In vitro conservation of mango (Mangifera indica L.) Ubá and Carlota cvs. through culturing immature embryos.

The in vitro rescue of immature embryos can be employed both for preservation of mango germplasm and rescue of hybrids produced from crosses to obtain traits of interest. The objective of this work was to establish a protocol for in vitro rescue of immature embryos aiming to preserve mango germplasm. Immature embryos of two mango varieties, 'Carlota' and 'Ubá', were inoculated in MS/2 medium supplemented with 100 mg L-1 of cysteine, 0.5 mg L-1 of gibberellic acid (AG3) and 30 g L-1 of sucrose. The experimental design was completely randomized with 30 repetitions, each composed of two embryos/flask. After in vitro growth for 84 days, some of the plantlets were transferred for acclimatization. The parameters evaluated were plant height, number of leaves and leaf, stem and root dry weight. The germination began on the sixth day after seeding, and the immature embryos presented a high oxidation rate, with 60% oxidized after 72 hours. The plantlets from immature embryos showed better development than those from aborted embryos. The results showed the viability of rescuing mango embryos in the immature phase and of their in vitro conservation for a period of 12 months.

Mutational analysis of the feline immunodeficiency virus matrix protein.

To study the process of feline immunodeficiency virus (FIV) assembly, we examined the suitability of the vaccinia vector system to reproduce FIV particle formation. To this end, we constructed a recombinant vaccinia virus carrying the FIV gag gene. Biochemical and electron microscopy analyses of cells infected with this recombinant virus showed that the FIV Gag polyprotein self-assembled into lentivirus-like particles that were released into the culture medium. As a first step in the identification of molecular determinants in FIV Gag that are involved in virus assembly, we performed a site-directed mutagenesis analysis of the N-terminal matrix (MA) domain of the FIV Gag precursor. To this end, a series of amino acid substitutions and small in-frame deletions were introduced into the FIV MA and the mutated FIV gag gene constructs were expressed by means of the vaccinia system. Characterization of the assembly phenotype of these FIV Gag mutants led to the identification of amino acidic regions within the MA domain that are necessary for efficient transport of the Gag precursor to the plasma membrane and particle assembly. Our results reveal the role that the FIV MA plays in virus morphogenesis and contribute to the understanding of the assembly process in non-primate lentiviruses.

Domains in the simian immunodeficiency virus gp41 cytoplasmic tail required for envelope incorporation into particles.

The mechanism by which lentivirus envelope (Env) glycoproteins are packaged into budding virions is poorly understood. Simian immunodeficiency virus (SIV) contains an Env protein with an unusually long cytoplasmic tail. To investigate the role of this domain in the incorporation of the SIV Env into virions, we generated a series of SIV Env mutants carrying small in-frame deletions within the cytoplasmic domain. The effects of these mutations on Env synthesis, processing, and association with Gag particles were analyzed by means of the vaccinia virus expression system. All of the mutant Env glycoproteins were synthesized and processed in a manner similar to that of the wild-type Env. However, deletions affecting domains C-terminal to residue 832 in the SIV Env protein significantly impaired Env incorporation into particles. Cell surface biotinylation assays showed that this phenotype could not be attributed to inefficient cell surface expression of the Env mutants. Furthermore, when the Env deletion mutants were tested for their ability to mediate virus entry in single-cycle infectivity assays, those mutations that impaired Env incorporation also caused a severe defect in virus infectivity. Our results suggest that domains in the C-terminal third of the SIV Env protein are required for Env incorporation into particles and Env-mediated virus entry.

Decreased scopolamine yield in field-grown Duboisia plants regenerated from hairy roots.

Hairy root cultures were obtained from hybrid clones of Duboisia myoporoides x D. leichhardtii following transformation by Agrobacterium rhizogenes strain A4. Shoots spontaneously regenerating from the hairy root cultures were rooted and transferred to soil. The plants displayed typical morphological alterations known as hairy root syndrome to varying degrees. PCR analysis confirmed that all transformed plants contained the rolA, rolB and rolC genes, irrespective of the degree of morphological alterations. A field test of the transformed regenerated plants revealed that those plants displaying the strongest hairy root syndrome symptoms had the highest content of the tropane alkaloid scopolamine. However, the overall scopolamine and hyoscyamine yield of all transformed plants was clearly reduced compared to untransformed control plants. These results demonstrate that the A. rhizogenes-transformed plants tested in this study do not provide a viable alternative to agricultural farming of hybrid clones of D. myoporoides x D. leichhardtii obtained by conventional breeding.

Small variations in the length of the cytoplasmic domain of the simian immunodeficiency virus transmembrane protein drastically affect envelope incorporation and virus entry.

