Sementes recalcitrantes: intolerantes a baixas temperaturas? Embriões recalcitrantes de Inga vera Willd. subsp. affinis (DC.) T. D. Penn. toleram temperatura sub-zero

Sementes recalcitrantes são intolerantes à dessecação e a baixas temperaturas, que são as principais formas de conservação de sementes e, portanto, são de difícil armazenamento. Inga vera subsp. affinis produz sementes denominadas recalcitrantes, uma vez que não toleram redução do teor de água para valores inferiores a 35%. Neste trabalho é demonstrada a tolerância desses embriões a temperatura de -2 ºC. Embriões de I. vera de diferentes estádios de maturação foram armazenados por até 45 dias nas temperaturas de 8 ºC a -18 ºC, com ou sem secagens prévias até -4,0 e -6,0 MPa. Os resultados permitiram observar que, embora nenhum tratamento tenha proporcionado resistância à temperatura de -18 ºC, a secagem dos embriões maduros a -4 MPa proporcionou maior tolerância à redução de temperatura até níveis de congelamento da água (-2 ºC).

Biomass and chemical composition of Nitella furcata subsp. mucronata var. mucronata f. oligospira (A. Braun) R. D. Wood (Chlorophyta, Characeae) in the littoral region of Ninféias Pond, São Paulo, Southeast Brazil

Temporal variation of Nitella furcata (Roxburgh ex Bruzelius) C. Agardh emend. R. D. Wood subsp. mucronata (A. Braun) R. D. Wood var. mucronata f. oligospira (A. Braun) R. D. Wood biomass and chemical composition were studied at the Ninféias Pond (23°38'18.9" S, 46°37'16.3" W), a mesotrophic reservoir located in the Parque Estadual das Fontes do Ipiranga Biological Reserve, Municipality of São Paulo, Southeast Brazil. Plants were collected monthly from October 1996 to October 1997 at three fixed stations of reservoir's littoral region. Charophyte biomass spatial distribution pattern did not vary significantly throughout the study period at all sampling stations. As to seasonal variation, the highest average values of the total alga biomass (98.35-266.06 g m-2 DW) were registered during the rainy season, whereas lowest values (48.86-170.56 g m-2 DW) were in the dry season. P values varied from 23.8 to 225.2 mg m-2 and C from 139 to 353 mg m-2. During the rainy season, greatest air and water temperature, rain precipitation, turbidity and dissolved inorganic nitrogen values were measured, constituting the best conditions for charophyte growth. Water temperature and nutrient availability in the reservoir played a decisive role towards growth and accumulation of algal biomass.

Dinâmica espaço-temporal de Melocactus ernestii subsp. ernestii (Cactaceae) no Nordeste do Brasil

Melocactus ernestii Vaupel subsp. ernestii ocorre em afloramentos rochosos descontínuos no leste do Brasil. O presente estudo investigou o padrão espaço-temporal dos eventos de mortalidade e sobrevivência de plântulas da M. ernestii e a relação com as plantas perenes, a partir da utilização de parcelas, alocadas com auxílio de trenas, onde foram tomadas as coordenadas cartesianas do centróide dos estádios de desenvolvimento, com posterior análises de padrões de ponto baseado na função K de Ripley. Foram mapeados nas parcelas 686 indivíduos da M. ernestii. O padrão espacial variou em função da escala, do estádio de desenvolvimento e do grau de intemperização dos afloramentos. Os eventos de recrutamento e mortalidade foram espacialmente estruturados e influenciados por pulsos de precipitação sazonais. As plântulas emergiram a ca. 50 cm do centróide dos adultos. Das 136 plântulas monitoradas em 18 meses, 84% morreram. Os resultados mostraram que microsítios inicialmente eficazes na manutenção de sementes e promotores da germinação não necessariamente podem ser eficazes para o estabelecimento das plântulas. A magnitude das interações bióticas em ambientes estressantes é espécie-específica e indica que associação positiva entre plantas adultas e sobrevivência de plântulas não é consistente com o tempo. Este tipo de modelagem populacional permite melhor compreensão sobre o papel das interações positivas e negativas como forças estruturadoras de comunidades e pode ajudar a entender a complexidade das interações bióticas em gradientes ambientais severos.

