Chemical treatment of Escherichia coli. II. Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells

A method is presented for the direct extraction of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact Escherichia coli cells. Chemical treatment with 6M urea, 3 mM EDTA, and 20 mM dithiothreitol (DTT) at pH 9.0 proved an effective combination for extracting recombinant protein from intact cells. Comparable levels of Long-R-3-IGF-I were recovered by direct extraction as achieved by in vitro dissolution following mechanical disruption. However, the purity of directly extracted recombinant protein was lower due to contamination by bacterial cell components. The kinetics of direct extraction are described using a first-order equation with the time constant of 3 min. Urea appears important for permeabilization of the cell and dissolution of the inclusion body. Conversely, EDTA is involved in permeabilization of the cell wall and DTT enhances protein release. pH proved to be important with lower levels of protein release achieved at low pH values (

Gonadotrophin and prolactin secretion in castrated male sheep following subcutaneous or intracranial treatment with testicular hormones

Interactions between testosterone, estradiol, and inhibin in the control of gonadotrophin secretion in males are poorly understood. Castrated rams were treated with steroid-free bovine follicular fluid (bFF), testosterone, or estradiol and for 7 d (2 x 2 x 2 factorial design). Given independently, none of the exogenous hormones affected follicle-stimulating hormone (FSH) concentrations, but the combination of one or both steroids with bFF reduced FSH secretion. Testosterone and estradiol reduced luteinizing hormone (LH) pulse frequency (there was no synergism), and bFF had no effect. Plasma prolactin concentrations were not affected by any treatment. To locate the central sites of steroid action, castrated rams were bilaterally implanted in the preoptic area (POA), ventromedial nucleus (VMH), or arcuate nucleus (ARC). These implants did not affect FSH or prolactin concentrations, or LH pulse amplitude. The frequency of the LH pulses was not affected by testosterone in any site. Estradiol located in the ARC, but not the POA or VMH, decreased LH pulse frequency. In summary, FSH secretion is controlled by synergistic interactions between inhibin and estradiol or testosterone, whereas GnRH/LH pulse frequency is controlled by testicular steroids. Estradiol acts partly, at least, in the ARC, but the central site of action, testosterone remains unknown.

Serotonin-1A receptors in the dorsal periaqueductal gray matter mediate the panicolytic-like effect of pindolol and paroxetine combination in the elevated T-maze

The beta-adrenergic blocker and 5-HT(1A) receptor antagonist pindolol has been combined with selective serotonin reuptake inhibitors (SSRIs) in patients with depressive and anxiety disorders to shorten the onset of the clinical action and/or increase the proportion of responders. The results of a previous study have shown that pindolol potentiates the panicolytic effect of paroxetine in rats submitted to the elevated T-maze (ETM). Since reported evidence has implicated the 5-HT(1A) receptors of the dorsal periaqueductal gray matter (DPAG) in the panicolytic effect of antidepressants, rats treated with pindolol (5.0 mg/kg, i.p.) and paroxetine (1.5 mg/kg, i.p.) received a previous intra-DPAG injection of the selective 5-HT(1A) antagonist, WAY-100635 (0.4 mu g) and were submitted to the ETM. Pretreatment with WAY-100635 reversed the increase in escape latency, a panicolytic effect, determined by the pindolol-paroxetine combination. These results implicate the 5-HT(1A) receptors of the DPAG in the panicolytic effect of the pindolol-paroxetine combination administered systemically. They also give further preclinical support for the use of this drug combination in the treatment of panic disorder. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Bleeding patterns and menstrual-related symptoms with the continuous use of a contraceptive combination of ethinylestradiol and drospirenone: a randomized study

Background: The objective of this study was to compare bleeding patterns of women using a contraceptive combination of 30 mcg ethinylestradiol (EE) and 3 mg drospirenone (DRSP) continuously or cyclically. Menstrual-related symptoms were also evaluated. Study Design: This open, prospective, randomized study evaluated 78 women using the EE/DRSP combination continuously for 168 days or for six 28-day cycles, each followed by a 7-day hormone-free interval. A diary with pre-established scales was used to assess the frequency and intensity of bleeding and menstrual-related symptoms. Results: Amenorrhea increased with continuous use; 62.2% of women with continuous use were amenorrheic at the end of treatment (95% CI: 46.6-77.8%). Dysmenorrhea, headache, acne, nausea, edema and increased appetite improved significantly in the continuous-use group, and mastalgia and edema in the cyclic-use group (p<.05). Six subjects in the continuous-use group (15.4%) and three in the cyclic-use group (7.7%) discontinued due to adverse events. Conclusions: Continuous use was associated with amenorrhea and fewer menstrual-related symptoms compared to cyclic use. (C) 2010 Elsevier Inc. All rights reserved.