Simian immunodeficiency viruses (SIVs) have an envelope (Env) glycoprotein with an unusually long cytoplasmic domain of 164 amino acids. In this article, we have characterized a series of SIV Env truncation mutants in which the cytoplasmic domain was progressively shortened from its carboxyl terminus by 20 amino acids. Expression by means of the vaccinia virus system showed that all of the SIV Env mutants were expressed and processed into the surface and transmembrane (TM) subunits. When the ability of the Env mutants to associate with SIV Gag particles was examined, we found that deletion of 20 to 80 residues from the carboxyl terminus of the SIV TM cytoplasmic tail abrogated the incorporation of the Env glycoprotein into particles. By contrast, further truncation of the SIV TM protein by 100 to 140 amino acids restored the ability of the Env protein to associate with Gag particles. Interestingly, mutants bearing a 44- or 24-amino acid cytoplasmic domain were incorporated at levels significantly higher than those of the wild-type Env. Single-cycle infectivity assays showed that Env mutants bearing cytoplasmic tails of 144 to 64 amino acids were highly inefficient at mediating virus entry. By contrast, truncation of the cytoplasmic domain to 44 or 24 amino acids drastically enhanced virus infectivity with respect to that conferred by the full-length Env protein. Our results demonstrate that small variations in the length of the SIV Env cytoplasmic domain dramatically influence Env-mediated viral functions.

Cancer mortality and exposure to chemical carcinogens in the work place: an ecological study in the Valencian Community, Spain (1981-1995).

To evaluate the geographical distribution of the mortality from malignant tumours in relationship with exposure to chemical carcinogens in the work place, and to asses the possible association between these questions and the percentage of population employed in certain high-risk sectors, an ecological study in the Valencian Community (VC), Spain was carried out. Age-adjusted mortality rates for the total number of malignant tumours, lung, bladder, lymphomas and leukaemia during the periods 1981-1985 and 1991-1995 were calculated for the 34 geographical areas. The percentage of population in each area working in sectors in which they may be exposed to chemical carcinogens was obtained. The relationship between mortality on-the-job exposure was studied using linear regression methods. Large differences in cancer mortality were seen. In men, the geographical pattern was very stable and reveals a significant association with the distribution of certain high-risk jobs. Statistically significant positive correlations (p < 0.001) were found between cancer mortality and the percentage of the population working in metal, wood and furniture sectors. In contrast, a negative and statistically significant (p < 0.001) correlation was observed between cancer mortality and the percentage of the population working in agriculture. In conclusion, although the variability in cancer mortality in men was significantly associated with some occupational sectors in the VC, caution is needed when drawing conclusions about causation from ecological studies.

Determination of N-acetylcysteine in human plasma by liquid chromatography coupled to tandem mass spectrometry.

An analytical method for the determination of total N-acetylcysteine in human plasma has been developed, validated and applied to the analysis of samples from a phase I clinical trial. The analytical method consists of plasma digestion with dithiothreitol in order to reduce all the oxidized forms of N-acetylcysteine, and extraction with ethyl acetate followed by determination of levels by an LC-MS-MS method. The intra- and inter-assay precision and accuracy of this technique were good and the limit of quantitation was 50 ng/ml of plasma. The concentration working range was established between 50 ng/ml and 1000 ng/ml. This method has been used in the analysis of approximately 800 human plasma samples from a clinical study with 24 volunteers; the precision of the quality controls was in the range 8.7 to 13.4% and the accuracy was in the range -5.9 to 8.5%, expressed as the RSD and the relative error, respectively.

Simultaneous determination of paracetamol and chlorpheniramine in human plasma by liquid chromatography-tandem mass spectrometry.

An analytical method for the determination of paracetamol and chlorpheniramine in human plasma has been developed, validated and applied to the analysis of samples from a phase I clinical trial. The analytical method consists in the extraction of paracetamol and chlorpheniramine with diethyl ether, followed by the determination of both drugs by an LC-MS-MS method, using 2-acetamidophenol as internal standard. The intra-assay and inter-assay precision and accuracy of this technique were good and the limit of quantitation was 0.5 microg/ml of plasma for paracetamol and 0.2 ng/ml for chlorpheniramine. The concentration working range was established between 0.5 microg/ml and 25 microg/ml for paracetamol and between 0.2 ng/ml and 50 ng/ml for chlorpheniramine. This method has been used for analyzing more than 1200 human plasma samples from a clinical study with 24 volunteers.

Determination of the platelet activating factor antagonist 6-(2- chlorophenyl)-9-[(4-methoxyphenyl)thiocarbamoyl]-1-methyl-7,8,9,10- tetrahydro-4H-pyrido[4',3'-4,5]-thieno[3,2-f][1,2,4]triazolo[4, 3-a][1,4] diazepine in human plasma by liquid chromatography-thermospray mass spectrometry.

