Tratamiento de la epífora funcional con toxina botulínica tipo A frente a la tira tarsal en un ensayo aleatorizado / Treatment for functional epiphora with botulinum toxin-A versus lateral tarsal strip in a randomized trial

Objetivo Comparar la eficacia de la inyección de toxina botulínica A (TBA) en la glándula lagrimal con la tira tarsal lateral (TTL) en la epífora funcional. Material y método Ensayo clínico aleatorizado. Diseño secuencial, paralelo y no ciego. Pacientes de 18 años o más con epífora funcional con un mínimo de 3 en la escala de Munk (EM) se incluyeron a grupo de TBA o TTL. Los cambios en la EM, el test de Schirmer y la calidad de vida se evaluaron a la semana 6 y hasta la semana 30. Se obtuvo el tiempo medio sin epífora y los acontecimientos adversos. Resultados El análisis final incluyó 25 pacientes, 12 (21 ojos) se asignaron a TBA (5U/0,05mL) y 13 (20 ojos) a TTL. A la semana 6 hubo un mayor descenso en la EM en el grupo de TBA frente a TTL (−2.48 vs. −1.55, p=0,0152) y a la semana 12 (−2,68 vs. −1,69, p=0,0267). Se observó un descenso significativo en el test de Shirmer a las semanas 2, 12 y 30 con TBA. La calidad de vida mejoró después de ambas intervenciones, sin diferencias significativas. El tiempo medio sin epífora en el grupo TBA fue de 26,2 semanas (7,7-36,6) y en el grupo TTL de 24,8 semanas (6,7-37,6), p=0,9368. Se observó ptosis temporal en un 25% (3/12) en el grupo TBA y un 23% (3/13) de molestias de la cicatriz quirúrgica en el grupo TTL, p=0,722. Ningún acontecimiento adverso fue severo. Conclusión La inyección de TBA en la glándula lagrimal es efectiva y segura en el tratamiento de la epífora funcional, con mayor descenso en la EM a las 6 y 12 semanas comparado con la TTL (AU)

Objective To compare the efficacy of botulinum toxin A (BoNTA) injection into the lacrimal gland versus lateral tarsal strip (LTS) for functional epiphora Material and methods Randomized clinical trial. Sequential, parallel, non-blinded study design. Patients aged 18 years or older with functional epiphora and a minimum score of 3 in Munk Scale (MS) were randomized to BoNTA or LTS group. Changes in MS, Schirmer test and quality of life were assessed at week 6 and during follow-up until week 30. The mean time without epiphora and the adverse events were recorded. Results The final analysis included 25 patients, 12 (21 eyes) assigned to BoNTA (5U/0.05mL) and 13 (20 eyes) to LTS. At 6 weeks there was an improvement in the MS in BoNTA versus LTS group (−2.48 vs. −1.55, P=.0152) and at 12 weeks (−2.68 vs. −1.69, P=.0267). A significant decrease was noted in the Schirmer test at week 2, 12 and 30 with BoNTA. The quality of life improved after both interventions without statistical significance. The mean duration of effectiveness in BoNTA group was 26.2 weeks (range 7.7-36.6) and in LTS group was 24.8 weeks (range 6.7-37.6), P=.9368. The main adverse events were temporary eyelid ptosis in 25% (3/12) of the BoNTA group and surgical scar discomfort in 23% (3/13) of the LTS group, P=.722. No adverse events were classified as severe. Conclusion BoNTA injection into the lacrimal gland is a safe and effective treatment for functional epiphora, with a greater decrease in MS at 6 and 12 weeks compared with LTS (AU)

Treatment for functional epiphora with botulinum toxin-A versus lateral tarsal strip in a randomized trial.

