding property with a Kd value 1.2 × 10⁻⁵ M. From these two specific observations it appears probable that the biological function of mung bean actin like protein is to take part in cell division process directly or indirectly during the time of seedling development.