Photoinduced electron transfer fluorometric Hg(II) chemosensor based on a BODIPY armed with a tetrapod receptor.

From the great variety of BODIPY based-chemosensors able to determine Hg(2+), only a small portion has been applied to its determination in environmental and/or biological samples. The lack of studies on the analytical performance of the latter sensors makes interesting the development of investigations oriented to their possible analytical applications. The synthesis of a BODIPY derivative armed with a tetrapod receptor is described. The procedure is based on a previous publication, and the modifications performed to improve the synthesis include alternative procedures with different objectives, as the consecution of a multigram synthesis, improving the low yields of some of the previously proposed procedure steps, simplifying the experimental steps, achieving the desired purity requirements for use with analytical purposes, and enriching the characterization of the implied structures. The characteristics of its selectivity towards Hg(2+) have been investigated, and the OFF-ON fluorometric response, based on a photo-electron transfer (PET) mechanism, served as the base for the development of a method able to determine Hg(2+) in environmental waters at ng mL(-1) levels. The intrinsic fluorescence of the BODIPY core is inhibited and the probe exhibits a weak fluorescence (i.e. "OFF" state due to the deactivating PET effect). Upon complexation, Hg(2+) interacts with the lone-pair electrons on the nitrogen atoms of the receptor moiety so that the electronic transfer from the receptor to the photo-excited fluorophore is slowed down or switched off (i.e. "ON" state due to the suppression of the deactivating PET effect by coordination of the analyte to the probe). Regarding the complex photostability in aqueous solution, it is mandatory to conduct the experiments at darkness due to its photodegradation. The stoichiometry studies indicated a 1:2 relationship for the BODIPY-Hg(2+) complex. The high selectivity towards mercuric ions is considerably influenced by pH, being necessary to conduct the experiments in a pH value higher than 6. Calibration samples were prepared by adding appropriate amounts of Hg(2+) between 20.0-120.0 ng mL(-1), at a constant BODIPY concentration of 1 µmol L(-1). After agitating for 5 min at darkness, phosphate buffer (pH=7.50) was added, and it was diluted to the mark with water. Fluorescence measurements were carried out at 18 °C, exciting at 515 nm, and obtaining fluorescence emission at 538 nm. The method has been satisfactory applied to Hg(2+) determination in environmental water samples.

Evidence of exotoxin secretion of Piscirickettsia salmonis, the causative agent of piscirickettsiosis.

Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a disease which affects a variety of teleost species and that is particularly severe in salmonid fish. Bacterial-free supernatants, obtained from cultures of three isolates of Piscirickettsia salmonis, were inoculated in Atlantic salmon, Salmo salar L., and in three continuous cell lines in an effort to determine the presence of secretion of extracellular products (ECPs) by this microorganism. Although steatosis was found in some liver samples, no mortalities or clinical signs occurred in the inoculated fish. Clear cytotoxicity was observed after inoculation in the cell lines CHSE-214 and ASK, derived from salmonid tissues, but not in MDBK, which is of mammalian origin. The degree of cytotoxicity of the ECPs was different among the P. salmonis isolates tested. The isolate that evidenced the highest cytotoxicity in its ECPs exhibited only an intermediate virulence level after challenging fish with bacterial suspensions of the three P. salmonis isolates. Almost complete inhibition of the cytotoxic activity of ECPs was seen after proteinase K treatment, indicating their peptidic nature, and a total preclusion of the cytotoxicity was shown after their incubation at 50 °C for 30 min. Results show that P. salmonis can produce ECPs and at least some of them are thermolabile exotoxins that probably play a role in the pathogenesis of piscirickettsiosis.

Detection of emerging HIV variants in blood donors from urban areas of Cameroon.

