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.

Chemical composition, acetylcholinesterase inhibitory and antifungal activities of Pera glabrata (Schott) Baill. (Euphorbiaceae)

Pera glabrata (Schott) Baill. was selected for this study after showing a preliminary positive result in a screening of Atlantic Forest plant species in the search for acetylcholinesterase inhibitors and antifungal compounds. The bioassays were conducted with crude ethanol extract of the leaves using direct bioautography method for acetylcholinesterase and antifungal activities. This extract was partitioned with hexane, chloroform and ethyl acetate solvents. The active chloroform fraction was submitted to silica gel chromatography column affording 12 groups. Caffeine, an alkaloid, which showed detection limits of 0.1 and 1.0 µg for anticholinesterasic and antifungal activities, respectively, was isolated from group nine. After microplate analyses, only groups four, nine, 10, 11 and 12 showed acetylcholinesterase inhibitory activity of 40% or higher. The group 12 was purified by preparative layer chromatography affording four sub-fractions. Two sub-fractions from this group were analyzed by gas chromatography-mass spectrometry and gas chromatography-flame ionization detector. The first sub-fraction showed anticholinesterasic activity and contained two major compounds: 9-hydroxy-4-megastigmen-3-one (84%) and caffeine (6%). The second sub-fraction presented five major compounds identified as 9-hydroxy-4-megastigmen-3-one, isololiolide, (-) loliolide, palmitic acid and lupeol and did not show activity.

Multivariate analysis of pollen frequency of the native species Escallonia pulverulenta (Saxifragaceae) in Chilean honeys

The aim of this work was the identification of geographic zones suitable for the production of honeys in which pollen grains of Escallonia pulverulenta (Ruiz & Pav.) Pers. (Saxifragaceae) can be detected. The analysis of botanical origin of 240 honey samples produced between La Serena and Puerto Mont (the IV and X Administrative Regions of Chile), allowed the detection of pollen grains of E. pulverulenta in 46 Chilean honeys. The geographic distribution of the honeys studied is presented together with their affinities, through factor analysis and frequency tables. The study was based on the presence of E. pulverulenta pollen. Escallonia pulverulenta pollen percentages oscillated between 0.24% and 78.5%. Seventeen of the studied samples were designated as unifloral - i.e. samples showing more than 45% pollen of a determined plant species. Two of these corresponded to E. pulverulenta (corontillo, madroño or barraco) honeys. The remaining unifloral honeys correspond to 8 samples of Lotus uliginosus Schkuhr (birdsfoot trefoil), 2 samples of Aristotelia chilensis (Molina) Stuntz (maqui) and 1 sample of Escallonia rubra (Ruiz & Pav.) Pers. (siete camisas), Eucryphia cordifolia Cav. (ulmo or muemo), Weinmannia trichosperma Cav. (tineo), Rubus ulmifolius Schott (blackberry) and Brassica rapa L. (turnip). Honeys with different percentages of E. pulverulenta pollen - statistically analyzed through correspondence analysis - could be associated and assigned to one of three geographic types, defined on the basis of this analysis. The geographical type areas defined were the Northern Mediterranean Zone (samples from the IV Region), Central Mediterranean Zone (samples from the V to the VIII regions including two samples of unifloral Escallonia pulverulenta honey), and Southern Mediterranean Zone (samples from the IX Region).

Test of hypotheses about herbivory and chemical defences of Qualea parviflora (Vochysiaceae) in Brazilian Cerrado

(Test of hypotheses about herbivory and chemical defences of Qualea parviflora (Vochysiaceae) in Brazilian Cerrado). Qualea parviflora Mart. (Vochysiaceae), a widely distributed tree found in different habitats in Brazilian Cerrado (savanna), provides resources for a great variety of insects. In this study, we tested two hypotheses about plant investment in anti-herbivore defences along a fertility gradient in Cerrado: the carbon/nitrogen balance (CNBH) and resource availability (RAH). We also investigated how the pattern of herbivory varies through the year and among three types of vegetation in Brazilian Cerrado - campo sujo, cerrado sensu strictu and cerradão. Sampling was conducted in three types of vegetation and in rainy (January and November) and dry months (April and July). Damage on 20 completely expanded leaves, leaf nutrients, sclerophlylly, total phenols and tannins were recorded for each plant (n = 30). When leaves were young, less sclerophyllous, and with higher concentration of nutrients and tannins, damage by herbivores was about 7% in cerrado sensu stricto and 3% in campo sujo. Mature leaves did not show any significant difference on herbivory among habitats, that varied from 6 to 9%. Nutrient availability to plants is an important factor determining production of secondary metabolites in Q. parviflora, corroborating the CNBH. The absence of correlation between damaged leaf area and tannin concentration did not corroborate the RAH, suggesting that tannin production is not strongly influenced by herbivores on Q. parviflora.