Outcomes after multi-modality treatment of musculoskeletal tumours

Outcomes of treatment of musculoskeletal tumours are evaluated for effectiveness of chemotherapy protocols, function obtained after surgery and survival after treatment. Quality of life achieved after multi-modality treatment is dependent on a combination of all of these factors. Quality of life varies significantly along the treatment pathway, and continuously through the life of a patient. The patient's perception of outcome is based on the total effect of the disease and its treatment, rather than necessarily focussing on separate items of treatment. We have found that visual analogue scales can be used effectively to gauge the patient's perception of their quality of life. Such a method has shown that, overall, perceptions of quality of life seem to be better for those patients who have undergone successful limb salvage surgery when compared with those who have undergone amputation, but the differences are not as great as might be assumed.

Chemical treatment of Escherichia coli .1. Extraction of intracellular protein from uninduced cells

Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical disruption. A chemical treatment that destroys the integrity of the bacterial cell wall and could provide an alternative technique is examined in this study. Treatment with a combination of the chelating agent ethylenediaminetetraacetate (EDTA) (greater than 0.3 mM) and the chaotropic agent urea (6 M) is highly effective at releasing protein from uninduced E. coli. The 6 M urea in the presence of 3 mM EDTA can release cytoplasmic protein from both logarithmic-phase and stationary-phase E. coli cells at levels equivalent to mechanical disruption. The concentrations of the two chemical agents were the major variables affecting the maximum levels of protein release. Several minor variables and interactions were also identified. The kinetics of protein release is first order. For 2, 4, and 6 M urea with 3 mM EDTA, the time constant is approximately 2.5 min independent of urea concentration. Kinetics for 3 mM EDTA without urea is considerably slower, with a time constant of 12.3 min. (C) 1997 John Wiley & Sons, Inc.

Combination of N-acetylcysteine and metformin improves histological steatosis and fibrosis in patients with non-alcoholic steatohepatitis

Aim: There is no proven medical therapy for the treatment of non-alcoholic steatohepatitis (NASH). Oxidative stress and insulin resistance are the mechanisms that seem to be mostly involved in its pathogenesis. The aim of our study was to evaluate the efficacy of N-acetylcysteine (NAC) in combination with metformin (MTF) in improving the aminotransferases and histological parameters (steatosis, inflammation, hepatocellular ballooning, and fibrosis) after 12 months of treatment. Methods: Twenty consecutive patients (mean age 53 +/- 2 years [36-68] and body mass index [BMI] 29 [25-35]) with biopsy-proven NASH were enrolled in the study. NAC (1.2 g/day) and MTF (850-1000 mg/day) were given orally for 12 months. All patients underwent evaluation of serum aminotransferases, fasting lipid profile and serum glucose, anthropometric parameters, and nutritional status at 0 and 12 months. A low calorie diet was prescribed for all patients. Results: Serum alanine aminotransferase, high-density lipoprotein, insulin, and glucose concentrations and thehomeostasis model assessment-insulin resistance (HOMA-IR) index were reduced significantly at the end of study (P < 0.05). The BMI declined, but without statistical significance. Aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, cholesterol, and triglycerides levels were not altered with the treatment. Liver steatosis and fibrosis decreased (P < 0.05), but no improvement was noted in lobular inflammation or hepatocellular ballooning. The NASH activity score was significantly improved after treatment. Conclusion: Based on the biochemical and histological evidence in this pilot study, NAC in combination with MTF appears to ameliorate several aspects of NASH, including fibrosis. Further studies of this form of combination therapy are warranted to assess its potential efficacy.

