Célia Martins Cortez

Quenching of the intrinsic fluorescence of bovine serum albumin by chlorpromazine and hemin

The binding of chlorpromazine (CPZ) and hemin to bovine serum albumin was studied by the fluorescence quenching technique. CPZ is a widely used anti-psychotic drug that interacts with blood components, influences bioavailability, and affects function of several biomolecules. Hemin is an important ferric residue of hemoglobin that binds within the hydrophobic region of albumin with high specificity. Quenching of the intrinsic fluorescence of bovine serum albumin (BSA) was observed by selectively exciting tryptophan residues at 290 nm. Emission spectra were recorded in the range from 300 to 450 nm for each quencher addition. Stern-Volmer graphs were plotted, and the quenching constant estimated for BSA solution titrated with hemin at 25 masculine C was 1.44 (+/- 0.05) x 10(5) M(-1). Results showed that bovine albumin tryptophans are not equally accessible to CPZ, in agreement with the idea that polar or charged quenchers have more affinity for amino acid residues on the outer wall of the protein. Hemin added to albumin solution at a molar ratio of 1:1 quenched about 25% of their fluorescence. The quenching effect of CPZ on albumin-hemin solution was stronger than on pure BSA. This increase can be the result of combined conformational changes in the structure of albumin caused firstly by hemin and then by CPZ. Our results suggest that the primary binding site for hemin on bovine albumin may be located asymmetrically between the two tryptophans along the sequence formed by subdomains IB and IIA, closer to tryptophan residue 212.

One round of SELEX for the generation of DNA aptamers directed against KLK6.

Abstract Kallikrein-related peptidase 6 (KLK6) is an active serine protease that has been implicated in common pathologies, including neurodegenerative disorders such as Parkinson and Alzheimer disease and certain types of cancer. Antibodies, either polyclonal or monoclonal, that exhibit specificity for distinct members of the extended kallikrein family, including KLK6, were developed. With the exception of KLK3/PSA, the identification and generation of aptamers, as potential new tools with improved characteristics demanded for therapeutic and diagnostic applications, has not been explored for KLKs. Here, we report for the first time the identification of novel DNA aptamers against KLK6 that were isolated using a modified systemic evolution of ligands by exponential enrichment technique. The identified aptamers were characterized using fluorescence spectroscopy, competition ELISA, and quartz crystal microbalance, and two aptamers (008 and 022) were found to exhibit high affinity (Kd in the low nanomolar range) for KLK6. Aptamers were tested for their ability to bind to serum albumin, to demonstrate their specificity for their target, and the possible involvement of such proteins in the transport of aptamers into the bloodstream. The developed aptamers are expected to assist the development of novel diagnostic, biosensing, and therapeutic strategies.

Tension neck syndrome treated by acupuncture combined with physiotherapy: A comparative clinical trial (pilot study)

OBJECTIVE To evaluate the effect of acupuncture combined with physiotherapy in comparison with acupuncture and physiotherapy performed alone in different parameters; pain intensity, muscle tension, functional disability and muscle strength in the treatment of tension neck syndrome (TNS). DESIGN A prospective, comparative clinical trial. SETTING Acupuncture and Rehabilitation Department. BACKGROUND TNS can occur in computer users. Acupuncture has been one alternative treatment in physiotherapeutic rehabilitation of musculoskeletal disorders. SUBJECTS Forty-six patients with TNS. INTERVENTIONS Patients were allocated into three groups: Group-1 received physiotherapy (therapeutic exercises) combined with acupuncture; Group-2, acupuncture alone, and Group-3, physiotherapy alone; over a period of 10 weeks, with one or two sessions weekly. OUTCOME ASSESSMENT All patients had completed the protocols and were assessed using a visual analogue scale for pain intensity (VASpain) and muscle tension (VASmt), the Neck Disability Index: Brazilian Portuguese version for functional disability, and the cranio-cervical Flexion Test for isometric neck muscle strength (INMS); in the periods before treatment (baseline), after 10 weeks of treatment, and after 6 months of follow-up. RESULTS All groups showed significant improvement (p < 0.001) in these parameters after 10 weeks of treatment and after 6 months of follow-up. Group-1 was superior to Group-3 in pain and functional disability improvements (p<0.05); and Group-1 was superior to both Group-2 (p < 0.01) and Group-3 (p < 0.05) in INMS. After 6 months of follow-up, the improvements of all groups were maintained (p < 0.05). CONCLUSION The data suggested that acupuncture effect may facilitate and/or enhance physiotherapy performance in musculoskeletal rehabilitation for tension neck syndrome.