Germination, initial growth, and biomass allocation in three native Cerrado species

The parameters of germination, initial growth, and biomass allocation of three native plant species of Cerrado (Copaifera langsdorffii, Dipteryx alata and Kielmeyera coriacea) were established. The species had germination percentages above 88% and average germination times longer than 139 hours. The average time for the opening of the first leaf pair was more than 538 hours for all three species. The average root length of C. langsdorffii and D. alata seedlings after 80 days of growth was around 40cm, four times larger than the average shoot length (<10cm), although the root and shoot biomasses were similar for both species. The average root length (>20cm) of K. coriacea seedlings was four times larger than the average shoot length (<5cm), and the root biomass was 243% greater than the shoot biomass. Increase in seedling biomass was sustained primarily by the cotyledons in C. langsdorffii and D. alata, which acted as reserve organs and showed progressive weight reductions. Increase in seedling biomass in K. coriacea was sustained primarily by photosynthesis, since the cotyledons showed no significant weight reduction, acting primarily as photosynthetic organs. The length of the root systems was at least four times larger than the length of the shoots parts in all three species. Higher investment in root length rather than in root biomass suggest that the initial growth of these species is primarily to ensure access to water resources, apparently putting off the function of the radicular system as storage organ.

Connexin domains relevant to the chemical gating of gap junction channels

Most cells exchange ions and small metabolites via gap junction channels. These channels are made of two hemichannels (connexons), each formed by the radial arrangement of six connexin (Cx) proteins. Connexins span the bilayer four times (M1-M4) and have both amino- and carboxy-termini (NT, CT) at the cytoplasmic side of the membrane, forming two extracellular loops (E1, E2) and one inner (IL) loop. The channels are regulated by gates that close with cytosolic acidification (e.g., CO2 treatment) or increased calcium concentration, possibly via calmodulin activation. Although gap junction regulation is still unclear, connexin domains involved in gating are being defined. We have recently focused on the CO2 gating sensitivity of Cx32, Cx38 and various mutants and chimeras expressed in Xenopus oocytes and studied by double voltage clamp. Cx32 is weakly sensitive to CO2, whereas Cx38 is highly sensitive. A Cx32 chimera containing the second half of the inner loop (IL2) of Cx38 was as sensitive to CO2 as Cx38, indicating that this domain plays an important role. Deletion of CT by 84% did not affect CO2 sensitivity, but replacement of 5 arginines (R) with sparagines (N) at the beginning of CT (C1) greatly enhanced the CO2 sensitivity of Cx32. This suggests that whereas most of CT is irrelevant, positive charges of C1 maintain the CO2 sensitivity of Cx32 low. As a hypothesis we have proposed a model that involves charge interaction between negative residues of the beginning of IL1 and positive residues of either C1 or IL2. Open and closed channels would result from IL1-C1 and IL1-IL2 interactions, respectively

pH titration of native and unfolded ß-trypsin: evaluation of the D D G0 titration and the carboxyl pK values

The stabilizing free energy of ß-trypsin was determined by hydrogen ion titration. In the pH range from 3.0 to 7.0, the change in free energy difference for the stabilization of the native protein relative to the unfolded one (D D G0 titration) was 9.51 ± 0.06 kcal/mol. An isoelectric point of 10.0 was determined, allowing us to calculate the Tanford and Kirkwood electrostatic factor w. This factor presented a nonlinear behavior and indicated more than one type of titratable carboxyl groups in ß-trypsin. In fact, one class of carboxyl group with a pK = 3.91 ± 0.01 and another one with a pK = 4.63 ± 0.03 were also found by hydrogen ion titration of the protein in the folded state

