Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies with Aspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellow peroba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium berghei in mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.     

Interleukin-1, tumor necrosis factor-alpha,and transforming growth factor-beta 1 and integrative meniscal repair: influences on meniscal cell proliferation and migration

Introduction  

Interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are up-regulated in injured and osteoarthritic knee joints. IL-1 and TNF-α inhibit integrative meniscal repair; however, the mechanisms by which this inhibition occurs are not fully understood. Transforming growth factor-β1 (TGF-β1) increases meniscal cell proliferation and accumulation, and enhances integrative meniscal repair. An improved understanding of the mechanisms modulating meniscal cell proliferation and migration will help to improve approaches for enhancing intrinsic or tissue-engineered repair of the meniscus. The goal of this study was to examine the hypothesis that IL-1 and TNF-α suppress, while TGF-β1 enhances, cellular proliferation and migration in cell and tissue models of meniscal repair.     

The Caulobacter crescentus DNA-(adenine-N6)-methyltransferase CcrM methylates DNA in a distributive manner

The specificity and processivity of DNA methyltransferases have important implications regarding their biological functions. We have investigated the sequence specificity of CcrM and show here that the enzyme has a high specificity for GANTC sites, with only minor preferences at the central position. It slightly prefers hemimethylated DNA, which represents the physiological substrate. In a previous work, CcrM was reported to be highly processive [Berdis et al. (1998) Proc. Natl Acad. Sci. USA 95: 2874-2879]. However upon review of this work, we identified a technical error in the setup of a crucial experiment in this publication, which prohibits making any statement about the processivity of CcrM. In this study, we performed a series of in vitro experiments to study CcrM processivity. We show that it distributively methylates six target sites on the pUC19 plasmid as well as two target sites located on a 129-mer DNA fragment both in unmethylated and hemimethylated state. Reaction quenching experiments confirmed the lack of processivity. We conclude that the original statement that CcrM is processive is no longer valid.     

Functional and morphological assessment of early impairment of airway function in a rat model of emphysema

The aim of this study was to evaluate airway structure-function relations in elastase-induced emphysema in rats. Sprague-Dawley rats were treated intratracheally with 50 IU porcine pancreatic elastase (PPE, n = 8) or saline (controls, n = 6). Six weeks later, lung volumes [functional residual capacity (FRC), residual volume (RV), and total lung capacity (TLC)] and low-frequency impedance parameters (Newtonian resistance, R(N); tissue damping; tissue elastance, H) were measured, and tracheal sounds were recorded during slow inflation to TLC following in vivo degassing. The lungs were fixed and stained for standard morphometry, elastin, and collagen. In the PPE group, FRC and RV were higher [4.53 ± 0.7 (SD) vs. 3.28 ± 0.45 ml; P = 0.003 and 1.06 ± 0.35 vs. 0.69 ± 0.18 ml; P = 0.036, respectively], and H was smaller in the PPE-treated rats than in the controls (1,344 ± 216 vs. 2,178 ± 305 cmH(2)O/l; P < 0.001), whereas there was no difference in R(N). The average number of crackles per inflation was similar in the two groups; however, the crackle size distributions were different and the lower knee of the pressure-volume curves was higher in the PPE group. Microscopic images revealed different alveolar size distributions but similar bronchial diameters in the two groups. The treatment caused a slight but significant decrease in the numbers of alveolar attachments, no difference in elastin and slightly increased mean level and heterogeneity of collagen in the bronchial walls. These results suggest that tissue destruction did not affect the conventionally assessed airway resistance in this emphysema model, whereas the alterations in the recruitment dynamics can be an early manifestation of impaired airway function.     

Modifications of ribonuclease A induced by p-benzoquinone

The nature of ribonuclease A (RNase) modifications induced by p-benzoquinone (pBQ) was investigated using several analysis methods. SDS-PAGE experiments revealed that pBQ was efficient in producing oligomers and polymeric aggregates when RNase was incubated with pBQ. The fluorescence behavior and anisotropy changes of the modified RNase were monitored for a series of incubation reactions where RNase (0.050 mM) was incubated with pBQ (0.050, 0.25, 0.50, 1.50 mM) at 37 °C in phosphate buffer (pH 7.0, 50 mM). The modified RNase exhibited less intense fluorescence and slightly higher anisotropy than the unmodified RNase. UV-Vis spectroscopy indicated that pBQ formed covalent bonds to the modified RNase. Confocal imaging analysis confirmed the formation of the polymeric RNase aggregates with different sizes upon exposure of RNase to high concentrations of pBQ. The interaction between the modified RNase and salts affecting biomineralization of salts was also investigated by scanning electron microscopy. Overall, our results show that pBQ can induce formation of both RNase adducts and aggregates thus providing a better understanding of its biological activity.     