Conservação e germinação in vitro de pólen de milho (Zea mays subsp. mays)

O armazenamento de pólen é uma importante ferramenta para programas de melhoramento de milho, permitindo preservar, sob condições artificiais, a viabilidade de gametas masculinos e ampliar as possibilidades de cruzamentos, independente da época do florescimento das variedades genitoras. O presente trabalho teve como objetivo avaliar meios de cultivo para a germinação in vitro de pólen de milho e analisar condições de conservação. Para avaliar viabilidade, foram testados seis meios de cultivo, contendo sacarose, ácido bórico, cloreto de cálcio hidratado e ágar. Para a conservação de pólen foram analisadas duas temperaturas (4 ºC e -20 ºC) e dois agentes desidratantes de pólen (sílica gel e cloreto de cálcio hidratado). Os altos valores de viabilidade do pólen no período de até 30 dias de armazenamento indicam que a desidratação em sílica gel e armazenamento a 4 ºC conservam a viabilidade de pólen de milho. O meio de cultivo que proporcionou a maior taxa de germinação in vitro foi com a composição de 0,7% de ágar, 17% de sacarose, 0,01% de ácido bórico e 0,03% de cloreto de cálcio hidratado.

DNA fingerprinting of Mycobacterium tuberculosis from patients with and without AIDS in Rio de Janeiro

Isolates of Mycobacterium tuberculosis derived from patients with AIDS from a single hospital in Rio de Janeiro were typed using a standardized RFLP technique detecting IS6110 polymorphism. Nineteen isolates were obtained from 15 different patients. Eleven distinct IS6110 patterns were found, with 4 banding patterns shared by 2 patients. The clustering value of 53% was much higher in comparison with clustering of M. tuberculosis strains from TB patients without clinical signs for HIV infection from randomly selected health centers. We present these results as preliminary data on M. tuberculosis strain polymorphism in Brazil and on the higher risk for recent transmission amongst patients with AIDS

Expression of the Mycobacterium bovis P36 gene in Mycobacterium smegmatis and the baculovirus/insect cell system

In the present study we evaluated different systems for the expression of mycobacterial antigen P36 secreted by Mycobacterium bovis. P36 was detected by Western blot using a specific antiserum. The P36 gene was initially expressed in E. coli, under the control of the T7 promoter, but severe proteolysis prevented its purification. We then tried to express P36 in M. smegmatis and insect cells. For M. smegmatis, we used three different plasmid vectors differing in copy number and in the presence of a promoter for expression of heterologous proteins. P36 was detected in the cell extract and culture supernatant in both expression systems and was recognized by sera from M. bovis-infected cattle. To compare the expression level and compartmentalization, the MPB70 antigen was also expressed. The highest production was reached in insect cell supernatants. In conclusion, M. smegmatis and especially the baculovirus expression system are good choices for the production of proteins from pathogenic mycobacteria for the development of mycobacterial vaccines and diagnostic reagents.

McFarland nephelometer as a simple method to estimate the sensitivity of the polymerase chain reaction using Mycobacterium tuberculosis as a research tool

Polymerase chain reaction (PCR) has been widely investigated for the diagnosis of tuberculosis. However, before this technique is applied on clinical samples, it needs to be well standardized. We describe the use of McFarland nephelometer, a very simple approach to determine microorganism concentration in solution, for PCR standardization and DNA quantitation, using Mycobacterium tuberculosis as a model. Tuberculosis is an extremely important disease for the public health system in developing countries and, with the advent of AIDS, it has also become an important public health problem in developed countries. Using Mycobacterium tuberculosis as a research model, we were able to detect 3 M. tuberculosis genomes using the McFarland nephelometer to assess micobacterial concentration. We have shown here that McFarland nephelometer is an easy and reliable procedure to determine PCR sensitivity at lower costs.

