Increase in skeletal muscle protein content by the ß-2 selective adrenergic agonist clenbuterol exacerbates hypoalbuminemia in rats fed a low-protein diet

This investigation examined how the nutritional status of rats fed a low-protein diet was affected when the animals were treated with the ß-2 selective agonist clenbuterol (CL). Males (4 weeks old) from an inbred, specific-pathogen-free strain of hooded rats maintained at the Dunn Nutritional Laboratory were used in the experiments (N = 6 rats per group). CL treatment (Ventipulmin, Boehringer-Ingelheim Ltd., 3.2 mg/kg diet for 2 weeks) caused an exacerbation of the symptoms associated with protein deficiency in rats. Plasma albumin concentrations, already low in rats fed a low-protein diet (group A), were further reduced in CL rats (A = 25.05 ± 0.31 vs CL = 23.64 ± 0.30 g/l, P<0.05). Total liver protein decreased below the level seen in either pair-fed animals (group P) or animals with free access to the low-protein diet (A = 736.56 ± 26 vs CL = 535.41 ± 54 mg, P<0.05), whereas gastrocnemius muscle protein was higher than the values normally described for control (C) animals (C = 210.88 ± 3.2 vs CL = 227.14 ± 1.7 mg/g, P<0.05). Clenbuterol-treated rats also showed a reduction in growth when compared to P rats (P = 3.2 ± 1.1 vs CL = -10.2 ± 1.9 g, P<0.05). This was associated with a marked decrease in fat stores (P = 5.35 ± 0.81 vs CL = 2.02 ± 0.16 g, P<0.05). Brown adipose tissue (BAT) cytochrome oxidase activity, although slightly lower than in P rats (P = 469.96 ± 16.20 vs CL = 414.48 ± 11.32 U/BAT x kg body weight, P<0.05), was still much higher than in control rats (C = 159.55 ± 11.54 vs CL = 414.48 ± 11.32 U/BAT x kg body weight, P<0.05). The present findings support the hypothesis that an increased muscle protein content due to clenbuterol stimulation worsened amino acid availability to the liver and further reduced albumin synthesis causing exacerbation of hypoalbuminemia in rats fed a low-protein diet.

Limited-sampling strategy models for estimating the pharmacokinetic parameters of 4-methylaminoantipyrine, an active metabolite of dipyrone

Bioanalytical data from a bioequivalence study were used to develop limited-sampling strategy (LSS) models for estimating the area under the plasma concentration versus time curve (AUC) and the peak plasma concentration (Cmax) of 4-methylaminoantipyrine (MAA), an active metabolite of dipyrone. Twelve healthy adult male volunteers received single 600 mg oral doses of dipyrone in two formulations at a 7-day interval in a randomized, crossover protocol. Plasma concentrations of MAA (N = 336), measured by HPLC, were used to develop LSS models. Linear regression analysis and a "jack-knife" validation procedure revealed that the AUC0-¥ and the Cmax of MAA can be accurately predicted (R²>0.95, bias <1.5%, precision between 3.1 and 8.3%) by LSS models based on two sampling times. Validation tests indicate that the most informative 2-point LSS models developed for one formulation provide good estimates (R²>0.85) of the AUC0-¥ or Cmax for the other formulation. LSS models based on three sampling points (1.5, 4 and 24 h), but using different coefficients for AUC0-¥ and Cmax, predicted the individual values of both parameters for the enrolled volunteers (R²>0.88, bias = -0.65 and -0.37%, precision = 4.3 and 7.4%) as well as for plasma concentration data sets generated by simulation (R²>0.88, bias = -1.9 and 8.5%, precision = 5.2 and 8.7%). Bioequivalence assessment of the dipyrone formulations based on the 90% confidence interval of log-transformed AUC0-¥ and Cmax provided similar results when either the best-estimated or the LSS-derived metrics were used.

The leaves of green plants as well as a cyanobacterium, a red alga, and fungi contain insulin-like antigens

We report the detection of insulin-like antigens in a large range of species utilizing a modified ELISA plate assay and Western blotting. We tested the leaves or aerial parts of species of Rhodophyta (red alga), Bryophyta (mosses), Psilophyta (whisk ferns), Lycopodophyta (club mosses), Sphenopsida (horsetails), gymnosperms, and angiosperms, including monocots and dicots. We also studied species of fungi and a cyanobacterium, Spirulina maxima. The wide distribution of insulin-like antigens, which in some cases present the same electrophoretic mobility as bovine insulin, together with results recently published by us on the amino acid sequence of an insulin isolated from the seed coat of jack bean (Canavalia ensiformis) and from the developing fruits of cowpea (Vigna unguiculata), suggests that pathways depending on this hormone have been conserved through evolution.

Ureases display biological effects independent of enzymatic activity: Is there a connection to diseases caused by urease-producing bacteria?

Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein) that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori.