Adiponectin Levels during Low‐ and High‐Fat Eucaloric Diets in Lean and Obese Women

Objective: Adiponectin influences insulin sensitivity (S_I) and fat oxidation. Little is known about changes in adiponectin with changes in the fat content of eucaloric diets. We hypothesized that dietary fat content may influence adiponectin according to an individual's S_I. Research Methods and Procedures: We measured changes in adiponectin, insulin, glucose, and leptin in response to high‐fat (HF) and low‐fat (LF) eucaloric diets in lean (n = 10) and obese (n = 11) subjects. Obese subjects were further subdivided in relation to a priori S_I. Results: We found significantly higher insulin, glucose, and leptin and lower adiponectin in obese vs. lean subjects during both HF and LF. The mean group values of these measurements, including adiponectin (lean, HF 21.9 ± 9.8; LF, 20.8 ± 6.6; obese, HF 10.0 ± 3.3; LF, 9.5 ± 2.3 ng/mL; mean ± SD), did not significantly change between HF and LF diets. However, within the obese group, the insulin‐sensitive subjects had significantly higher adiponectin during HF than did the insulin‐resistant subjects. Additionally, the change in adiponectin from LF to HF diet correlated positively with the obese subjects’ baseline S_I. Discussion: Although in lean and obese women, group mean values for adiponectin did not change significantly with a change in fat content of a eucaloric diet, a priori measured S_I in obese subjects predicted an increase in adiponectin during the HF diet; this may be a mechanism that preserves S_I in an already obese group.     

Comparative aspects of the density and diameter of Haversian canals in the diaphyseal compact bone of man and dog

By using an original method, the authors injected the system of canals and then studied microscopically their network in the femoral diaphysis of man and dog. In the human bone, the density varies between 1.09 and 2.63 canals per mm2, with an average of 1.83; in dogs the limits of variation are 1.98--2.86 canals per mm2. The canal calibre is of 68.8 microns in man as against 48.5 microns in dogs. Both parameters present a wider variability in the femoral compact bone of man than in dog. By calculation it was found that the canal density in dogs is very significantly increased as compared to man; on the contrary, the diameter presents in dogs very significantly decreased values as compared to those of the diaphyseal compact bone of man.     

Adsorption of ethylenediaminetetraacetic dianhydride modified oxalate decarboxylase on calcium oxalate

We used ethylenediaminetetraacetic acid dianhydride (EDTAD) to modify oxalate decarboxylase (OXDC) to improve its adsorption on calcium oxalate stones. The modified sites were identified by Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and the adsorption mechanism of the EDTAD-modified OXDC on calcium oxalate (CaOx) was investigated. We investigated adsorption time, initial enzyme concentration, temperature and solution pH on the adsorption process. Data were analyzed using kinetics, thermodynamics and isotherm adsorption models. UPLC-MS showed that EDTAD was attached to OXDC covalently and suggested that the chemical modification occurred at both the free amino of the side chain and the α-NH₂ of the peptide. The adsorption capacity of the EDTAD-OXDC on calcium oxalate was 53.37% greater than that of OXDC at the initial enzyme concentration of 5 mg/ml, pH = 7.0, at 37° C. The modified enzyme (EDTAD-OXDC) demonstrated improved oxalate degradation activity at pH 4.5?6.0. Kinetic data fitting analysis suggested a pseudo second order kinetic model. Estimates of the thermodynamic parameters including ΔG⁰, ΔH⁰ and ΔS⁰ of the adsorption process showed it to be feasible, spontaneous and endothermic. Isotherm data fitting analysis indicated that the adsorption process is reduced to monolayer adsorption at a low enzyme concentration and to multilayer adsorption at a high enzyme concentration. It may be possible to apply OXDC to degradation of calcium oxalate stones.     

Antioxidant activity,acetylcholinesterase and tyrosinase inhibitory potential of Pulmonaria officinalis and Centarium umbellatum extracts

In this study several investigations and tests were performed to determine the antioxidant activity and the acetylcholinesterase and tyrosinase inhibitory potential of Pulmonaria officinalis and Centarium umbellatum aqueous extracts (10% mass) and ethanolic extracts (10% mass and 70% ethanol), respectively. Moreover, for each type of the prepared extracts of P. officinalis and of C. umbellatum the content in the biologically active compounds – polyphenols, flavones and proanthocyanidins was determined. The antioxidant activity was assessed using two methods, namely the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and reducing power assay. The analyzed plant extracts showed a high acetylcholinesterase and tyrosinase inhibitory activity in the range of 72.24–94.24% (at the highest used dose – 3 mg/mL), 66.96% and 94.03% (at 3 mg/mL), respectively correlated with a high DPPH radical inhibition – 70.29–84.9% (at 3 mg/mL). These medicinal plants could provide a potential natural source of bioactive compounds and could be beneficial to the human health, especially in the neurodegenerative disorders and as sources of natural antioxidants in food industry.