Effects of diazepam on Mycobacterium bovis-induced infection in hamsters

The in utero exposure of hamsters to low doses of diazepam results in impaired host defense against Mycobacterium bovis during adulthood. Delayed developmental immunotoxicity, however, represents a specific situation that might not be general. The present experiment was undertaken to investigate the effects of diazepam on hamster resistance to M. bovis using adult animals. The effects of diazepam treatment on serum cortisol levels were also studied. Adult hamsters (N = 10 for each group) were treated with diazepam (E1 = 1.0, E2 = 2.0 or E3 = 3.0 mg kg-1 day-1 subcutaneously) or with control solution (C) for 30 days. Seven days after the beginning of the treatment, the animals received identical inoculum concentrations of M. bovis. Hamsters treated with the higher (2.0 and 3.0 mg kg-1 day-1) doses of diazepam exhibited: 1) increased granuloma areas in the liver (C = 1.81 ± 1.39, E2 = 10.29 ± 4.64 and E3 = 15.80 ± 4.82) and lung (C = 0.54 ± 0.55, E2 = 6.28 ± 3.85 and E3 = 6.31 ± 3.56) and 2) increased scores of M. bovis colony-forming units isolated from liver (C = 2.0, E2 = 3.0 and E3 = 3.5), lung (C = 1.0, E2 = 3.0 and E3 = 3.5) and spleen (C = 1.0, E2 = 2.5 and E3 = 4.0). These effects were dose dependent, and were not detected or were less severe in animals treated with the lowest (1.0 mg/kg) dose of diazepam as well as in those of the control group. Furthermore, diazepam treatment (3.0 mg kg-1 day-1 for 30 days) increased (E3 = 71.32 ± 2.99; N = 10) the serum levels of cortisol compared to control hamsters (C = 22.61 ± 2.75; N = 10). The present data, that demonstrate an impaired defense against M. bovis in adult hamsters treated with diazepam, were tentatively explained on the basis of a direct and/or indirect action of diazepam on the cytokine network. The effects may be related to stimulation of peripheral benzodiazepine receptor binding sites (PBR) by macrophages and/or lymphocytes, or they may be mediated by PBR stimulation of the adrenals.

Effect of the injection of an extract of Ascaris suum on macrophage activation during the early phase of Mycobacterium bovis BCG infection in C57Bl/6 mice

Injection of an Ascaris suum extract (Asc) affects both the humoral and cellular immune responses to unrelated antigens when it is co-administered with these antigens. In the present study we evaluated the effect of Asc on macrophage activation in the early phase of Mycobacterium bovis BCG (Pasteur strain TMCC 1173) infection in C57Bl/6 mice. C57Bl/6 mice were injected intraperitoneally (ip) with 0.1 mg BCG (BCG group) or BCG plus 1 mg Asc (BCG + Asc group). The peritoneal exudates were obtained at 2, 7 and 14 days after infection. The numbers of IFN-g-secreting cells were assessed by the ELISPOT assay. Nitric oxide (NO) production was measured by the Griess method and by the evaluation of NADPH diaphorase activity in the peritoneal exudates. The administration of Asc extract increased NADPH diaphorase activity (2 days: control = 0, BCG = 7%, BCG + Asc = 13%, and Asc = 4%; 7 days: control = 4, BCG = 13%, BCG + Asc = 21%, and Asc = 4.5%) and TNF-a levels (mean ± SD; 2 days: control = 0, BCG = 169 ± 13, BCG + Asc = 202 ± 37, and Asc = 0; 7 days: control = 0, BCG = 545 ± 15.5, BCG + Asc = 2206 ± 160.6, and Asc = 126 ± 26; 14 days: control = 10 ± 1.45, BCG = 9 ± 1.15, BCG + Asc = 126 ± 18, and Asc = 880 ± 47.67 pg/ml) in the early phase of BCG infection. Low levels of NO production were detected at 2 and 7 days after BCG infection, increasing at 14 days (mean ± SD; 2 days: control = 0, BCG = 3.7 ± 1.59, BCG + Asc = 0.82 ± 0.005, Asc = 0.48 ± 0.33; 7 days: control = 0, BCG = 2.78 ± 1.54, BCG + Asc = 3.07 ± 1.05, Asc = 0; 14 days: control = 0, BCG = 9.05 ± 0.53, BCG + Asc = 9.61 ± 0.81, Asc = 10.5 ± 0.2 (2 x 106) cells/ml). Furthermore, we also observed that Asc co-injection induced a decrease of BCG-colony-forming units (CFU) in the spleens of BCG-infected mice during the first week of infection (mean ± SD; 2 days: BCG = 1.13 ± 0.07 and BCG + Asc = 0.798 ± 0.305; 7 days: BCG = 1.375 ± 0.194 and BCG + Asc = 0.548 ± 0.0226; 14 days: BCG = 0.473 ± 0.184 and BCG + Asc = 0.675 ± 0.065 (x 102) CFU). The present data suggest that Asc induces the enhancement of the immune response in the early phase of BCG infection.