The U.S. Food and Drug Administration has licensed several assays for use in donor testing and management of persons with human immunodeficiency virus (HIV) infection. However, the performance of these assays for detection and quantitation of emerging HIV genetic variants has not been studied extensively. We tested 240 human plasma specimens collected from two urban blood centers in Cameroon where HIV genetic diversity and recombinant HIV strains are highly prevalent, using several FDA licensed assays. The testing record in Cameroon indicated that 149 specimens were HIV antibody positive and 91 specimens were negative using a rapid HIV-1/2 antibody assay in routine use in Cameroon blood centers. Both sets of samples were evaluated in the FDA laboratory using four ELISA tests for HIV-1 group M, HIV-1 group O, and HIV-2 antibodies, one IFA for HIV-1 antibody, one Western blot for HIV-1, one HIV-1 p-24 antigen assay, and three nucleic acid tests (NAT). Our results indicate that the assays had high sensitivity for detection of emerging genetic variants, although a small number of samples harboring circulating recombinant forms (CRFs) found in Cameroon were not always consistently detected by a few assays. These findings may be due to the evolving genetic diversity of HIV strains in Cameroon.

Radioactive and stable metal bioaccumulation, crystalline compound and siderophore detection in Clavariadelphus truncatus.

137Cs and 40K activity concentrations and stable elements have been measured in Clavariadelphus truncatus collected in Mexico. Iron-chelating compounds of siderophore-type was also studied in the species. 137Cs and 40K were determined in soil and mushroom samples with HpGe gamma-ray spectrometry. Macro- and micro-elemental concentrations were determined by XRF and ICP-MS. Siderophore detection was obtained with a colorimetric assay and X-ray diffraction analysis was performed using a Siemens D5000 diffractometer. 137Cs geometric mean concentration in C. truncatus was 26 times higher as compared with other Mexican edible mushroom species, while 40K showed stability. Soil-C. truncatus concentration ratio for 137Cs and other micro-elements such as Cs, Rb and Pb were also higher than other Mexican edible species. The 137Cs committed effective dose due to the ingestion of C. truncatus was 8 x 10(-6) Sv year(-1). The main crystalline structure found in C. truncatus was D-Mannitol.

Metal-chelating compounds produced by ectomycorrhizal fungi collected from pine plantations.

AIMS: To investigate the in vitro production of metal-chelating compounds by ectomycorrhizal fungi collected from pine plantations in southern Chile. METHODS AND RESULTS: Scleroderma verrucosum, Suillus luteus and two isolates of Rhizopogon luteolus were grown in solid and liquid modified Melin-Norkans (MMN) media with and without iron addition and the production of iron-chelating compounds was determined by Chrome Azurol S (CAS) assay. The presence of hydroxamate and catecholate-type compounds and organic acids was also investigated in liquid medium. All isolates produced iron-chelating compounds as detected by CAS assay, and catecholates, hydroxamates as well as oxalic, citric and succinic acids were also detected in all fungal cultures. Scleroderma verrucosum produced the greatest amounts of catecholates and hydroxamates whereas the highest amounts of organic acids were detected in S. luteus. Nevertheless, the highest catecholate, hydroxamate and organic acid concentrations did not correlate with the highest CAS reaction which was observed in R. luteolus (Yum isolate). CONCLUSIONS: Ectomycorrhizal fungi produced a variety of metal-chelating compounds when grown in liquid MMN medium. However, the addition of iron to all fungi cultures reduced the CAS reaction, hydroxamate and organic acid concentrations. Catecholate production was affected differently by iron, depending on the fungal isolate. SIGNIFICANCE AND IMPACT OF THE STUDY: The ectomycorrhizal fungi described in this study have never been reported to produce metal-chelating compound production. Moreover, apart from some wood-rotting fungi, this is the first evidence of the presence of catecholates in R. luteolus, S. luteus and S. verrucosum cultures.

Effect of cereal brans on Lentinula edodes growth and enzyme activities during cultivation on forestry waste.