INTRODUCTION: To compare the efficacy of botulinum toxin A (BoNTA) injection into the lacrimal gland versus lateral tarsal strip (LTS) for functional epiphora. METHODS: Randomized clinical trial. Sequential, parallel, non-blinded study design. Patients aged 18 years or older with functional epiphora and a minimum score of 3 in Munk Scale (MS) were randomized to BoNTA or LTS group. Changes in Munk scale, Schirmer test (ST) and quality of life (QoL) were assessed at week 6 and during follow-up until week 30. The mean time without epiphora and the adverse events (AE) were recorded. RESULTS: The final analysis included 25 patients, 12 (21 eyes) assigned to BoNTA (5U/0.05 mL) and 13 (20 eyes) to LTS. At 6 weeks there was an improvement in the MS in BoNTA versus LTS group (-2.48 vs -1.55, P = .0152) and at 12 weeks (-2.68 vs -1.69, P = .0267). A significant decrease was noted in the ST at week 2, 12 and 30 with BoNTA. The QoL improved after both interventions without statistical significance. The mean duration of effectiveness in BoNTA group was 26.2 weeks (range 7.7-36.6) and in LTS group was 24.8 weeks (range 6.7-37.6), P = .937. The main AE were temporary eyelid ptosis in 25% (3/12) of the BoNTA group and surgical scar discomfort in 23% (3/13) of the LTS groups, P = .722. No AE were classified as severe. CONCLUSION: BoNTA injection into the lacrimal gland is a safe and effective treatment for functional epiphora, with a greater decrease in MS at 6 and 12 weeks compared with LTS.

miR-711 upregulation induces neuronal cell death after traumatic brain injury.

Traumatic brain injury (TBI) is a leading cause of mortality and disability. MicroRNAs (miRs) are small noncoding RNAs that negatively regulate gene expression at post-transcriptional level and may be key modulators of neuronal apoptosis, yet their role in secondary injury after TBI remains largely unexplored. Changes in miRs after controlled cortical impact (CCI) in mice were examined during the first 72 h using miR arrays and qPCR. One selected miR (711) was examined with regard to its regulation and relation to cell death; effects of miR-711 modulation were evaluated after CCI and using in vitro cell death models of primary cortical neurons. Levels of miR-711 were increased in the cortex early after TBI and in vitro models through rapid upregulation of miR-711 transcription (pri-miR-711) rather than catabolism. Increases coincided with downregulation of the pro-survival protein Akt, a predicted target of miR-711, with sequential activation of forkhead box O3 (FoxO3)a/glycogen synthase kinase 3 (GSK3)α/ß, pro-apoptotic BH3-only molecules PUMA (Bcl2-binding component 3) and Bim (Bcl2-like 11 (apoptosis facilitator)), and mitochondrial release of cytochrome c and AIF. miR-711 and Akt (mRNA) co-immunoprecipitated with the RNA-induced silencing complex (RISC). A miR-711 hairpin inhibitor attenuated the apoptotic mechanisms and decreased neuronal death in an Akt-dependent manner. Conversely, a miR-711 mimic enhanced neuronal apoptosis. Central administration of the miR-711 hairpin inhibitor after TBI increased Akt expression and attenuated apoptotic pathways. Treatment reduced cortical lesion volume, neuronal cell loss in cortex and hippocampus, and long-term neurological dysfunction. miR-711 changes contribute to neuronal cell death after TBI, in part by inhibiting Akt, and may serve as a novel therapeutic target.

High Grade Penetrating Pancreatic Trauma - Case Report and Review of the Literature.

INTRODUCTION: The pancreatic injuries have fortunately a low frequency, but when present associate multiple intraabdominal lesions, and carry a significant morbidity and mortality. The aim of this study is to underline the significant morbidity associated with high grade pancreatic injuries. CASE REPORT: Female patient, 36 years old, with penetrating abdominal trauma due to domestic violence was referred to our center from a regional county hospital, after multiple laparotomies, hemodynamically unstable, with multiple organ failure. Abdominal clinical exam revealed evisceration, with massive pancreatic leakage at the level of the median laparotomy and through the stabbing wounds from the right flank. Emergency Computed Tomography showed multiple intraabdominal collections, with laceration of the liver, right kidney and pancreatic head. Abdominal exploration was decided. After a thorough abdominal debridement was revealed a deep laceration of the pancreatic head, with active extravasation of pancreatic secretion, correlating with a grade IV injury. Peritoneal lavage and large drainage of the lesser and greater peritoneal cavity was performed. The postoperative recovery was uneventful, with progressive decrease in pancreatic fistula output and discharge after 35 days. CONCLUSIONS: High grade pancreatic traumas associate a significant morbidity. Efficient drainage of the pancreatic head injuries and patients management in high volume centers for pancreatic surgery maximize the survival rate.