Oral Azithromycin for Treatment of Posterior Blepharitis

Purpose: To evaluate the effects of oral azithromycin in patients with posterior blepharitis. Methods: Twenty-six eyes of 13 patients with posterior blepharitis diagnosed by a qualified ophthalmologist were enrolled in this study. Patients were instructed to use oral azithromycin 500 mg per day for 3 days in 3 cycles with 7-day intervals. Subjective clinical outcomes were graded and scored 1 day before and 30 days after the end of the treatment (53 days after initiating the treatment) based on severity scores of: (1) eyelid debris; (2) eyelid telangiectasia; (3) swelling of the eyelid margin; (4) redness of the eyelid margin; and (5) ocular mucus secretion. For the assessment of global efficacy, patients were asked by the investigator to rate the subjective symptoms (eyelid itching, ocular itching, eyelid hyperemia, ocular hyperemia, ocular mucus secretion, photophobia, foreign body sensation, and dry eye sensation) on a scale of 0 (no symptoms) to 5 (severe symptoms). Break-up time, Schirmer I test, corneal fluorescein staining score, and rose bengal staining score were also performed in all patients. Results: All clinical outcomes scoring showed statistically significant improvement after oral azithromycin, except for eyelid swelling. Average subjective symptom grading improved statistically after treatment with oral azithromycin, except for eyelid hyperemia, photophobia, and foreign body sensation. Average tear film break-up time values showed statistically significant improvement after the treatment with oral azithromycin. No statistically significant improvement was observed on average values of Schirmer I test, corneal fluorescein staining score, and rose bengal staining score. Conclusions: The combination of multiple clinical parameters shown in this study supports the clinical efficacy of pulsed oral azithromycin therapy for the management of posterior blepharitis.

An Eight-week, Multicentric, Randomized, Interventional, Open-label, Phase 4, Parallel Comparison of the Efficacy and Tolerability of the Fixed Combination of Timolol Maleate 0.5%/Brimonidine Tartrate 0.2% Versus Fixed Combination of Timolol Maleate 0.5%/Dorzolamide 2% in Patients With Elevated Intraocular Pressure

Purpose: To compare the efficacy and tolerability of the fixed combination of timolol maleate 0.5%/brimonidine tartrate 0.2% versus fixed combination of timolol maleate 0.5%/dorzolamide 2% in patients with elevated intraocular pressure (IOP) over 8 weeks. Patients and Methods: This 8-week, multicentric. interventional, randomized, open-label, parallel group study was conducted Lit 4 centers in Brazil and 1 center in Argentina. Patients with open-angle glaucoma or ocular hypertension were randomized to receive bilaterally fixed combination of brimonidine/timolol maleate 0.5% or fixed combination of dorzolamide 2%/timolol 0.5% twice daily at 8:00 AM and 8:00 PM. A modified diurnal tension curve (8:00 AM 10:30 AM, 02:00 PM, and 4:00 PM) followed by the water drinking test (WDT), which estimates IOP peak of diurnal tension curve, were performed in the baseline and week-8 visits. Adverse events data were recorded at each visit. Results: A total of 210 patients were randomized (brimonidine/timolol, n = 111; dorzolamide/timolol, n = 99). Mean baseline IOP was 23.43 +/- 3.22 mm Hg and 23.43 +/- 4.06 mm Hg in the patients treated with brimonidine/timolol and dorzolamide/timolol, respectively (P = 0.993). Mean diurnal IOP reduction after 8 weeks were 7.02 +/- 3.06 mm Hg and 6.91 +/- 3.67 mm Hg. respectively (P = 0.811). The adjusted difference between groups (analysis of covariance) Lit week 8 was not statistically significant (P = 0.847). Mean baseline WDT peak was 27.79 +/- 4.29 mm Hg in the brimonidine/timolol group and 27.68 +/- 5.46 mm Hg in the dorzolamide/timolol group. After 8 weeks of treatment, mean WDT peaks were 20.94 +/- 3.76 mm Hg (P < 0.001) and 20.98 +/- 4.19 (P < 0.001), respectively. The adjusted difference between groups (analysis of covariance) was not statistically significant (P = 0.469). No statistical difference in terms of adverse events was Found between groups. Conclusions: Both fixed combinations were capable of significantly reducing the mean diurnal IOP, mean diurnal peak, and mean WDT peak after 8 weeks of treatment. Also, both fixed combinations are well tolerated with few side effects.

Quetiapine versus clomipramine in the augmentation of selective serotonin reuptake inhibitors for the treatment of obsessive-compulsive disorder: a randomized, open-label trial

After 12 weeks of selective serotonin reuptake inhibitor (SSRI) monotherapy with inadequate response, 10 patients received clomipramine and 11 received quetiapine as augmentation agents of the SSRI. The primary outcome measure was the difference between initial and final scores of the YaleBrown Obsessive-Compulsive Scale (Y-BOCS), rated in a blinded fashion, and the score of clinical global improvement (CGI-I). Statistical analyses were performed using nonparametric tests to evaluate treatment efficacy and the difference between treatment groups. Percentile plots were constructed with YBOCS scores from the clomipramine and quetiapine groups. Considering response a >= 35% reduction in the initial Y-BOCS score plus a rating of `much improved` or `very much improved` on CGI-I, four of eleven quetiapine patients and one out of ten clomipramine patients were classified as responders. The mean final Y-BOCS score was significantly lower than baseline in the quetiapine augmentation group (P = 0.023), but not in the clomipramine augmentation group (P = 0.503). The difference between groups showed a trend towards significance only at week 4, the mean Y-BOCS score being lower for those receiving quetiapine (P = 0.052). A difference between groups was also observed at week 4 according to percentile plots. These results corroborate previous findings of quetiapine augmentation efficacy in obsessive-compulsive disorder (OCD). Clomipramine augmentation did not produce a significant reduction in Y-BOCS scores. Higher target maximum dosages might have yielded different results.