Plasma cortisol levels in captive wild felines after chemical restraint

Eight Panthera onca (Po), 13 Felis concolor (Fc), 7 Felis yagouaroundi (Fy), 7 Felis tigrina (Ft) and 5 Felis pardalis (Fp) specimens from São Paulo State zoos were used. All animals were restrained with darts containing 10 mg/kg ketamine and 1 mg/kg xylazine. Venous blood samples were collected as soon as possible (within 15-20 min) and serum was frozen until the time for cortisol quantification. Cortisol was determined using a solid phase radioimmunoassay with an intra-assay coefficient of 8.51%. Data were analyzed statistically by the Kruskal-Wallis test, followed by Dunn's multiple comparisons test, and the one-sample t-test, with the level of significance set at P<0.05. Data are reported as means ± SEM. Cortisol levels differed among the captive felines: Po = 166 ± 33a, Fc = 670 ± 118b, Fy = 480 ± 83b, Ft = 237 ± 42ab, Fp = 97 ± 12a nmol/l (values followed by different superscript letters were significantly different (P<0.001)). Since most of the veterinary procedures on these species involve chemical restraint, these results show the necessity of preventive measures in order to minimize the effect of restraint stress on more susceptible species

Steps in the formation of neurites and synapses studied in cultured leech neurons

Leech neurons in culture have provided novel insights into the steps in the formation of neurite outgrowth patterns, target recognition and synapse formation. Identified adult neurons from the central nervous system of the leech can be removed individually and plated in culture under well-controlled conditions, where they retain their characteristic physiological properties, grow neurites and form specific chemical or electrical synapses. Different identified neurons develop distinctive outgrowth patterns that depend on their identities and on the molecular composition of the substrate. On native substrates, the patterns displayed by these neurons reproduce characteristics from the adult or the developing neurons. In addition, the substrate may induce selective directed growth between pairs of neurons that normally make contact in the ganglion. Upon contact, pairs of cultured leech neurons form chemical or electrical synapses, or both types depending on the neuronal identities. Anterograde and retrograde signals during membrane contact and synapse formation modify the distribution of synaptic terminals, calcium currents, and responses to 5-hydroxytryptamine.

Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis.

The use of protein structure/activity relationships in the rational design of stable particulate delivery systems

The recombinant heat shock protein (18 kDa-hsp) from Mycobacterium leprae was studied as a T-epitope model for vaccine development. We present a structural analysis of the stability of recombinant 18 kDa-hsp during different processing steps. Circular dichroism and ELISA were used to monitor protein structure after thermal stress, lyophilization and chemical modification. We observed that the 18 kDa-hsp is extremely resistant to a wide range of temperatures (60% of activity is retained at 80ºC for 20 min). N-Acylation increased its ordered structure by 4% and decreased its ß-T1 structure by 2%. ELISA demonstrated that the native conformation of the 18 kDa-hsp was preserved after hydrophobic modification by acylation. The recombinant 18 kDa-hsp resists to a wide range of temperatures and chemical modifications without loss of its main characteristic, which is to be a source of T epitopes. This resistance is probably directly related to its lack of organization at the level of tertiary and secondary structures.

Antinociceptive activity of Hypericum caprifoliatum and Hypericum polyanthemum (Guttiferae)

The aim of the present study was to assess the analgesic activity of the aerial parts of two Hypericum species native to Southern Brazil, H. caprifoliatum and H. polyanthemum. The antinociceptive effect of the H. polyanthemum cyclohexane extract (POL; 180 mg/kg) and of the H. caprifoliatum methanol (MET) and cyclohexane (CH) extracts (90 mg/kg) was evaluated in the hot-plate (ip and po) and writhing (po) tests using male Swiss CF1 mice weighing 22-27 g (N = 10 per group). All extracts displayed antinociceptive effects in the hot-plate test (MET ip = 48%, MET po = 39%, CH ip = 27%, CH po = 50%, POL ip = 74%, and POL po = 49% compared to control). Pretreatment with naloxone (2.5 mg/kg, sc) abolished the effects of CH and POL, and partially prevented the analgesia induced by MET administered by the ip (but not by the po) route. POL and CH (po) significantly reduced the number of writhes induced by acetic acid, while MET was ineffective in this regard. We conclude that the antinociceptive effects of the H. caprifoliatum (CH) and H. polyanthemum (POL) hexane extracts seem to be mediated by the opioid system. Moreover, the antinociceptive activity of the H. caprifoliatum MET extract seems to depend on at least two chemical substances (or groups of substances) with distinct pharmacokinetic profiles and mechanisms of action. Only the naloxone-insensitive component of MET activity showed good bioavailability following oral administration.