Traumatic Tricuspid Valve Rupture after Blunt Chest Trauma - A Case Report and Review of the Literature.

INTRODUCTION: Despite the high frequency of thoracic injuries secondary to traffic related accidents, the blunt cardiac valve rupture is extremely rare. METHOD: Case report and review of the literature using PubMed/MEDLINE and EMBASE databases. RESULT: A 38 year old female patient, victim of car accident was admitted. On primary survey the patient was conscious, cooperative and hemodynamic and respiratory stable. On secondary survey was found a bilateral open leg fracture and a seat belt sign. Whole body Computed Tomography revealed minimal haemorrhagic contusion of the cortex, left hemopneumothorax and right pneumothorax, bilateral rib fractures, liver contusion, left femoral neck fracture and fracture to the lumbar spinal column. After bilateral pleurostomy, the patient becomes hemodynamically unstable, but with no signs of external bleeding. The transthoracic echocardiography revealed an acute severe tricuspid regurgitation with hepatic veins reflux. After orthopaedic surgeries, the tricuspid valve rupture was managed by replacing the valve with a bioprostheses. The hospital stay was 122 days. CONCLUSION: Only a high index of suspicion may reveal blunt cardiac lesions as a cause for hemodynamic instability in acute setting.

Massive intraoperative bleeding after laparoscopic assisted abdominoperineal resection: a case report and systematic review of the literature.

INTRODUCTION: The laparoscopic-assisted abdominoperineal resection (LAPR) has been proved to be associated with a shorter postoperative recovery, with equivalent oncological results and similar survival when compared with conventional open surgery, for patients with low rectal cancer. METHOD: Case report of a massive intraoperative bleeding during LAPR and systematic review of the English language literature, using PubMed Medline, ISI Thopmson, OVID and EMBASE databases. RESULTS: 58 years old patient admitted in emergency setting or rectal bleeding. Rectal examination revealed a protruding,frail tumor, located 2 cm from the anal verge. Total colono scopy revealed an infiltrative, protruding tumor, situated at 2 cm from the anal verge, with a 5 cm cranial extension,without any additional colonic lesions. Computed Tomography showed a 4,5 cm circumferential rectal wall thickening, without any enlarged mesorectal or abdominal lymph nodes. The patient was transported to operating room for a LAPR. During final hemostasis, at the level of perineal surgical wound, an acute massive bleeding occurred from the presacral vessels with severe blood loss. This bleeding couldnot be managed laparo scopicaly and conversion to laparotomywas decided, with pelvic packing. At 48 hours after the initial surgical approach, the tamponing packs were removed, without signs of active bleeding. There were applied haemostatic agents and the perineal wound was sutured, without further rbleeding during in-hospital stay. CONCLUSIONS: A rapid and effective control of the presacral bleeding is mandatory to prevent a fatal outcome. Pelvic packing remains a life-saving procedure and the treatment of choice in severe cases.

Epidemiology of diverticular disease -- systematic review of the literature.

