Lactobacillus acidophilus Autolysins Inhibit Helicobacter pylori In Vitro

Antibacterial activity of 17 strains of lactobacilli was tested against 10 strains of H. pylori. The inhibition observed was related to the acid production and the low pH attained. No relationship between CagA phenotype of H. pylori strains and tolerance to lactic acid was observed. In mixed cultures, L. acidophilus CRL 639 showed an autolytic behavior after 24 h of culture. At this moment, H. pylori CCUG17874 showed a decrease of 2 log-cycle, and no viable count was detected after 48 h. The bactericidal effect of L. acidophilus CRL 639 in mixed cultures is related to a proteinaceous compound released after cell lysis. Received: 19 June 2000 / Accepted: 12 July 2000     

Characterization of INTA 51-3, a New Atypical Strain of Bacillus thuringiensis from Argentina

Several isolates of Bacillus thuringiensis native to Argentina obtained in a nationwide screening program showed atypical crystal morphology. One of these strains, INTA 51-3, was further characterized in order to determine other features like protein composition of its parasporal crystal, plasmid pattern, identification of cry genes and toxicological properties. B. thuringiensis INTA 51-3 (serovar tohokuensis) had an amorphous inclusion containing a major protein component of ca. 130 kDa. After trypsin digestion of solubilized crystals, SDS-PAGE resolved a unique protease-resistant peptide of ca. 90 kDa. The plasmid pattern from INTA 51-3 resembled that of the standard strain HD-1. However, Southern analysis showed no hybridization to fragments of cry1Aa, cry2Aa, cry3A, and cry11A genes. Degenerate primers were used for identification of the cry1 genes by PCR. Nevertheless, the presence of cry1 type gene(s) in B. thuringiensis INTA 51-3 was confirmed. Highly concentrated crystal suspensions showed to be weakly toxic only to lepidopteran species. Received: 23 May 2000 / Accepted: 26 June 2000     

Inhibition ofras oncogene: A novel approach to antineoplastic therapy

The most frequently detected oncogene alterations, both in animal and human cancers, are the mutations in the ras oncogene family. These oncogenes are mutated or overexpressed in many human tumors, with a high incidence in tumors of the pancreas, thyroid, colon, lung and certain types of leukemia. Ras is a small guanine nucleotide binding protein that transduces biological information from the cell surface to cytoplasmic components within cells. The signal is transduced to the cell nucleus through second messengers, and it ultimately induces cell division. Oncogenic forms of p21(ras) lead to unregulated, sustained signaling through downstream effectors. The ras family of oncogenes is involved in the development of both primary tumors and metastases making it a good therapeutic target. Several therapeutic approaches to cancer have been developed pointing to reducing the altered gene product or to eliminating its biological function: (1) gene therapy with ribozymes, which are able to break down specific RNA sequences, or with antisense oligonucleotides, (2) immunotherapy through passive or active immunization protocols, and (3) inhibition of p21(ras) farnesylation either by inhibition of farnesyl transferase or synthesis inhibition of farnesyl moieties.     

Squid nerve Na+/Ca2+ exchanger expressed in Saccharomyces cerevisiae: Up-regulation by a phosphorylated cytosolic protein (ReP1–NCXSQ) is identical to that of native exchanger in situ

