Relevance of Hydrodynamic Effects for the Calculation of Outer Surface Potential of Biological Membrane Using Electrophoretic Data.

In this paper, we present the results of a study on the influence of hydrodynamic effects on the surface potentials of the erythrocyte membrane, comparing two different models formulated to simulate the electrophoretic movement of a biological cell: the classical Helmholtz-Smoluchowski model and a model presented by Hsu et al. (1996). This model considers hydrodynamic effects to describe the distribution of the fluid velocity. The electric potential equation was obtained from the non-linear Poisson-Boltzmann equation, considering the spatial distribution of electrical charges fixed in glycocalyx and cytoplasmic proteins, as well as electrolyte charges and ones fixed on the surfaces of lipidic bilayer. Our results show that the Helmholtz-Smoluchowski model is not able to reflect the real forces responsible to the electrophoretic behavior of cell, because it does not take account the hydrodynamic effects of glycocalyx. This charged network that covers cellular surface constitutes a complex physical system whose electromechanical characteristics cannot be neglected. Then, supporting the hypothesis of other authors, we suggest that, in electrophoretic motion analyses of cells, the classical model represents a limiting case of models that take into account hydrodynamic effects to describe the velocity distribution of fluid.

Effects of prepubertal corticosterone treatment on urinary bladder.

PURPOSE: The aim of this work was to analyze the bladder wall modifications after a chronic treatment with high doses of corticosterone in prepubertal rats. METHODS: This study included 26 male rats assigned into four groups: T30 was treated with corticosterone until 29 days of age and killed at day 30, while T65 group received the same treatment but was killed at day 65. Each group had its own control group (C30 and C65). For treated animals, daily intraperitoneal injections of corticosterone (20 mg/Kg) were administered between 7th and 29th day of life. Bladders were removed and collagen, smooth muscle, elastic fibers system, vascular density and epithelium were analyzed by morphometrical methods, immunofluorescence, and biochemistry. RESULTS: Vascular density in lamina propria was reduced by 40% (p<0.05) in group T65. Collagen organization was altered in T30 and T65, although total collagen concentration was unchanged. The T65 group had an increase in elastic system fibers. There was no difference in epithelial height and cell density between the groups. Concerning the smooth muscle fibers density we observed a 19% increase (p<0.05) in the T65 group. CONCLUSION: Prepubertal administration of corticosterone induces structural modifications in the bladder of rats in a medium term analysis.

Anti-heparanase aptamers as potential diagnostic and therapeutic agents for oral cancer.

Heparanase is an endoglycosidase enzyme present in activated leucocytes, mast cells, placental tissue, neutrophils and macrophages, and is involved in tumour metastasis and tissue invasion. It presents a potential target for cancer therapies and various molecules have been developed in an attempt to inhibit the enzymatic action of heparanase. In an attempt to develop a novel therapeutic with an associated diagnostic assay, we have previously described high affinity aptamers selected against heparanase. In this work, we demonstrated that these anti-heparanase aptamers are capable of inhibiting tissue invasion of tumour cells associated with oral cancer and verified that such inhibition is due to inhibition of the enzyme and not due to other potentially cytotoxic effects of the aptamers. Furthermore, we have identified a short 30 bases aptamer as a potential candidate for further studies, as this showed a higher ability to inhibit tissue invasion than its longer counterpart, as well as a reduced potential for complex formation with other non-specific serum proteins. Finally, the aptamer was found to be stable and therefore suitable for use in human models, as it showed no degradation in the presence of human serum, making it a potential candidate for both diagnostic and therapeutic use.

Modeling the electric potential across neuronal membranes: the effect of fixed charges on spinal ganglion neurons and neuroblastoma cells.

We present a model for the electric potential profile across the membranes of neuronal cells. We considered the resting and action potential states, and analyzed the influence of fixed charges of the membrane on its electric potential, based on experimental values of membrane properties of the spinal ganglion neuron and the neuroblastoma cell. The spinal ganglion neuron represents a healthy neuron, and the neuroblastoma cell, which is tumorous, represents a pathological neuron. We numerically solved the non-linear Poisson-Boltzmann equation for the regions of the membrane model we have adopted, by considering the densities of charges dissolved in an electrolytic solution and fixed on both glycocalyx and cytoplasmic proteins. Our model predicts that there is a difference in the behavior of the electric potential profiles of the two types of cells, in response to changes in charge concentrations in the membrane. Our results also describe an insensitivity of the neuroblastoma cell membrane, as observed in some biological experiments. This electrical property may be responsible for the low pharmacological response of the neuroblastoma to certain chemotherapeutic treatments.