AIMS: To develop strategies for increasing the growth of Lentinula edodes in eucalyptus residues. To this end, we have examined the effects of cereal brans additions on production of mycelial biomass and enzymes. METHODS AND RESULTS: Three isolates of the mushroom shiitake, L. edodes (Berk. Pegler), were evaluated for enzyme and ergosterol production on eucalyptus residue supplemented with 5, 10, 15 and 20% (w/w) of soya, wheat or rice brans. Nitrogen imput on eucalyptus residues accelerated mycelial growth by supplying the L. edodes with this limiting nutrient. High levels of enzymes activities were produced in eucalyptus residues supplemented by soya bran. Comparison of cellulose and xylanase production with manganese peroxidase (MnP) at 20% soya bran indicated that hydrolytic enzymes, but oxidative enzymes were reduced. CONCLUSIONS: Mycelial growth measurements revealed that eucalyptus residues supplemented with cereal brans supported fast growth of L. edodes, indicating that mycelium extension is related to the bioavailability of nitrogen. The type and concentration of nutrient supplement has a considerable effect both on substrate colonization and on the type of hydrolytic and oxidative enzymes produced. These characteristics may be useful for mushroom growing. SIGNIFICANCE AND IMPACT OF THE STUDY: Lentinula edodes is commercially important for edible mushroom production and supplements which enhance growth and enzymes production might also be beneficial for mushroom yields.

Use of CAS-agar plate modified to study the effect of different variables on the siderophore production by Aspergillus.

AIMS: To evaluate the suitability of chrome azurol S (CAS) agar plate assay as a quantitative methodology for siderophore production. METHODS AND RESULTS: Aspergillus species (A. flavus, A. niger, A. tamarii) were inoculated in the CAS-agar plates and the siderophores production was determined and expressed as CAS-reaction rate (mm per day). All the species showed positive CAS reaction with different rates depending on culture conditions and A. flavus showed the highest CAS-reaction rate. The siderophore production in solid medium expressed as CAS-reaction rate was correlated with siderophore production in liquid medium. CONCLUSIONS: The use of CAS-agar plate assay was modified and the evaluation of CAS reaction in mm per day made it possible to study and quantify the effect of several variables on the siderophore production by Aspergillus fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe the CAS-agar plate assay as a quantitative methodology, which make it possible to select and evaluate the siderophore production by several microorganisms (fungi and bacteria) according to different culture conditions.

The effect of antiretroviral therapy on HTLV infection.

No effective treatment for TSP/HAM has been described so far. Interventions with corticosteroids, plasmapheresis, interferon and, more recently, with antiretroviral drugs have been tried with poor results. The main HTLV replication mechanism is thought to be through clonal expansion of HTLV-infected cells, which excludes the involvement of the reverse transcriptase (RT) enzyme. However, a virological and clinical improvement has been noticed in HTLV-I carriers suffering from TSP/HAM receiving zidovudine or lamivudine. Herein, we describe the virological and clinical outcome in two TSP/HAM patients infected with HTLV-I treated with zidovudine plus lamivudine, and in two HTLV-II/HIV-1 co-infected patients receiving triple combinations including lamivudine. While, one TSP/HAM patient experienced a 2 log decrease in HTLV-I proviral load, an increase of 1 log was observed in another patient after several months of treatment with zidovudine plus lamivudine. The two HTLV-II/HIV-1 co-infected patients showed an initial increase in HTLV-II proviral load after beginning HAART followed by a slight decline a few months later. Plasma HIV-1 RNA fell to <50 copies/ml in both patients after beginning therapy. None of the four HTLV positive patients developed genetic changes at the conserved YMDD domain within their respective RT genes, which could be related to lamivudine resistance. No clinical improvement was observed in one TSP/HAM patient after more than 1 year on treatment with nucleoside analogues. The inhibition of the HTLV RT along with the cytostatic effect of some nucleoside analogues, including zidovudine, could reduce HTLV replication, and therefore reduce HTLV proviral load. The clinical consequences of this effect need to be further examined.

