Pteridine concentrations differ between insectary-reared and field-collected Anopheles albimanus mosquitoes of the same physiological age.

Biopterin, isoxanthopterin and 6-pterincarboxylic acid were identified in the head of the malaria vector mosquito Anopheles albimanus Weidemann (Diptera: Culicidae) by HPLC. Total pteridine concentrations (TPC) were estimated in heads, body parts (BP: abdomen, legs and wings) and whole bodies of insectary-reared and field-collected females, by spectrofluorometry, to investigate whether they could be used for age determination. Pteridine concentrations diminished with age in both mosquito groups. TPC correlated with chronological age in insectary-reared sugar-fed females (heads: r2 = 0.35, BP: r2 = 0.34, P < 0.001), but lower correlation occurred in blood-fed females (heads: r2 = 0.22, BP: r2 = 0.27). TPC differed among females of the same age fed with blood at different times (P < 0.05), indicating that bloodmeals modify the diminution rate of pteridines with age. Nevertheless, a polynomial significant correlation was documented for TPC and the number of ovipositions (heads: r2 = 0.24, BP: r2 = 0.27, whole body: r2 = 0.52, P < 0.001) in insectary-reared mosquitoes. This correlation was lower in field-collected mosquitoes (heads: r2 = 0.14, BP: r2 = 0.10, P < 0.05), which showed a remarkable pteridine increase in one-parous females. The correlation of TPC in whole body with physiological age was much less (r2 = 0.03). These observations indicate that TPC determination by spectrofluorometry is not a reliable method to estimate the age of An. albimanus females from the feral population.

Mg2+, Mn2+, and Co2+ stimulate Entamoeba histolytica to produce chitin-like material.

The mechanism of Entamoeba histolytica cyst cell wall synthesis is not well understood. Previous research has shown that cyst-like structures formed in the presence of chitin synthase cofactors (Mg2+, Mn2+, and Co2+) resist 1% sodium dodecyl sulfate lysis (RCLS), whereas those formed in the absence of cofactors (CLS) do not, and trophozoites are immediately destroyed. This suggests that E. histolytica is able to synthesize chitin, initiating a differentiation process under axenic conditions. To test this hypothesis, polysaccharide hydrolysates from E. histolytica trophozoites, CLS, or RCLS were analyzed with high-performance liquid chromatography. The major components found in all 3 preparations were N-acetylglucosamine (NAG) and glucose (GLC), with RCLS possessing 129 and 180 times more NAG and 2.4 and 2.0 more GLC than trophozoites and CLS, respectively. After 36 hr of incubation with chitinase (16 U/ml) in a hypotonic medium (50 mOsm/kg), 68% of RCLS was lysed, and 100% lost affinity for calcofluor white M2R. The RCLS polysaccharides bound wheat germ agglutinin and appeared as long and thin or short and thick fibers. Accordingly, Mg2+, Mn2+, and Co2+ stimulated E. histolytica to synthesize a chitin-like material.

Mycolic acid index susceptibility method for Mycobacterium tuberculosis.

A rapid drug susceptibility test to measure the susceptibility of Mycobacterium tuberculosis to isoniazid (INH) and rifampin (RIF) using clinical isolates and a newly defined mycolic acid index (MAI) was evaluated. A total of 200 clinical isolates of M. tuberculosis were tested for susceptibility or resistance to INH and RIF by the MAI susceptibility and indirect-proportion methods. Overall, there was agreement between the two methods for 398 (99.5%) of the 400 total tests. Specifically, the sensitivity of the MAI susceptibility method for INH and RIF was 97.6 and 100%, respectively. The specificity and positive predictive value were 100% for both drugs, and the negative predictive value for INH and RIF was 98.3 and 100%, respectively. In conclusion, the MAI susceptibility method described here can be used for rapid drug susceptibility testing of M. tuberculosis clinical isolates within 5 days after clinical isolates are incubated in the presence or absence of an antituberculosis drug.