INTRODUCTION: Associated with the Western diet and life style,diverticular disease is affecting more and more developing countries worldwide. Recent studies show an increase in incidence of the disease at young age, that raises the risk of complications, along with major consequences for the patient but also for the healthcare system. METHOD: Systematic review of the literature with US National Library of Medicine and National Institutes of Health International PubMed Medline, using abstracts and articles available in PubMed Medline, Cochrane databases searching for ("Diverticulosis, Colonic epidemiology" [MeSH] OR"Diverticulosis, Colonic etiology" [MeSH] OR "Diverticulosis,Colonic genetics" [MeSH] OR "Diverticulosis, Colonic history" [MeSH]). RESULTS: Even from the rise of diverticular disease as a public healthcare problem, at the end of the previous century, it was associated with a diet rich in refined sugars, lacking vegetable fibres. The higher incidence in countries like U.S.A., Canada, United Kingdom and the northern states compared with its rare occurrence in the sub-Saharan African continent, strengthen the anterior assumptions. In regions like Asia, the disease pattern is characterized by are latively low incidence of colonic diverticular disease, with distribution of diverticula mainly on the right colon. The different incidence by sex and age show the possible existence of hormonal protective factors. Studies from countries with a rich ethnic diversity, bring into question the probable genetic predisposition to diverticular disease, fact backed-up by the few studies on twins and 1st degree relatives available in the literature. DISCUSSION: The rising incidence of colonic diverticular disease in Romania makes our country adhere the epidemiologic model existing in countries with a close socio-economic status.Although with a lower incidence than countries that have adopted a Western diet, Romania is likely to encounter a public health problem, if certain measures to identify and minimise the population exposure to risk factors are not taken.

SELENIUM STATUS IN AUTOIMMUNE THYROIDITIS.

UNLABELLED: Selenium (Se) is an important element that exerts its effects through selenoproteins. The thyroid gland has the highest Se concentration and specific selenoprotein enzymes families are crucial in the thyroid hormone metabolism. There is little evidence on the link between Se and thyroid autoimmune disease, therefore future studies are required to elucidate the nature of this associ ation. AIM: To evaluate the Se status in euthyroid subjects with autoimmune thyroiditis. MATERIAL AND METHODS: From January 2014 to January 2015 we recruited 100 consecutive euthyroid subjects with autoimmune thyroiditis, living in the same region and with normal iodine intake. Serum concentrations of Se, thyroid antibodies (antithyroperoxidase--TPOAb--and antithyroglobulin--TgAb), thyroid-stimulating hormone (TSH), and thyroid ultrasound were performed in all patients. RESULTS: Mean age of the study group was 48.87 ± 12.83 years, range: 18-82 years. Since thyroid pathology is more frequent in the 5th - 6th decades of life we selected the age of 50 for the comparative analysis of the results (51% of patients were under 50). No statistical age-group differences in antibody levels were found: mean TPOAb = 420.95 IU/ml, p = 0.840; mean TgAb = 327.98 IU/ml, p = 0.977. TSH mean was 2.14 [µIU/ml, with no significant age-group differences (p = 0.176). Se levels ranged between 8.05 - 998.50 µg/ with a mean value of 294.96 µg/L and no significant differences between age groups (p = 0.158). Thyroid ultrasound showed inhomogeneity in 89%, nodules in 35% of patients, and a mean thyroid volume of 11.72ml, with no significant age-group differences (p = 0.366). The low TSH levels were associated with low Se levels in 11.6% of cases, but the direct correlation was statistically insignificant (r = 0.116; R2 = 0.0161; p = 0.371). Depend ing on TSH percentiles, mean Selevels showed no significant differences, however pointing out the highest mean value at the 25th percentile (F = 0.441, df = 61, p = 0.646). A negative correlation trend was found between Se and TPOAb (r = -0.2276) or TgAb (r = -0.2190) but lacking statistical significance (p=0.099). CONCLUSION: Our results showed a weak negative correlation between Se and antithyroid antibodies, suggesting that selenium supplementation may improve the course of thyroid autoimmunity.

Rad51 overexpression and resistance to genotoxic agents. A study in the fission yeast Schizosaccharomyces pombe.

BACKGROUND: Many cancer cell lines have been found to overexpress the recombinase Rad51. The overexpression is associated with increased invasive potential and resistance to DNA-damaging therapeutic agents. This has been attributed to an increased capacity of cells overexpressing Rad51 to repair DNA lesions or to a genetic stabilization of the genome. AIM: As the explanations are somewhat controversial, we attempted to reproduce overexpression in the unicellular eukaryote Schizosaccharomyces pombe to have a simpler tool to study the problem of Rad51 overexpression and its induced resistance to DNA-damaging agents. METHODS: We used the nmt1 promoter inserted upstream of rad51 gene to induce its overexpression and studied the phenotype of the transformed strain, especially its sensitivity to camptothecin and hydroxyurea. RESULTS: We found that overexpression induced sensitivity to the two drugs even when it was associated with the deletion of a recombination mediator rad22/rad52 gene. However, when overexpression was associated with the deletion of the helicase-encoding fbh1 gene, the sensitivity to camptothecin was diminished.