Infrared coagulation versus rubber band ligation in early stage hemorrhoids

The ideal therapy for early stages of hemorrhoids is always debated. Some are more effective but are more painful, others are less painful but their efficacy is also lower. Thus, comfort or efficacy is a major concern. In the present randomized study, a comparison is made between infrared coagulation and rubber band ligation in terms of effectiveness and discomfort. One hundred patients with second degree bleeding piles were randomized prospectively to either rubber band ligation (N = 54) or infrared coagulation (N = 46). Parameters measured included postoperative discomfort and pain, time to return to work, relief in incidence of bleeding, and recurrence rate. The mean age was 38 years (range 19-68 years). The mean duration of disease was 17.5 months (range 12 to 34 months). The number of male patients was double that of females. Postoperative pain during the first week was more intense in the band ligation group (2-5 vs 0-3 on a visual analogue scale). Post-defecation pain was more intense with band ligation and so was rectal tenesmus (P = 0.0059). The patients in the infrared coagulation group resumed their duties earlier (2 vs 4 days, P = 0.03), but also had a higher recurrence or failure rate (P = 0.03). Thus, we conclude that band ligation, although more effective in controlling symptoms and obliterating hemorrhoids, is associated with more pain and discomfort to the patient. As infrared coagulation can be conveniently repeated in case of recurrence, it could be considered to be a suitable alternative office procedure for the treatment of early stage hemorrhoids.

Screening of antibacterial extracts from plants native to the Brazilian Amazon Rain Forest and Atlantic Forest

More than 20% of the world's biodiversity is located in Brazilian forests and only a few plant extracts have been evaluated for potential antibacterial activity. In the present study, 705 organic and aqueous extracts of plants obtained from different Amazon Rain Forest and Atlantic Forest plants were screened for antibacterial activity at 100 µg/ml, using a microdilution broth assay against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli. One extract, VO581, was active against S. aureus (minimum inhibitory concentration (MIC) = 140 µg/ml and minimal bactericidal concentration (MBC) = 160 µg/ml, organic extract obtained from stems) and two extracts were active against E. faecalis, SM053 (MIC = 80 µg/ml and MBC = 90 µg/ml, organic extract obtained from aerial parts), and MY841 (MIC = 30 µg/ml and MBC = 50 µg/ml, organic extract obtained from stems). The most active fractions are being fractionated to identify their active substances. Higher concentrations of other extracts are currently being evaluated against the same microorganisms.

Evaluation by blue native polyacrylamide electrophoresis colorimetric staining of the effects of physical exercise on the activities of mitochondrial complexes in rat muscle

Blue native polyacrylamide electrophoresis (BN-PAGE) is a technique developed for the analysis of membrane complexes. Combined with histochemical staining, it permits the analysis and quantification of the activities of mitochondrial oxidative phosphorylation enzymes using whole muscle homogenates, without the need to isolate muscle mitochondria. Mitochondrial complex activities were measured by emerging gels in a solution containing all specific substrates for NADH dehydrogenase and cytochrome c oxidase enzymes (complexes I and IV, respectively) and the colored bands obtained were measured by optique densitometry. The objective of the present study was the application of BN-PAGE colorimetric staining for enzymatic characterization of mitochondrial complexes I and IV in rat muscles with different morphological and biochemical properties. We also investigated these activities at different times after acute exercise of rat soleus muscle. Although having fewer mitochondria than oxidative muscles, white gastrocnemius muscle presented a significantly higher activity (26.7 ± 9.5) in terms of complex I/V ratio compared to the red gastrocnemius (3.8 ± 0.65, P < 0.05) and soleus (9.8 ± 0.9, P < 0.001) muscles. Furthermore, the complex IV/V ratio of white gastrocnemius muscle was always significantly higher when compared to the other muscles. Ninety-five minutes of exhaustive physical exercise induced a decrease in complex I/V and complex IV/V ratios after all resting times (0, 3 and 6 h) compared to control (P < 0.05), probably reflecting the oxidative damage due to increasing free radical production in mitochondria. These results demonstrate the possible and useful application of BN-PAGE-histochemical staining to physical exercise studies.