A sensitive and specific method for the determination of the platelet activating factor (PAF) antagonist 6-(2-chlorophenyl)-9-[(4-methoxyphenyl)-thiocarbamoyl]-1-methyl-7,8,9,10- tetrahydro-4H-pyrido[4',3'-4,5]thieno-[3,2-f][1, 2, 4]triazolo [4, 3-a][1, 4] diazepine (I) in human plasma is described. The target molecule was analyzed by high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) after extraction by ion-exchange chromatography. HPLC was carried out using a C18 column and the coupling to the MS was done by a thermospray (TSP) interface working in the direct ion-evaporation ionization mode in presence of 0.1 M ammonium acetate. Selected-ion monitoring (SIM) was carried out for the ion m/z 370 and its [M + 2]+ isotopic peak. Evaluation of the intensity matching of such ions has been used in the validation results. The method gives good accuracy and precision over the concentration range 1-200 ng/ml in human plasma.

Determination of the aza alkyl lysophospholipid 3-methoxy-2-N,N-methyloctadecylaminopropyloxyphosphorylcholine in rat plasma by liquid chromatography-particle beam-mass spectrometry.

A sensitive and specific method for the determination of the aza alkyl lysophospholipid (AALP) 3-methoxy-2-N,N-methyloctadecylaminopropyloxyphosphorylcholine (I) in rat plasma is described. The target molecule was analyzed by high-performance liquid chromatography (HPLC)-mass spectrometry (MS) after one single liquid-liquid extraction with chloroform-methanol (2:1, v/v). 1,2-Didecanoyl-sn-glycero-3-phosphocholine was used as internal standard. HPLC was carried out using a polymeric reversed-phase column; the coupling to the mass spectrometer was a particle beam (PB) interface, and the ionization method was electron impact (EI). This simple and rugged method permits the measurement of I in rat plasma in the range of 25 ng/ml-5 micrograms/ml with good accuracy and precision and is used in pharmacokinetic studies.

Convergent solid-phase peptide synthesis IX: application to the synthesis of peptides with repetitive sequences.

The synthesis of a 36 amino acid peptide containing six conserved repeats of Val-His-Leu-Pro-Pro-Pro which corresponds to the glutelin-2 protein of maize has been carried out using a convergent approach. The protected single sequence repeat has been synthesized using a combination of a 9-fluorenylmethyloxycarbonyl (Fmoc) protection scheme and a p-alkoxybenzyl resin. The final peptide, as well as the different intermediate peptides, has been characterized by fast atom bombardment mass spectrometry (FAB-MS) and by co-elution with another sample obtained by stepwise continuous flow synthesis.

Determination of the enantiomeric purity of synthetic peptides by gas chromatography-mass spectrometry.

Hydrolysis using 2H-labelled HCl and H2O, derivatization of free amino acids as N.O.S-trifluoroacetyl isobutyl esters, separation by gas chromatography on a chiral stationary phase and detection by mass spectrometry in selected-ion monitoring mode have been used in order to determine the enantiomeric purity of several synthetic peptides. Chromatographic separation has been optimized for proline, whose two enantiomers are difficult to resolve under standard conditions. Electron impact and methane chemical ionization mass spectra and chromatographic resolution of unnatural amino acids, such as 3-(1-naphthyl)-alanine and p-chlorophenylalanine, are reported. For both natural and unnatural amino acids selected-ion monitoring of the different fragmentation peaks has been carried out. The results are interpreted from the point of view of whether or not the fragments contain a hydrogen atom on the alpha-carbon, and a comparison between electron impact and methane chemical ionization has been carried out. The main advantage of the latter method is that a quasimolecular ion can be observed for all the amino acids studied.

Evaluation of practical chromatographic procedures for identification of clinical isolates of mycobacteria.

After experimental conditions were established, 366 strains of mycobacteria belonging to 23 different species were studied for fatty acids, secondary alcohols, and mycolic acid cleavage products by capillary gas-liquid chromatography. Additionally, the mycolic acid pattern was studied by thin-layer chromatography. Capillary gas-liquid chromatography allowed direct identification of the following Mycobacterium spp.: M. kansasii, M. marinum, M. szulgai, M. xenopi, M. malmoense, and M. gordonae. The patterns of mycolic acid methyl esters recorded for the test strains of M. chelonae and M. agri may be of value in the identification of these species. Moreover, the combined use of the two chromatographic techniques provided precise identification of the M. tuberculosis complex, M. simiae, M. fallax, M. triviale, and M. chelonae-like organisms. A minimal set of biochemical tests is usually required to obtain identification to the species level when chromatographic procedures alone are not sufficient. Under the reported experimental conditions, thin-layer chromatography and capillary gas-liquid chromatography are rapid and very useful techniques for the identification of mycobacteria.