Trypanosoma cruzi infection and/or administration of the nonsteroidal anti-inflammatory nimesulide increase the number of colonic crypts overexpressing metallothioneins in rat colon carcinogenesis

Trypanosoma cruzi infection and nonsteroidal anti-inflammatory drugs inhibit colorectal carcinogenesis by mechanisms not completely known and metallothionein proteins (MTs) may be involved in this process. Sixty-six male Wistar rats weighing 90 to 120 g were randomly divided into seven groups (GI to GVII). GI, GII and GIII animals were subcutaneously infected with 200,000 trypomastigote forms of the Y strain of T. cruzi. After 8 weeks, GI, GII, GIV, and GVI were injected with one weekly subcutaneous dose of 12 mg/kg dimethylhydrazine for 4 weeks. In sequence, GI, GIV and GV were treated with nimesulide (10 mg/kg per dose, five times per week for 8 weeks). Groups I, III, IV, and VI had 12 animals, and each of the other groups had 6 animals. All the animals were euthanized 8 weeks after the last dimethylhydrazine injection. The colons were fixed and processed for MT immunohistochemistry. The index of MT-overexpressing colonic crypts (MTEC) was estimated as the percentage of MT-stained crypts in relation to the total number of crypts scored. Five hundred crypts per animal were scored. Data were analyzed by the Kruskal-Wallis test followed by the Dunn test. There was an increase in MTEC index in the groups either infected with T. cruzi or treated with nimesulide or both infected and treated when compared to control (401, 809, and 1011%, respectively). We suggest that the increased formation of MTEC may be related to the protection against carcinogenesis provided both by T. cruzi infection and nimesulide.

Chlorpheniramine facilitates inhibitory avoidance in teleosts submitted to telencephalic ablation

The present study investigated the involvement of H(1) histaminegic receptor on the acquisition of inhibitory avoidance in Carassius auratus submitted to telencephalic ablation. The fish were submitted to telencephalic ablation 5 days before the experiment. The inhibitory avoidance procedure included 1 day for habituation, 3 days for training composed of 3 trials each (1st day: T1, T2, T3; 2nd day: 2T1, 2T2, 2T3; 3rd day: 3T1, 3T2, 3T3) and 1 day for test. On training days, the fish were placed in a white compartment, after 30 s the door was opened. When the fish crossed to a black compartment, a weight was dropped (aversive stimuli). Immediately after the third trial, on training days, the fish received, intraperitoneally, one of the pharmacological treatments (saline (N = 20), 8 (N = 12) or 16 (N = 13) µg/g chlorpheniramine, CPA). On the test day, the time to cross to the black compartment was determined. The latency of the saline group increased significantly only on the 3rd trial of the 2nd training day (mean ± SEM, T1 (50.40 ± 11.69), 2T3 (226.05 ± 25.01); ANOVA: P = 0.0249, Dunn test: P < 0.05). The group that received 8 µg/g CPA showed increased latencies from the 2nd training day until the test day (T1 (53.08 ± 17.17), 2T2 (197.75 ± 35.02), test (220.08 ± 30.98); ANOVA: P = 0.0022, Dunn test: P < 0.05)). These results indicate that CPA had a facilitating effect on memory. We suggest that the fish submitted to telencephalic ablation were able to learn due to the local circuits of the mesencephalon and/or diencephalon and that CPA interferes in these circuits, probably due an anxiolytic-like effect.

Muscle strength but not functional capacity is associated with plasma interleukin-6 levels of community-dwelling elderly women

The association of plasma interleukin-6 (IL-6) levels, muscle strength and functional capacity was investigated in a cross-sectional study of community-dwelling elderly women from Belo Horizonte, Brazil. Elderly people who present controlled chronic diseases with no negative impact on physical, psychosocial and mental functionality are considered to be community-dwelling. Psychological and social stress due to unsuccessfully aging can represent a risk for immune system disfunctions. IL-6 levels, isokinetic muscle strength of knee flexion/extension, and functional tests to determine time required to rise from a chair and gait velocity were measured in 57 participants (71.21 ± 7.38 years). Serum levels of IL-6 were measured in duplicate and were performed within one single assay (mouse monoclonal antibody against IL-6; High-Sensitivity, Quantikine®, R & D Systems, USA; intra-assay coefficient of variance = 6.9-7.4%; interassay coefficient of variance = 9.6-6.5%; sensitivity = 0.016-0.110 pg/mL; mean = 0.039 pg/mL). Muscle strength was assessed with the isokinetic dynamometer Biodex System 3 Pro®. After the Shapiro-Wilk normality test was applied, correlations were investigated using Spearman and Kruskal-Wallis tests. Post hoc analysis was performed using the Dunn test. A significant negative correlation was observed between plasma IL-6 levels (1.95 ± 1.77 pg/mL) and muscle strength for knee flexion (70.70 ± 21.14%; r = -0.265; P = 0.047) and extension (271.84 ± 67.85%; r = -0.315; P = 0.017). No significant correlation was observed between IL-6 levels and the functional tests (time to rise from a chair = 14.65 ± 2.82 s and gait velocity = 0.95 ± 0.14 m/s). These results suggest that IL-6 is associated with reduced muscle strength.