This work shows, for the first time, a properly metabolically regulated squid nerve Na⁺/Ca²⁺ exchanger (NCXSQ1) heterologous expressed in Saccharomyces cerevisiae. The exchanger was fused to the enhanced green fluorescence protein (eGFP) on its C-terminus and had two tags, a Strep-tag II and 6 histidines, added to the N-terminal region (ST–6HB–NCXSQ1–eGFP). The eGFP fluorescence signal co-localized with that of the plasma membrane marker FM1-43 in whole cells that displayed an uptake of Ca²⁺ with the expected characteristics of the reverse Na⁺/Ca²⁺ exchange mode. Similar to squid nerve membrane vesicles, inside-out yeast plasma membrane vesicles (ISOV) showed a Ca²⁺_i regulation of the forward mode that was modulated by previously phosphorylated regulatory cytosolic protein (ReP1–NCXSQ). On the other hand, a close association between NCXSQ1 and ReP1–NCXSQ, estimated by co-immunoprecipitation, was independent of ReP1–NCXSQ phosphorylation. An additional crucial observation was that in proteoliposomes containing only the ST–6HB–NCXSQ1–eGFP protein, Na⁺/Ca²⁺ exchange was stimulated by phosphorylated ReP1–NCXSQ; i.e., this up-regulation needs no other requirement besides the lipid membrane and the exchanger protein. Finally, this work provides a potential approach to obtain enough purified NCXSQ1 for structural and biochemical studies which have been delayed due to the lack of sufficient material.     

Mitochondrial Profiling of Acute Myeloid Leukemia in the Assessment of Response to Apoptosis Modulating Drugs

BH3 profiling measures the propensity of transformed cells to undergo intrinsic apoptosis and is determined by exposing cells to BH3-mimicking peptides. We hypothesized that basal levels of prosurvival BCL-2 family proteins may modulate the predictive power of BH3 profiling and termed it mitochondrial profiling. We investigated the correlation between cell sensitivity to apoptogenic agents and mitochondrial profiling, using a panel of acute myeloid leukemias induced to undergo apoptosis by exposure to cytarabine, the BH3 mimetic ABT-199, the MDM2 inhibitor Nutlin-3a, or the CRM1 inhibitor KPT-330. We found that the apoptogenic efficacies of ABT-199 and cytarabine correlated well with BH3 profiling reflecting BCL2, but not BCL-XL or MCL-1 dependence. Baseline BCL-2 protein expression analysis increased the ability of BH3 profiling to predict resistance mediated by MCL-1. By utilizing engineered cells with overexpression or knockdown of BCL-2 family proteins, Ara-C was found to be independent, while ABT-199 was dependent on BCL-XL. BCL-2 and BCL-XL overexpression mediated resistance to KPT-330 which was not reflected in the BH3 profiling assay, or in baseline BCL-2 protein levels. In conclusion, mitochondrial profiling, the combination of BH3 profiling and prosurvival BCL-2 family protein analysis, represents an improved approach to predict efficacy of diverse agents in AML and may have utility in the design of more effective drug combinations.     

Functional characterization of the CDF transporter SMc02724 (SmYiiP) in Sinorhizobium meliloti: Roles in manganese homeostasis and nodulation

In bacteria, membrane transporters of the cation diffusion facilitator (CDF) family participate in Zn^2 +, Fe^2 +, Mn^2 +, Co^2 + and Ni^2 + homeostasis. The functional role during infection processes for several members has been shown to be linked to the specificity of transport. Sinorhizobium meliloti has two homologous CDF genes with unknown transport specificity. Here we evaluate the role played by the CDF SMc02724 (SmYiiP). The deletion mutant strain of SmYiiP (ΔsmyiiP) showed reduced in vitro growth fitness only in the presence of Mn^2 +. Incubation of ΔsmyiiP and WT cells with sub-lethal Mn^2 + concentrations resulted in a 2-fold increase of the metal only in the mutant strain. Normal levels of resistance to Mn^2 + were attained by complementation with the gene SMc02724 under regulation of its endogenous promoter. In vitro, liposomes with incorporated heterologously expressed pure protein accumulated several transition metals. However, only the transport rate of Mn^2 + was increased by imposing a transmembrane H⁺ gradient. Nodulation assays in alfalfa plants showed that the strain ΔsmyiiP induced a lower number of nodules than in plants infected with the WT strain. Our results indicate that Mn^2 + homeostasis in S. meliloti is required for full infection capacity, or nodule function, and that the specificity of transport in vivo of SmYiiP is narrowed down to Mn^2 + by a mechanism involving the proton motive force.     