A single strain of Mycobacterium bovis bacillus Calmette-Guérin (BCG) grown in two different media evokes distinct humoral immune responses in mice

Two attenuated bacillus Calmette-Guérin (BCG) preparations derived from the same Moreau strain, Copenhagen but grown in Sauton medium containing starch and bacto-peptone (onco BCG, O-BCG), or asparagine (intradermal BCG, ID-BCG), exhibited indistinguishable DNA sequences and bacterial morphology. The number of viable bacilli recovered from spleen, liver and lungs was approximately the same in mice inoculated with the vaccines and was similarly reduced (over 90%) in mice previously immunized with either BCG vaccine. The humoral immune response evoked by the vaccines was, however, distinct. Spleen cell proliferation accompanying the growth of bacilli in tissue was significantly higher in mice inoculated with O-BCG. These cells proliferated in vitro upon challenge with the corresponding BCG extract. Previous cell treatment with mAb anti-CD4 T cells abolished this effect. Anti-BCG antibodies, as assayed either in serum by ELISA or by determining the number of antibody-producing spleen cells by the spot-ELISA method, were significantly higher in mice inoculated with ID-BCG. Anti-BCG antibodies were detected in all immunoglobulin classes, but they were more prevalent in IgG with the following distribution among its isotypes: IgG1>(IgG2a = IgG2b)>IgG3. When some well-characterized Mycobacterium tuberculosis antigens were used as substitutes for BCG extracts in ELISA, although antibodies against the 65-kDa and 96-kDa proteins were detected significantly, antibodies against the 71-kDa, 38-kDa proteins and lipoarabinomannan were only barely detected or even absent. These results indicate that BCG bacilli cultured in Sauton-asparagine medium permitted the multiplication of bacilli, tending to induce a stronger humoral immune response as compared with bacilli grown in Sauton-starch/bacto-peptone-enriched medium.

Further biochemical characterization of Mycobacterium leprae laminin-binding proteins

It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells. Searching for M. leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp) and ribosomal proteins S4 and S5 as potential adhesins involved in M. leprae-Schwann cell interaction. Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells. In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5. The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays. However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins. The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells. Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the mycobacteria growth indicator tube (MGIT) system

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has increased the need for rapid drug susceptibility tests, which are needed for adequate patient treatment. The objective of the present study was to evaluate the mycobacteria growth indicator tube (MGIT) system to detect multidrug-resistant M. tuberculosis strains. The MGIT system was compared with two standard methods (proportion and resistance ratio methods). One hundred clinical M. tuberculosis isolates [25 susceptible to isoniazid (INH) and rifampicin (RIF), 20 resistant to INH, 30 resistant to INH-RIF, and 25 resistant to INH-RIF and other drugs] obtained in the State of São Paulo were tested for INH and RIF susceptibility. Full agreement among the tests was found for all sensitive and all INH-resistant strains. For RIF-resistant strains results among the tests agreed for 53 (96.4%) of 55 isolates. Results were obtained within 6 days (range, 5 to 8 days), 28 days and 12 days when using MGIT, the proportion method and the resistance ratio methods, respectively. The MGIT system presented an overall agreement of 96% when compared with two standard methods. These data show that the MGIT system is rapid, sensitive and efficient for the early detection of multidrug-resistant M. tuberculosis.