Effects of prepubertal corticosterone treatment on urinary bladder

PURPOSE: The aim of this work was to analyze the bladder wall modifications after a chronic treatment with high doses of corticosterone in prepubertal rats. METHODS: This study included 26 male rats assigned into four groups: T30 was treated with corticosterone until 29 days of age and killed at day 30, while T65 group received the same treatment but was killed at day 65. Each group had its own control group (C30 and C65). For treated animals, daily intraperitoneal injections of corticosterone (20 mg/Kg) were administered between 7th and 29th day of life. Bladders were removed and collagen, smooth muscle, elastic fibers system, vascular density and epithelium were analyzed by morphometrical methods, immunofluorescence, and biochemistry. RESULTS: Vascular density in lamina propria was reduced by 40% (p<0.05) in group T65. Collagen organization was altered in T30 and T65, although total collagen concentration was unchanged. The T65 group had an increase in elastic system fibers. There was no difference in epithelial height and cell density between the groups. Concerning the smooth muscle fibers density we observed a 19% increase (p<0.05) in the T65 group. CONCLUSION: Prepubertal administration of corticosterone induces structural modifications in the bladder of rats in a medium term analysis. .

Effects of chronic stress on penile corpus cavernosum of rats.

The objective of this study was to investigate structural changes in the penile corpus cavernosum of prepubertal chronically stressed rats. Eight Wistar rats were assigned into the stress group (SG) and were submitted to 2 hours of tube restraint daily, from the fourth to the ninth week of life. Another 7 rats were used as the control group (CG). All animals were weighed weekly. At day 64, animals were sacrificed by anesthetic overdose, blood was collected for testosterone concentration by radioimmunoassay, and penis and adrenal were collected. Adrenal mass index and testosterone serum levels were used to assess the efficacy of the stress stimulus. The surface density of connective tissue and smooth muscle fibers of corpus cavernosum were measured on Masson trichromic-stained slices. Picrosirius red-stained slices were assessed under polarized light for different types of collagen. The Student's t test was applied for mean comparisons, with P < .05 considered significant. Testosterone serum concentrations decreased and adrenal mass index increased, confirming the effectiveness of the stress protocol. Smooth muscle fibers of corpus cavernosum decreased from 14.07% (CG) to 8.98% (SG) (P = .02), and connective tissue increased from 53.66% (CG) to 64.47% (SG) (P = .01). Also, there was a higher level of type I collagen in the SG animals compared with the CG. Stress stimuli induced structural changes in the corpus cavernosum of rats suggestive of penile fibrosis, which may play a role in erection dysfunction.

Acupuncture at "Zusanli" (St.36) and "Sanyinjiao" (SP.6) Points on the Gastrointestinal Tract: A Study of the Bioavailability of (99m)Tc-Sodium Pertechnetate in Rats.

The objective of this study is to investigate the differences of acupuncture effect between the Zusanli (St.36) and Sanyinjiao (SP.6) points on the gastrointestinal-tract (GIT) segment performed by the bioavailability of (99m)Tc-sodium-pertechnetate (Na(99m)TcO(4)) in rats. Male Wistar rats (n = 21) were allocated into three groups of seven each. Group 1 was treated by acupuncture bilaterally at St.36; Group 2 at SP.6; and Group 3 was untreated (control). After 10 min of needle insertion in anesthetized rats, 0.3 mL of Na(99m)TcO(4) (7.4 MBq) was injected via ocular-plexus. After 20 min, the exitus of animals was induced by cervical-dislocation and GIT organs isolated. However, immediately before the exitus procedure, blood was collected by cardiac-puncture for blood radio-labeling (BRL). The radioactivity uptake of the blood constituents was calculated together with the GIT organs by a well gamma counter. The percentage of injected dose per gram of tissue (%ID/g) of Na(99m)TcO(4) was calculated for each GIT organs, while BRL was calculated in %ID. According to the one-way ANOVA, the stomach, jejunum, ileum from the treated groups (Group 1 and Group 2) had significant differences compared to the controls (Group 3). However, between the treated groups (Group 1 and Group 2), there were significant differences (P < .05) in the stomach, jejunum, ileum, cecum, transverse and rectum. In BRL analysis, Group 2 showed significant increase and decrease of the insoluble and soluble fractions of the blood cells, respectively (P < .0001). The authors suggest that St.36 may have a tendency of up-regulation effect on GIT, whereas SP.6, down-regulation effect. However, further rigorous experimental studies to examine the effectiveness of acupuncture in either acupuncture points need to be carried out.

Effect of Zusanli (ST.36) electroacupuncture at two frequencies on the bioavailability of (99m)Tc-sodium pertechnetate and on labeling of blood constituents in rats.