Monitoring the response to antiretroviral therapy in HIV-1 group O infected patients using two new RT-PCR assays.

Failure to recognise infection caused by human immunodeficiency virus type 1 (HIV-1) group O variants has been described using both serological and genetic procedures. Moreover, monitoring the response to antiretroviral therapy is a difficult task in patients infected with HIV-1 group O since commercial tests are not available so far for the quantitation of this virus. In this study, the virological response to antiretroviral therapy were assessed in five HIV-1 group O-infected patients living in Spain by using two new and different RT-PCR methods (MUPROVAMA and LCx). Twenty-four plasma samples belonging to these five patients were selected. As reference, p24 antigenaemia levels and CD4+ cell counts were used. All samples yielded positive viral load values using MUPROVAMA (range: 138 to 595,500 HIV-RNA copies/ml) and 23 of 24 using LCx (range: < 178 to 98,356 HIV-RNA copies/ml). Overall, the results obtained using both assays showed a good correlation among themselves, and in respect to p24 antigenaemia and CD4+ cell counts. However, the values provided by LCx were significantly lower (0.33 logs on average) than those provided by MUPROVAMA. In conclusion, both the highly sensitive MUPROVAMA and LCx Quantitative assays might represent an useful tool for guiding the decision on when start treatment and for monitoring the response to antiretroviral therapy in HIV-1 group O-infected patients.

[Spastic paraparesias by HTLV-1: early identification of a new case. Review of the Spanish casuistics]. / Paraparesia espástica por HTLV-I: identificación temprana de un nuevo caso. Revisión de la casuística española.

This is a study of the early identification of a new case of tropical spastic paraparesis/HLTV-1-associated myelopathy in a Spanish patient not previously reported on. The patient is 48 years of age, and he has been living in Sierra Leone for the past seventeen years, where he has worked as a surgeon. On first examination, he had been suffering from symptoms of a spastic paraparesis for the previous five months. This diagnosis was confirmed using serological tests (EIA and Western-blot) on the blood and cerebrospinal fluid (CSF). We have studied the cases outlined in medical literature, paying special attention to the time elapsed between the onset of symptoms and the date on which the diagnosis was made.

Paraparesia espástica por HTLV-I: identificación temprana de un nuevo caso. Revisión de la casuística española / Spastic paraparesias by HTLV-1: early identification of a new case. Review of the Spanish casuistics

Presentamos la identificación temprana de un nuevo caso de paraparesia espástica asociada al virus HTLV-I. El paciente, un español de 48 años, había desarrollado su actividad quirúrgica durante los últimos 17 años en Sierra Leona. Presentaba un síndrome de paraparesia espástica sin alteración esfinteriana de 5 meses de evolución. El diagnóstico se confirmó mediante la determinación de anticuerpos frente al HTLV-I en sangre y líquido cefalorraquídeo (EIA y Western blot). Hemos revisado en la bibliografía los casos descritos, prestando especial atención al tiempo transcurrido entre el inicio de los síntomas y la fecha en que se estableció el diagnóstico (AU)

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In vivo fluctuation of HTLV-I and HTLV-II proviral load in patients receiving antiretroviral drugs.

HTLV-I and HTLV-II infect T lymphocytes. A high HTLV-I proviral load in peripheral blood mononuclear cells (PBMCs) has been associated with a higher risk of neurologic disease. For HTLV-II, large numbers of infected lymphocytes might contribute to accelerate the immunodeficiency and increase the risk of neuropathy in HTLV-II/HIV-1 coinfected people. We have examined the impact of antiretroviral drugs on HTLV proviral load, testing longitudinal samples collected from 1 HTLV-I infected patient suffering HTLV-I-associated myelopathy (HAM), and two HTLV-II/ HIV-1 coinfected subjects. The HAM patient showed a reduction greater than 2 log in the peripheral proviral load after being treated with zidovudine and lamivudine. In contrast, potent antiretroviral treatment in HIV-1/HTLV-II coinfected carriers produced an initial increase in the HTLV proviral load, which was followed by a reduction greater than 1 log thereafter. In conclusion, antiretroviral drugs seem to reduce HTLV proviral load, although in HIV-1 coinfected persons a transient increase in HTLV proviral load could reflect the rapid blocking of HIV-1 replication occurring in response to therapy, thus causing an increase in the number of circulating T lymphocytes carrying HTLV proviral DNA.