Effects of losartan, in monotherapy or in association with hydrochlorothiazide, in chronic nephropathy resulting from losartan treatment during lactation

Fanelli C, Fernandes BH, Machado FG, Okabe C, Malheiros DM, Fujihara CK, Zatz R. Effects of losartan, in monotherapy or in association with hydrochlorothiazide, in chronic nephropathy resulting from losartan treatment during lactation. Am J Physiol Renal Physiol 301: F580-F587, 2011. First published June 8, 2011; doi:10.1152/ajprenal.00042.2011.-We recently standardized a model (L(Lact)) of severe chronic kidney disease based on impaired nephrogenesis by suppression of angiotensin II activity during lactation (Machado FG, Poppi EP, Fanelli C, Malheiros DM, Zatz R, Fujihara CK. Am J Physiol Renal Physiol 294: F1345-F1353, 2008). In this new study of the L(Lact) model, we sought to gain further insight into renal injury mechanisms associated with this model and to verify whether the renoprotection obtained with the association of the angiotensin II receptor blocker losartan (L) and hydrochlorothiazide (H), which arrested renal injury in the remnant kidney model, would provide similar renoprotection. Twenty Munich-Wistar dams, each nursing six pups, were divided into control, untreated, and L(Lact) groups, given losartan (L; 250 mg.kg(-1).day(-1)) until weaning. The male LLact offspring remained untreated until 7 mo of age, when renal functional and structural parameters were studied in 17 of them, used as pretreatment control (L(Lact)Pre), and followed no further. The remaining rats were then divided among groups L(Lact) + V, untreated; L(Lact) + L, given L (50 mg.kg(-1).day(-1)) now as a therapy; L(Lact) + H, given H (6 mg.kg(-1).day(-1)); and L(Lact) + LH, given L and H. All parameters were reassessed 3 mo later in these groups and in age-matched controls. At this time, L(Lact) rats exhibited hypertension, severe albuminuria, glomerular damage, marked interstitial expansion/inflammation, enhanced cell proliferation, myofibroblast infiltration, and creatinine retention. L monotherapy normalized albuminuria and prevented hypertension and the progression of renal injury, inflammation, and myofibroblast infiltration. In contrast to the remnant model, the LH combination promoted only slight additional renoprotection, perhaps because of a limited tendency to retain sodium in L(Lact) rats.

Inflammatory and functional effects of increasing asthma treatment with formoterol or double dose budesonide

Adding a long-acting beta(2)-agonist to inhaled corticosteroids (ICS) for asthma treatment is better than increasing ICS dose in improving clinical status, although there is no consensus about the impact of this regimen on inflammation. In this double-blind, randomized, parallel group study, asthmatics with moderate to severe disease used budesonide (400 mcg/day) for 5 weeks (run-in period); then they were randomized to use budesonide (800 mcg/day - BUD group) or budesonide plus formoterol (400 mcg and 24 mcg/day, respectively - FORMO group) for 9 weeks (treatment period). Home PEF measurements, symptom daily reporting, spirometry, sputum induction (for differential cell counts and sputum cell cultures), and hypertonic saline bronchial challenge test were performed before and after treatments. TNF-alpha, IL-4 and eotaxin-2 levels in the sputum and cell culture supernatants were determined. Morning and night PEF values increased in the FORMO group during the treatment period (p < 0.01), from 435 +/- 162 to 489 +/- 169 and 428 +/- 160 to 496 +/- 173 L/min, respectively. The rate of exacerbations in the FORMO group was lower than in the BUD group (p < 0.05). Neutrophil counts in sputum increased in both groups (p < 0.05) and leukocyte viability after 48 h-culture increased in the FORMO group (p < 0.05). No other parameter changed significantly in either group. This study showed that adding formoterol to budesonide improved home PEF and provided protection from exacerbations, although increase of leukocyte viability in cell culture may be a matter of concern and needs further investigation. (C) 2008 Elsevier Ltd. All rights reserved.