Identification of Mycobacterium bovis antigens by analysis of bovine T-cell responses after infection with a virulent strain

Purification and characterization of individual antigenic proteins are essential for the understanding of the pathogenic mechanisms of mycobacteria and the immune response against them. In the present study, we used anion-exchange chromatography to fractionate cell extracts and culture supernatant proteins from Mycobacterium bovis to identify T-cell-stimulating antigens. These fractions were incubated with peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle in lymphoproliferation assays. This procedure does not denature proteins and permits the testing of mixtures of potential antigens that could be later identified. We characterized protein fractions with high stimulation indices from both culture supernatants and cell extracts. Proteins were identified by two-dimensional gel electrophoresis followed by N-terminal sequencing or MALDI-TOF. Culture supernatant fractions containing low molecular weight proteins such as ESAT6 and CFP10 and other proteins (85B, MPB70), and the novel antigens TPX and TRB-B were associated with a high stimulation index. These results reinforce the concept that some low molecular weight proteins such as ESAT6 and CFP10 play an important role in immune responses. Also, Rv3747 and L7/L12 were identified in high stimulation index cell extract fractions. These data show that protein fractions with high lymphoproliferative activity for bovine PBMC can be characterized and antigens which have been already described and new protein antigens can also be identified in these fractions.

Humoral response to low molecular weight antigens of Mycobacterium tuberculosis by tuberculosis patients and contacts

Much effort has been devoted to the identification of immunologically important antigens of Mycobacterium tuberculosis and to the combination of target antigens to which antibodies from serum of tuberculous patients could react specifically. We searched for IgG antibodies specific for antigens of 45 to 6 kDa obtained after sonication of the well-characterized wild M. tuberculosis strain in order to detect differences in the antibody response to low molecular weight antigens from M. tuberculosis between patients with pulmonary tuberculosis and contacts. Specific IgG antibodies for these antigens were detected by Western blot analysis of 153 serum samples collected from 51 patients with confirmed pulmonary tuberculosis. Three samples were collected from each patient: before therapy, and after 2 and 6 months of treatment. We also analyzed 25 samples obtained from contacts, as well as 30 samples from healthy individuals with known tuberculin status, 50 samples from patients with other lung diseases and 200 samples from healthy blood donors. The positive predictive value for associated IgG reactivity against the 6-kDa and 16-kDa antigens, 6 and 38 kDa, and 16 and 38 kDa was 100% since simultaneous reactivity for these antigens was absent in healthy individuals and individuals with other lung diseases. This association was observed in 67% of the patients, but in only 8% of the contacts. The humoral response against antigens of 16 and 6 kDa seems to be important for the detection of latent tuberculosis since the associated reactivity to these antigens is mainly present in individuals with active disease.

PCR identification of Mycobacterium tuberculosis complex in a clinical sample from a patient with symptoms of tuberculous spondylodiscitis

A 42-year-old male complaining of thoracic spine pain was admitted to the hospital for evaluation. An X-ray and computer tomography of the thoracic spine showed spondylodiscitis of the L3 lumbar and L2-L3 intervertebral disk. The tuberculin skin test (PPD) was strongly positive. A radioscopy-guided fine needle aspirate of the affected area was cultured but did not reveal the cause of the disease. Two biopsy attempts failed to reveal the cause of the disease by culturing or by acid-fast-resistant staining (Ziehl Neelsen) of the specimens. A third biopsy also failed to detect the infectious agent by using microbiological procedures, but revealed the presence of a 245-bp amplicon characteristic of the Mycobacterium tuberculosis complex after PCR of the sample. The result demonstrates the efficacy of PCR for the identification of M. tuberculosis in situations in which conventional diagnosis by culturing techniques or direct microscopy is unable to detect the microorganism. Following this result the patient was treated with the antituberculous cocktail composed by rifampicin, pirazinamide and isoniazid during a six-month period. At the end of the treatment the dorsalgia symptoms had disappeared.