Trauma registry -- a necessity of modern clinical practice.

INTRODUCTION: Traumas represent the cause of 10 % of deaths in the entire world. The successful development of trauma systems, including the use of trauma registries, played a significant part in lowering the mortality and the disabilities due to injuries resulted from trauma. METHOD: Review of the literature using computerized database of National Library of Medicine and the International Institutes of Health MEDLINE using PubMed interface. There were selected the articles that address the issue of trauma registry from the different world trauma systems. RESULTS: Trauma registries have developed once they were introduced in centers and trauma systems in the United States of America in 1970. First trauma database processed on computers was created in 1969 in Cook County Hospital in Chicago. This database became the prototype of trauma registry in Illinois which started gathering information from 50 designated hospitals across the entire state in 1971.Countries with limited resources were able to start useful trauma registers. Continuous financing and dedicated personnel inside the team are two essential factors in the success of a trauma registry. NISS (New Injury Severity Score) higher than 15 is a widely used inclusion criteria in the trauma register. Exclusion is represented by patients admitted at over 24 hours after the accident, those declared dead before hospital arrival or with no signs of life on arrival in hospital. In addition, it is recommended that asphyxia,drowning and burns to be excluded. CONCLUSION: The improvements regarding the treatment of multi-traumatized people in developing countries depend on establishing and performance of trauma systems, where trauma registry represents a part of these systems infrastructure.

Rad52 protein function study by fluorescence tagging.

Rad52 protein plays a significant role in DNA lesions repair by homologous recombination in eukariotic cells. Human Rad52 function somewhat overlaps with BRCA2 and has a role in cell survival in the absence of BRCA1-BRCA2 mediated recombination. Additional Rad52 function analysis and intracellular localization studies are probably necessary. We present a method for Rad22 protein tagging, a Schizosaccharomyces pombe Rad52 homologue, by Crerecombinase-mediated cassette exchange (RMCE) using the versatile pAW8 plasmid. Rad22 protein was C-termini yEGFP tagged; the resulting strain was analyzed by fluorescence microscopy. The yEGFP signal was observed (Rad22 foci) for 7.5 microM camptothecin, 0.005% methyl methanesulfonate, and 4 mM hydroxyurea treated cells. The RMCE method was efficient, and the presence of tagged Rad22 protein was confirmed by Western-Blot and fluorescence microscopy.

The effect of pioglitazone on non-pregnant female genital tract in experimental diabetes mellitus.

UNLABELLED: The effects of pioglitazone, a very used drug in the treatment of non-insulin dependent diabetes mellitus, were tested at the level of ovary of non-pregnant female rats. MATERIAL AND METHODS: The experiment was performed on three groups of adult non-pregnant female rats. group 1 was a control group (and did not receive any substance), group 2 received streptozotocin 60 mg/kg i.p. (single administration), and group 3 received streptozotocin 60 mg/kg i.p. (single administration) and pioglitazone 5 mg/kg/day p.o., daily for 8 weeks. The plasma glucose, cholesterol and triglyceride levels were determined before drugs administration and during the experiment. After 8 weeks the animals were anesthetized and sacrificed. The ovaries were examined by optical microscopy. A morphometric evaluation was performed. The obtained data were statistically interpreted by ANOVA test. RESULTS: After 8 weeks of treatment the plasma glucose and triglyceride levels were significantly lower in the pioglitazone treated group compared to the streptozotocin only group. In the pioglitazone group the number of primordial and primary ovarian follicles was significantly higher than in streptozotocin only group. CONCLUSIONS: The results showed a partial protective action of pioglitazone on ovary in nonpregnant diabetic female rats.

[Assessment of some biophysical parameters of human erythrocyte by FT-IR spectroscopy--preliminary study]. / Evaluarea unor parametri biofizici ai eritrocitului uman prin spectroscopie FT-IR--studiu preliminar.