OBJECTIVES: A study was performed on the effects of stimulation at Zusanli-point (ST.36) by electroacupuncture (EA) at two frequencies on the bioavailability of (99m)Tc-sodium pertechnetate (Na(99m)TcO(4)) in rats. METHODS: Forty Wistar rats were divided into four groups: untreated control, treated by manual acupuncture at ST.36 bilaterally, treated by EA at 2 Hz at ST.36 bilaterally, and the same site at 100 Hz bilaterally. Na(99m)TcO(4) (7.4 MBq) was administrated via the ocular-plexus and, 20 minutes before sacrifice, blood was withdrawn for radiolabeling assay (BRL). In the bioavailability analysis, organs and tissues were isolated, their radioactivity determined, and the percentage of injected dose per gram of organ or tissue (%ID/g) and the %ID were calculated for each organ or tissue (%ID/ot). For BRL, the plasma and blood cells isolated, and the fractions also precipitated with 5% trichloroacetic acid to separate the soluble and insoluble fractions; these were assessed as percentage of injected dose (%ID) in blood (%ID/b). RESULTS: The results showed significant differences in the %ID/g in some organs and tissues in comparison with controls; lung (p = 0.0013), spleen (p = 0.0085), pancreas (p = 0.0167), liver (p = 0.0003), stomach (p < 0.0001), small-intestine (p = 0.0181), large-intestine (p = 0.04099), urinary-bladder (p = 0.0271), thyroid (p < 0.0001), muscle (p = 0.0187); %ID/ot in spleen (p = 0.0349); and %ID/b in blood sample (p = 0.0235). In the blood labeling analyses, EA in either frequency significantly increased insoluble fraction/blood cells (p < 0.0001). CONCLUSIONS: These findings suggested that acupuncture procedures at ST.36 could modulate responses in some organs, tissues, and blood in rats. Further rigorous experimental studies to examine the effectiveness in either acupuncture therapy need to be pursued.

Chlorpromazine interactions to sera albumins. A study by the quenching of fluorescence.

Binding of chlorpromazine (CPZ) and hemin (Hmn) to human (HSA) and bovine (BSA) serum albumin was studied by fluorescence quenching technique. Intrinsic fluorescences of BSA and HSA were measured by selectively exciting their tryptophan residues. Gradual quenching was observed by titration of both proteins with CPZ and Hmn. CPZ is a widely used anti-psychosis drug that causes severe side effects and strongly interacts with biomembranes, both in its lipidic and proteic regions. CPZ also interacts with blood components, influences bioavailability, and affects the function of several biomolecules. Albumin plays an important role in the transport and storage of hormones, ions, fatty acids and others substances, including CPZ, affecting the regulation of their plasmatic concentration. Hmn is an important ferric residue of hemoglobin that binds within the hydrophobic region of albumin with great specificity. Hmn added to HSA and BSA solutions at a molar ratio of 1:1 quenched about half of their fluorescence. Stern-Volmer plots obtained from experiments carried out at 25 and 35 degrees C showed the quenching of fluorescence of HSA and BSA by CPZ to be a collisional phenomenon. Hmn quenches fluorescence by a static process, which specifically indicates the formation of a complex. Our results suggest the prime binding site for CPZ and Hmn on both HSA and BSA to be near tryptophan residues.

Methyl parathion interaction with human and bovine serum albumin.

Methyl parathion (MP; O,O-dimethyl O-p-nitrophenyl phosphorothioate) is an organophosphorous compound still largely used in agriculture and fish hatcheries. This pesticide is not quite selective and is potentially toxic for both vertebrates and invertebrates. Its mechanism of acute toxicity is the inhibition of the enzyme acetylcholinesterase in nervous tissue. Binding of pesticides to plasma proteins is one of many factors that influence their distribution and elimination. The free concentration available for toxic action can be effectively reduced for pesticides with high binding to plasma proteins, although the affinity of pesticides to plasma proteins is often lower than for the enzyme targets. Several different transport proteins exist in blood plasma, but albumin only is able to bind a wide diversity of xenobiotics reversibly with high affinity. It was already known that parathion (ethyl parathion) exhibits a high affinity to human and bovine serum albumins. We studied interactions of methyl parathion with these albumins by using fluorescence quenching techniques. We selectively excited the fluorescence of tryptophan residues with a 290 nm wavelength light, and observed quenching by titrating human and bovine serum albumin solutions with methyl parathion. Stern-Volmer graphs were plotted and quenching constants were estimated. Our results pointed to the formation of complexes of methyl parathion with albumins. Association constants at 25 degrees C were 3.07 x 10(4) (1.2 x 10(3))M(-1) for human serum albumin, and 1.96 x 10(4) (+/- 4.5 x 10(2))M(-1) for bovine serum albumin. At 37 degrees C, they were 1.08 x 10(4) (+/- 2.0 x 10(2))M(-1) for human serum albumin, and 8.16 x 10(3) (+/- 1.9 x 10(2))M(-1) for bovine serum albumin. Results also suggest that the primary binding site for methyl parathion on albumin is close to tryptophan residues 214 of human serum albumin and 212 of bovine serum albumin.