Drug Susceptibility of Mycobacterium tuberculosisThrough the Mycolic Acid Index.

The methods accepted to determine antimicrobial drug susceptibility of mycobacteria are based on the determination of the microorganisms' growth on solid or liquid medium containing a specified concentration of a single drug.

Mg, Mn, and Co ions enhance the formation of Entamoeba histolytica cyst-like structures resistant to sodium dodecyl sulfate.

BACKGROUND: Entamoeba histolytica forms cyst-like structures (CLS) in PEHPS but not in TYS-33 medium. Sodium dodecyl sulfate [(SDS (0.1%)] dissolves most of them in 10 min, but not natural cysts. Chitin is responsible mainly for cyst wall resistance. Its synthesis depends on Mg(2)+, Mn(2)+, or Co(2)+, whose action is interactive. With the aid of the Simplex method, we analyzed the effect of 20 blends of these cations to find the one that, when added to PEHPS, produced the highest proportion of CLS resistant to 1% SDS (RCLS). METHODS: The concentration of Mg(2)+, Mn(2)+, and Co(2)+ was determined in PEHPS and TYI-S-33 with a flame atomic absorption spectrometer. The proportion of RCLS produced in PEHPS with each ion blend was tested. The CLS and RCLS affinity to fluorescein wheat germ agglutinin (WGA/FITC), which binds chitin, was determined. RESULTS: PEHPS contained a similar concentration of Co(2)+ (0.52 microM) and 3.4 and 1.6 times more Mg(2)+ (798 microM) and Mn(2)+ (3.15 microM) than TYI-S-33, respectively. The proportion of RCLS increased gradually in PEHPS until reaching 3.6 +/- 1.43% with MgCl(2) 1.22 mM, MnCl(2) 14.44 mM, and CoCl(2) 19.44 mM (ion blend No. 20). Both CLS and RCLS bound WGA/FITC. The RCLS formed in the presence of ion blend No. 20 appeared wrinkled. CONCLUSIONS: Mg(2)+, Mn(2)+, and Co(2)+ enhanced the ability of PEHPS to form RCLS, possibly because these ions stimulated their chitin synthesis. Although ion blend No. 20 produced the highest proportion of RCLS, this high ion concentration may be toxic for encysting amebas.

Detecting residual bcr-abl transcripts in chronic myeloid leukaemia patients using coupled reverse transcriptase-polymerase chain reaction with rTth DNA polymerase.

The bcr-abl fusion gene is the hallmark of chronic myeloid leukaemia (CML) and presumably the cause of its development. Accordingly, long-term disappearance of the bcr-abl gene after intensive therapy suggests that a patient is probably cured of CML. The diagnostic protocol based on coupling of two enzymatic reactions, reverse transcription (RT) and nested polymerase chain reaction (nPCR), for the detection of bcr-abl transcripts in peripheral blood provides a powerful tool for minimal residual CML detection. We have developed a new detection protocol using rTth DNA polymerase as the only enzyme catalysing both reactions for simplifying CML diagnosis. We demonstrate its efficacy investigating residual leukaemic cells in the peripheral blood of 10 patients. This assay offers several advantages over the use of conventional RT-PCR, being more sensitive, faster, less prone to false positives since no opening of the tube is required between the two reactions and requires no special oils or waxes. Our simple assay for bcr-abl chimeric transcripts detection is a practical addition to the diagnostic evaluation of the patient with chronic myeloid leukaemia.

Identification of mycobacteria by mycolic acid pattern.