Clearance of human herpesvirus type 8 viraemia in HIV-1-positive patients with Kaposi's sarcoma treated with liposomal doxorubicin. Caelyx/KS Spanish Study Group.

OBJECTIVE: To assess the impact of liposomal doxorubicin on human herpesvirus type 8 (HHV-8) cell viraemia in HIV-infected patients with Kaposi's sarcoma. DESIGN: Prospective, non-controlled, multicenter study. METHODS: The presence of HHV-8 DNA was investigated by polymerase chain reaction in peripheral blood mononuclear cells from 46 HIV-positive patients with Kaposi's sarcoma. Samples were tested at baseline and every 3 months during treatment with liposomal doxorubicin. CD4 cell counts, plasma HIV RNA, and clinical outcome were recorded at baseline and at follow-up visits. RESULTS: HHV-8 sequences were detected in 32 (70%) patients at baseline. No significant differences were found between subjects with HHV-8 positive and negative results. The proportion of patients with positive HHV-8 viraemia decreased to 38% (10 of 26) after 3 months of treatment with liposomal doxorubicin (P < 0.01). Overall, 12 of 22 (57%) subjects with positive HHV-8 cell viraemia at baseline became negative during the treatment period. However, in one of them HHV-8 reappeared 8 months later despite being on therapy. On the other hand, six of eight subjects with negative HHV-8 at baseline remained negative thereafter. There were no significant changes in plasma HIV RNA, total lymphocyte, or CD4 cell counts during the treatment period. Clinical response of Kaposi's sarcoma to liposomal doxorubicin and clearance of HHV-8 viraemia did not correlate well. CONCLUSIONS: HHV-8 cell viraemia significantly decreased during treatment with liposomal doxorubicin in HIV-infected patients with Kaposi's sarcoma, although the clinical response and HHV-8 clearance did not correlate well.

Prevalence of HTLV infection in pregnant women in Spain.

OBJECTIVE: To estimate the prevalence of HTLV infection among pregnant women in Spain. METHODS: A commercial ELISA incorporating HTLV-I and HTLV-II antigens was used for HTLV antibody screening. Repeatedly reactive samples were further examined by western blot. Moreover, confirmation with PCR was performed when cells were available. RESULTS: 20,366 pregnant women in 12 different Spanish cities were tested in a 3 year period (July 1996 to August 1999). 32 samples were repeatedly reactive by ELISA, and 10 of them were confirmed as positive by western blot (eight for HTLV-II and two for HTLV-I). In addition, three of 13 women who had an indeterminate western blot pattern yielded positive results for HTLV-II by PCR. All 11 HTLV-II infected women had been born in Spain, and all but one were former drug users. Seven of them were coinfected with HIV-1. One HTLV-I infected woman was from Peru, where HTLV is endemic and where she most probably was infected during sexual intercourse. CONCLUSION: The overall prevalence of HTLV infection among pregnant women in Spain is 0.064% (13/20,366), and HTLV-II instead of HTLV-I is the most commonly found variant. A strong relation was found among HTLV-II infection and specific epidemiological features, such as Spanish nationality and injecting drug use. Although HTLV-II can be vertically transmitted, mainly through breast feeding, both the low prevalence of infection and its lack of pathogenicity would not support the introduction of HTLV antenatal screening in Spain.

Human immunodeficiency virus type 2 infection in Spain. The HIV-2 Spanish Study Group.