Efficacy and tolerance of metronidazole and miconazole nitrate in treatment of vaginitis

Objective: To evaluate the efficacy and tolerability of a vaginal pessary containing 750 mg of metronidazole and 200 mg of miconazole nitrate used daily for 7 days in the treatment of vaginitis. Methods: Ninety-two women with vaginitis participated in this phase 3 study using one vaginal pessary daily for 7 days. Gynecological and microbiological evaluations were carried out prior to and following treatment. Results: Reductions occurred in symptoms and signs of vaginitis. Clinical cure rate was 87.7%, while the cure rates according to microscopy and Candida albicans culture were 81.8% and 73.9%, respectively. The cure rate for bacterial vaginosis was 75% and culture of Gardnerella vaginalis turned negative in 63.6% of cases following treatment. The medication was well tolerated. Conclusion: Use of a combination of 750 mg of metronidazole and 200 mg of miconazole in a single daily application was found to be effective in the treatment of the most common causes of vaginitis. (c) 2008 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

Treatment of Hepatocellular Carcinoma With Liver Transplantation: A Single-Center Experience From Brazil

Introduction. Orthotopic liver transplantation (OLT) is the treatment of choice of hepatocellular carcinoma (HCC) for patients with cirrhosis, mainly those with early HCC. Herein we have present the clinical characteristics and outcomes of cirrhotic patients with HCC who underwent OLT from cadaveric donors in our institution. Methods. From May 2001 to May 2009, we performed 121 OLT including 24 patients (19.8%) with cirrhosis and HCC within the Milan criteria. In 4 cases, HCC was an incidental finding in the explants. Results. The patients` average age was 55 +/- 10 years, including 82% men. Fifty percent of patients were Child class B or C. The average Model for End Stage Liver Disease for Child A, B, and C categories were 11, 15, and 18, respectively. The HCC diagnosis was made by 2 dynamic images in 16 cases; 1 dynamic image plus alphafetoprotein >400 ng/mL in 4; and 4 by histologic confirmation. Twenty patients received a locoregional treatment before OLT: 6 percutaneous ethanol injection, 9 transarterial chemoembolization, 1 transarterial embolization, and 4 a combination of these modalities. The median follow-up after OLT was 19.7 months (range, 1-51). A vascular invasion was observed in the explant of 1 patient, who developed an HCC recurrence and succumbed at 8 months after OLT. Two further patients, without vascular invasion or satellite tumor displayed tumor recurrences at 7 and 3 months after OLT, and death at 2 and 1 month after the diagnosis. The remaining 25 patients have not shown a tumor recurrence. Conclusion. In the present evaluation, OLT patients with early HCC and no vascular invasion showed satisfactory results and good disease-free survival. Strictly following the Milan criteria for liver transplantation in patients with HCC greatly reduces but does not completely avoid, the chances of tumor recurrence.

Influence of dentin on pH of 2% chlorhexidine gel and calcium hydroxide alone or in combination

The aims of endodontic treatment in cases of apical periodontitis are to reduce as much as possible the number of microorganisms inside the root canal system and to inactivate toxins produced by them. Most of the times, these objectives are not achieved solely by chemomechanical preparation, and intracanal dressing may be necessary. In these cases, calcium hydroxide is used as a root canal dressing due to its well-known and recognized antimicrobial activity. Chlorhexidine has a wide spectrum of antimicrobial activity and its association with calcium hydroxide has been recommended in an attempt to amplify antimicrobial effects of calcium hydroxide. It is also known that dentin exerts a buffering effect under wide pH variations, and may be responsible for decreasing the antimicrobial activity of drugs inside the root canal. The objectives of this study were to assess the pH of 2% chlorhexidine gel and calcium hydroxide alone or in combination, as well as the influence of dentin on the pH of these compounds. Dentin powder was obtained from bovine teeth and added as 1.8% to the volume of the medications. All substances were individually stored in plastic flasks, in triplicate. A pH meter was used at five different moments to assess pH in viscous medium: immediately after preparation and after 24 h, and 7, 14, and 21 days. Results were analyzed by paired Student`s t-test. Statistically significant differences were observed in the 2% chlorhexidine gel group alone or associated with calcium hydroxide and added of dentin powder (P < 0.05). Mean pH values indicated the influence of dentin powder because of a significant increase in pH. Calcium hydroxide with propylene glycol as the vehicle always showed high pH, demonstrating that this compound was not affected by the presence of dentin.