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 Students 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.

Effects of Immobilization Stress on Kidneys of Wistar Male Rats: A Morphometrical and Stereological Analysis

This paper verifies the morphological changes induced by immobilization stress on the kidney of rats by using stereological methods. Fifteen 4-week-old Wistar male rats were randomly assigned to control (n = 7) and stressed (n = 8) groups. Stress stimuli were performed over 5 weeks by immobilization of the rats for 2 h daily in a rigid opaque plastic cylinder that restrained their movements. Increases in the adrenal mass index (p < 0.05) and decreases in serum testosterone levels (p < 0.05) demonstrated the efficacy of the stressor stimuli. Stressed rats presented diminished body weight gain when compared to controls (p < 0.05). The mean values of kidney weight, kidney volume, kidney volume index and glomerular volume density were significantly lower in the stressed group (p < 0.05); nevertheless, no significant difference was found in the cortical/medullar ratio or in the volume-weighted mean glomerular volume. The number of glomeruli per kidney was 45% lower in the stressed group (p < 0.0001), but no change in serum creatinine levels was found. However, the morphological alterations may have serious implications predisposing individuals to renal disease and hypertension in adult life.

The interaction of methyl-parathion with serum and albumin of the neo-tropical fish Piaractus mesopotamicus

The interaction of methyl-parathion with serum and albumin of pacu, Piaractus mesopotamicus, was studied, using the fluorescence quenching technique. Pacu is a neo-tropical fish specie inhabitant of rivers from western regions of Brazil. Methyl-parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate) is an organophosphorous pesticide still used in agriculture and fish farming in many countries. The quenching of fluorescence can be mathematically expressed by the Stern-Volmer equation to calculate quenching constants. Stern-Volmer curves analysis is able to give important information about the pesticide-albumin interaction. Our results showed that the serum quenching reached 10% when the molar ratio of pesticide/albumin was about 7:1 for the three temperatures of the experiment. For the pure albumin quenching of 10%, methyl-parathion concentrations were 6, 7 and 9 times higher than albumin at 20, 25 and 30 degrees C, respectively. The calculated Stern-Volmer constants at 25 degrees C were 9.73x10(3)(+/-4.9x10(2))M(-1) for serum and 9.20x10(3)(+/-2.0x10(2))M(-1) for albumin. It was observed that albumin quenching is the phenomenon contributing to the quenching of the pacu serum fluorescence for methyl-parathion concentration lower than 10microM, suggesting that the protein is the most important carrier for the pesticide in serum.

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 99mTc-sodium-pertechnetate (Na99mTcO4) 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 Na99mTcO4 (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 Na99mTcO4 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.

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.

A simple model for circadian timing by mammals

Circadian timing is structured in such a way as to receive information from the external and internal environments, and its function is the timing organization of the physiological and behavioral processes in a circadian pattern. In mammals, the circadian timing system consists of a group of structures, which includes the suprachiasmatic nucleus (SCN), the intergeniculate leaflet and the pineal gland. Neuron groups working as a biological pacemaker are found in the SCN, forming a biological master clock. We present here a simple model for the circadian timing system of mammals, which is able to reproduce two fundamental characteristics of biological rhythms: the endogenous generation of pulses and synchronization with the light-dark cycle. In this model, the biological pacemaker of the SCN was modeled as a set of 1000 homogeneously distributed coupled oscillators with long-range coupling forming a spherical lattice. The characteristics of the oscillator set were defined taking into account the Kuramotos oscillator dynamics, but we used a new method for estimating the equilibrium order parameter. Simultaneous activities of the excitatory and inhibitory synapses on the elements of the circadian timing circuit at each instant were modeled by specific equations for synaptic events. All simulation programs were written in Fortran 77, compiled and run on PC DOS computers. Our model exhibited responses in agreement with physiological patterns. The values of output frequency of the oscillator system (maximal value of 3.9 Hz) were of the order of magnitude of the firing frequencies recorded in suprachiasmatic neurons of rodents in vivo and in vitro (from 1.8 to 5.4 Hz).

Computer modeling of a spinal reflex circuit

We used a set of properties of the interactions among the spinal neurons in order to develop a computer model for a spinal reflex circuit. The model equations take into account the synaptic characteristics of the somatodendritic membrane of neurons in a morphofunctional unity of the spinal reflex activity. This model is based on the idea that the responses of spinal alpha-motoneurons to a sensorial stimulation can be modulated by the serial activation of a motor command chain. We developed a Fortran program for simulating a physiological situation. The results are discussed in terms of available experimental data for the motoneuron firing rate.