The first cases of human immunodeficiency virus type 2 (HIV-2) infection in Spain were identified in 1988, in 3 African immigrants living in Barcelona. Since then, up to December 1998, 92 individuals with HIV-2 infection have been reported in Spain. Most are adult men, infected through heterosexual contacts, originating from West African countries, and currently living in the largest urban Spanish cities. Fifteen individuals have developed AIDS, meanwhile the rest remain asymptomatic. For 22 subjects, HIV-2 subtyping was performed on proviral DNA, 16 being infected with subtype A (8 Spanish born and 8 African immigrants) and the remaining with subtype B (two Spanish born and 4 originating from Equatorial Guinea). Coinfection with HIV-1 was demonstrated in 9 individuals. In conclusion, HIV-2 is currently circulating in Spain with a low prevalence and without evidence for increase over time.

Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay.

A well-known and widely used method for detection of siderophore production by microorganisms in solid medium is the universal chrome azurol S (CAS)-agar plate assay. However, the high toxicity of CAS-blue agar medium caused by the presence of a detergent impedes its utilization with many varieties of fungi and Gram-positive bacteria. To solve this problem, a modification of the CAS-agar plate assay was made by incorporating the CAS-blue dye in a medium with no contact with the microorganisms tested. Half of each plate used in our experiments was filled with the most appropriate culture medium for each type of microorganism and the other half with CAS-blue agar. This modification allowed us to study several strains of fungi (basidiomycetes, deuteromycetes, ascomycetes and zygomycetes) and bacteria (Gram positive and negative), some of them appearing for the first time in the literature. All the microorganisms grew properly and reacted in different manners to the CAS assay. Some strains of wood-decaying basidiomycetes (mainly white-rot fungi) and Aspergillus species produced the fastest color-change reactions in the CAS-blue agar. This modified method could facilitate optimization of culture conditions, since both CAS-blue agar and growth medium were prepared and added in the Petri plate separately.

Isolation and partial characterization of an extracellular low-molecular mass component with high phenoloxidase activity from Thermoascus aurantiacus.

An extracellular low-molecular mass component (LMMC) with catalytic properties was isolated from liquid cultures containing wheat bran of ascomycete thermophilic Thermoascus aurantiacus. The partially purified LMMC showed very high activity with typical phenoloxidase substrates in the absence of hydrogen peroxide at acidic pH (2.8). However, in this pH range, the phenoloxidase (PO) activity was quickly lost. The LMMC showed a high optimum temperature (80 degrees C) and an elevated thermostability. The molecular mass of the component estimated by gel filtration chromatography was 530 Da. IR and 1H- and 13C-NMR spectra indicated the presence of hydroxamic acid moiety. Qualitative determination of metal ions by several techniques revealed the presence of mainly iron associated with this structure. Iron may be the responsible for the ability for catalyze oxidation reactions, such as o-dianisidine oxidation, by the LMMC. These results suggested the existence of a hydroxamate-type metal-binding component, most likely hydroxamate siderophore. In addition, the chrome azurol S (CAS) universal assay for noncomplexed siderophores detection revealed the production of these compounds by T.aurantiacus in solid and liquid media.

[7 new cases of HIV-2 infection recognized in Madrid in 1996 and 1997]. / Siete casos nuevos de infección por VIH-2 reconocidos en Madrid en 1996 y 1997.

The infection with human immunodeficiency virus type-2 (HIV-2) is endemic in Western Africa, where it is responsible for many of AIDS cases. In Spain, 72 cases with HIV-2 until December 1997 had been reported, mainly in African immigrants. Seven of these cases (4 males and 3 females) were identified during 1996 and 1997. Five came from Western Africa and other two were Spanish, one from Alicant and the other from Vigo. Only one out of the seven patients presented clinical manifestations (Pneumocystis carinii pneumonia); the remaining patients were asymptomatic. Viral isolates from six of these patients were genetically characterized: three were subtype A, whereas those from Equatorial Guinea patients corresponded to HIV-2 subtype B.