UNLABELLED: Based on the biochemical composition of erythrocyte membrane and asymmetric distribution of phospholipids, proteins, and cholesterol, FT-IR spectroscopy can monitor the distribution and interaction pattern of membrane constituents. MATERIAL AND METHODS: The study series included 21 clinically healthy subjects aged between 20 and 60 years. Complete blood counts were performed and the serum biochemical compounds (cholesterol, triglycerides, total protein, glucose) were determined. RESULTS AND DISCUSSIONS: The parameters that can be assessed by erythrocyte FT-IR in relation with the biochemical factors that may influence membrane fluidity are: degree of fatty acids unsaturation, saturated fatty acids/unsaturated fatty acids ratio, cholesterol/phospholipids ratio, and phospholipids/protein ratio. Based on the obtained results, in the assessment of membrane status the following vibration modes were selected as spectral parameters: vibration associated valence bond (=CH), asymmetric valence CH2 groups, modes associated with P=O bond and amine bands I and II specific for proteins. Other parameters, such as v(C-O) specific to glucose, may provide additional information on glucose metabolic status. CONCLUSIONS: By correlating biochemical markers with these spectral parameters information on red cell membrane status, status that may reflect different pathological processes, can be obtained.

Biocompatibility testing of chitosan hydrogels.

UNLABELLED: Chitosan is a linear, natural cationic polysaccharide comprising beta-1,4 linked glucosamine and N-acetyl-D-glucosamine. Hydrogels of chitosan were prepared by crosslinking with varying amounts of glutaraldehyde. It can be used as a bacteriostatic, fungistatic and coating agent, and the gels and suspensions may play the role of carriers for slow release or controlled delivery of drugs, as an immobilizing medium and an encapsulation material. MATERIAL AND METHODS: Chitosan and glutaraldehyde were used to prepare the hydrogels and their characteristics were investigated by Fourier transform infrared (FT-IR) spectroscopy and the inhibitory effect on cellular growth was tested by chemiluminescence assay. The hemolytic activity was also determined by direct contact with human blood and the concentration of hemoglobin was spectrophotometrically measured. RESULTS AND DISCUSSION: Chitosan hydrogels have no inhibitory effect on cell growth, and hemolytic action below 1%, which means good blood compatibility; therefore they are promising materials.

Effects of dietary flaxseed supplementation on renal oxidative stress in experimental diabetes.

UNLABELLED: Previous results demonstrate that experimental diabetes mellitus is accompanied by increased oxidant stress within glomeruli. Evidence are emerging that dietary flaxseed supplementation can have beneficial effects on oxidative stress. MATERIALS AND METHODS: Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) in male Golden Syrian hamsters, and both diabetic and control groups were fed either standard diet, or standard diet supplemented with flaxseed (15 g/100 g diet), for 20 weeks. At the end of the study, blood samples and renal homogenates were used for determination of oxidative stress markers. RESULTS: STZ-induced diabetes in hamsters substantially increased malondialdehyde levels along with corresponding decrease in the antioxidants levels. Supplementation of flaxseed resulted in the decrease in serum and renal homogenate malondialdehyde levels. The activities of antioxidant enzymes, total glutathione (tGSH) and superoxiddismutase (SOD) were also concomitantly raised to near normal levels by flaxseed supplementation diabetic hamsters. CONCLUSION: Dietary flaxseed supplementation in diabetes mellitus may have beneficial effects on diabetic nephropathy evolution by reducing the levels of oxidative stress and increasing the antioxidant defense systems.

In vivo protein transduction to the CNS.