BACKGROUND: Tuberculosis caused by Mycobacterium tuberculosis is a public health problem which has increased in importance during the last 12 years, due in part to the increasing number of cases caused by the association of acquired immunodeficiency syndrome (AIDS) and the appearance of multiple drug-resistant strains. Other mycobacteria which are often indistinguishable from tuberculosis have also increased. METHODS: Mycolic acid patterns were obtained from 53 clinical isolates of sputum, cerebrospinal fluid, bronchial washing, corneal ulcer, and bone marrow, as well as from 11 acid-fast stain smear-positive clinical specimens. Standardized mycolic acid extraction method was used to ensure the maximal extraction of mycolic acid derivatives to enhance the sensitivity of the method. A chromatographic column different from what others have employed and a different gradient elution from those reported in the literature were used, making a correlation between retention times for the chromatographic peaks obtained in this study and those previously reported for mycolic acid patterns from a strain of Mycobacterium avium necessary. Then, a comparison of retention times of mycolic acid pattern obtained in this study and those previously reported in the literature was carried out. Strains were identified as Mycobacterium tuberculosis complex, Mycobacterium avium complex, Mycobacterium fortuitum, Mycobacterium chelonae and Mycobacterium kansasii in less than 24 hours. RESULTS: In direct analysis of acid-fast stain smearpositive from 1+ to 4+ specimens, mycolic acid patterns were identified as Mycobacterium tuberculosis complex, Mycobacterium avium complex, Mycobacterium chelonae, and Mycobacterium kansasii, with a strong signal even in light 1+ positive samples. CONCLUSIONS: The results showed that identification of mycobacteria through mycolic acid pattern is a rapid, sensitive, and very useful method for identification of mycobacteria in the early diagnosis of the mycobacteriosis.

Determination of drug susceptibility of Mycobacterium tuberculosis through mycolic acid analysis.

In the present work a rapid method to determine the susceptibility of Mycobacterium tuberculosis to isoniazid and streptomycin by determining levels of mycolic acids by high-performance liquid chromatography (HPLC) was developed. Mycobacterial growth kinetics in the presence and absence of antituberculosis drugs was characterized by evaluating the total area corresponding to mycolic acid peaks (TAMA). Results show a linear relationship between the logarithm of CFU per milliliter and TAMA and show that it is possible to detect growth inhibition of M. tuberculosis in the presence of isoniazid or streptomycin by using HPLC in 3 and 4 days, respectively.

Natural anthracenone subcellular distribution and effects on NADPH-cytochrome P450 reductase microsomal activity.

Natural anthracenone subcellular distribution and effects on NADPH-cytochrome P450 reductase microsomal activity. Subcellular distribution study of a natural anthracenone (T-514) isolated from Karwinskia humboldtiana showed to be homogeneous on subcellular (nuclear, mitochondrial, peroxisomal and microsomal) fractions prepared from rat liver treated with an acute dose of T-514. These results indicate that T-514 can pass easily through subcellular compartment membranes and an absence of selectivity for some subcellular organelles. A significant increase of protein on liver homogenates and NADPH-cytochrome P450 reductase microsomal activity indicates that T-514 may act as a microsomal enzymatic inducer. In addition, this enzymatic specific activity increment could be due to the interaction of T-514 with the microsomal redox cycling.

[Pharmacokinetic design for the preclinical phase. Application to the study of an anthracenone]. / Diseño farmacocinético de fase preclínica. Aplicación al estudio de una antracenona.

T-514 is a toxic substance of Karwinskia humboldtiana which has been described as a possible anticancer agent. An animal model (New Zeland white rabbit) was selected for pharmacokinetic studies that would allow the performance of surgical techniques to catheterize central vessels in order to obtain blood samples at short time intervals. A nonlinear regression analysis method was used to fit the plasmatic concentration data to a multiexponential mathematical function; a computer program based on Marquant's iterating algorithm was used to fit the experimental curves. In the present work we show the advantage of introducing two catheters of different diameters in the jugular vein using modifications of the Seldinger and Braunüle techniques: a catheter of small diameter (18 G) is used to introduce the drug and a larger one (16 G) to sample at different times. The larger diameter catheter facilitates a rapid sampling of blood (two mL/s) which is essential to determine the initial phase of the distribution process (alpha phase). The behavior of T-514 corresponded to a two-compartment model with a biexponential equation C = 0.35 e-0.13(t-2) + 0.17 e-0.03(t-2).