Agentes de micosis endémicas en un área rural de Argentina: estudio seroepidemiológico en perros

BackgroundThree fungal species causing human disease, namely Paracoccidioides brasiliensis, Histoplasma capsulatum and Coccidioides sp., are endemic in different areas of Argentina. Rates of infection in domestic dogs have been used in other Latin American countries as indicators of the presence of these pathogens in a given area. We used such an approach to investigate the epidemiological relevance of paracoccidiodomycosis, histoplasmosis and coccidioidomycosis in our country.AimTo investigate the presence of P. brasiliensis, H. capsulatum and Coccidioides sp. in a rural area of Argentina called Interfluvio Teuco-Bermejito, located in Chaco province.MethodsWe applied Western Blotting to determine the presence of specific antibodies in sera from 89 domestic dogs inhabiting the area. Antibodies against the following extra-cellular fungal antigens were investigated: gP43 of P. brasiliensis, H/M of H. capsulatum and 120, 82 and 48 kDa antigen bands of Coccidioides sp.ResultsSpecific antibodies against H. capsulatum were found in 9/89 (10%) sera: 8 reacted against both H and M antigens and 1 only reacted against antigen M. Of these 9 sera, one showed additional anti-gp43 activity and another reacted against all the fungal antigens tested.ConclusionsThis is the first study using dog infection to assess the presence of endemic fungal pathogens in Argentina. Our results suggest that H. capsulatum is the main dimorphic fungal pathogen in the Interfluvio Teuco-Bermejito area. Therefore, the diagnosis of histoplasmosis should be taken into account in patients living in this geographic region who show pulmonary or mucocutaneous symptoms compatible with the disease.     

Anti-malarial market and policy surveys in sub-Saharan Africa

Background

Malaria microscopy and rapid diagnostic tests are insensitive for very low-density parasitaemia. This insensitivity may lead to missed asymptomatic sub-microscopic parasitaemia, a potential reservoir for infection. Similarly, mixed infections and interactions between Plasmodium species may be missed. The objectives were first to develop a rapid and sensitive PCR-based diagnostic method to detect low parasitaemia and mixed infections, and then to investigate the epidemiological importance of sub-microscopic and mixed infections in Rattanakiri Province, Cambodia.

Methods

A new malaria diagnostic method, using restriction fragment length polymorphism analysis of the cytochrome b genes of the four human Plasmodium species and denaturing high performance liquid chromatography, has been developed. The results of this RFLP-dHPLC method have been compared to 1) traditional nested PCR amplification of the 18S rRNA gene, 2) sequencing of the amplified fragments of the cytochrome b gene and 3) microscopy. Blood spots on filter paper and Giemsa-stained blood thick smears collected in 2001 from 1,356 inhabitants of eight villages of Rattanakiri Province have been analysed by the RFLP-dHPLC method and microscopy to assess the prevalence of sub-microscopic and mixed infections.

Results

The sensitivity and specificity of the new RFLP-dHPLC was similar to that of the other molecular methods. The RFLP-dHPLC method was more sensitive and specific than microscopy, particularly for detecting low-level parasitaemia and mixed infections. In Rattanakiri Province, the prevalences of Plasmodium falciparum and Plasmodium vivax were approximately two-fold and three-fold higher, respectively, by RFLP-dHPLC (59% and 15%, respectively) than by microscopy (28% and 5%, respectively). In addition, Plasmodium ovale and Plasmodium malariae were never detected by microscopy, while they were detected by RFLP-dHPLC, in 11.2% and 1.3% of the blood samples, respectively. Moreover, the proportion of mixed infections detected by RFLP-dHPLC was higher (23%) than with microscopy (8%).

Conclusions

The rapid and sensitive molecular diagnosis method developed here could be considered for mass screening and ACT treatment of inhabitants of low-endemicity areas of Southeast Asia.