Proteins and peptides are useful research and therapeutic tools, however applications are limited because delivery to the desired location is not easily achievable. There are two hurdles in protein/peptide delivery to the brain: the blood-brain barrier and intracellular penetration. Penetration to both brain and the intracellular space can be achieved by adjusting hydrophilicity, and small molecule pharmacological agents have been successfully developed using this approach. But with proteins and peptides, it is difficult to modify the hydrophilicity without influencing biological functions. Trans-acting factor protein from the human immunodeficiency virus contains a highly conserved cationic peptide sequence necessary for transduction across the cell membrane. While trans-acting factor peptide has been used for in vitro protein transduction, its in vivo application is very limited because it is rapidly degraded by proteolysis. Polyethylenimine is a chemically synthesized small molecule cationization agent; the charge density is greater than a peptide-based cationic cluster such as trans-acting factor, and it is resistant to proteolysis in vivo. We first tested intracellular protein transduction following direct brain injection in mice using polyethylenimine-conjugated green fluorescence protein and beta-galactosidase (molecular weights 29 and 540 kDa, respectively). Polyethylenimine-conjugates penetrated to the intracellular space immediately surrounding the injection site within one hour. We further tested polyethylenimine-mediated protein transduction following intranasal administration, which bypasses the blood-brain barrier. Polyethylenimine-conjugates in pH 7.5 solution did not reach the brain, probably because the polyethylenimine-conjugates penetrated into the intracellular space where first exposed to the tissue, i.e. at the nasal mucosae. We temporarily reduced the electrostatic interaction between cationized polyethylenimine-conjugates and cellular surfaces by adjusting the pH to 4.5; solution rapidly reached the brain and penetrated to the intracellular space. This study suggests that polyethylenimine is a useful protein transduction agent in the brain in vivo, and adjusting cationic charge interaction can determine the extent of brain penetration.

Method for protein tagging in Schizosaccharomyces pombe.

Tagging is a useful method for the investigation of proteins. It allows the localization of the proteins in the cell, their purification in order to investigate their function and the determination of their expression. The aim of the present study was to tag the Rad32 protein of fission yeast (which is the homologue of Mre11 protein from humans) at its N-terminus. Rad32p as well as Mre11p are involved in the repair of DNA double strand breaks and in the DNA damage checkpoint. We carried out this tagging using the Cre-loxp recombination system. In a first step, a 2 kb DNA fragment was integrated upstream of the initiating codon of rad32 gene. This fragment encoded the TAP-tag (tandem affinity purification), a loxp site, a selectable marker (sup3-5), an exogenous promoter (nmt1) and a second loxp site, in this sequence. Following transformation of this DNA fragment into S. pombe cells, rad32 was under the control of the artificial promotor, which allows a controlled expression of the gene by thiamine. In a second step, the cells were transformed with a plasmid coding for Cre recombinase, which catalyses the excision of the DNA sequence between the two loxp sites, removing the marker and the artificial promotor. Thus the tag became attached to the rad32 gene upstream of the ATG, placing the gene under the control of its native promotor. The strain thus obtained will be subsequently used for evidencing the tagged protein by Western blotting and then for its purification in order to investigate its function.

[Deletion of two genes from the genome of fission yeast Schizosaccharomyces pombe. Genetic manipulation and phenotype study]. / Deletia a doua gene din genomul drojdiei fisipare Schizosaccharomyces pombe. Manipulare genetica si studiu fenotipic.

The aim of this study was to delete two genes from the genome of the fission yeast S. pombe in order to search for their functions in the cell. These genes are SPAC869.02c (MRI) and SPBC21C3.19 (MR2) and previous studies reported their significant induction after gamma irradiation. We carried out the deletions of the two genes and we replaced them with the selection marker ura4. Among the phenotype characteristics we tested the viability, the sexual behaviour and the radiosensitivity to ultraviolet and gamma irradiation. Our results indicate that MR1-deleted strain is sensitive to both UV and gamma irradiation, while the survival of the irradiated MR2-deleted strain doesn't appear to be influenced by the deletion. This suggests an involvement of MR1 gene in the adaptive response triggered by these types of genotoxic aggression. The comparison of MR1-d and MR2-d with the double deleted strains containing the deletion of MR1 or MR2 combined with the deletion of sty1 or rad3 genes led to a surprising result: the double mutants MR1-d sty1-d and MR1-d rad3-d were more resistant to both UV and gamma irradiation than the simple deleted strains sty1-d and rad3-d, respectively. This suggests a possible contribution of MR1 gene to the lethal process taking place in irradiated cells.

Anandamide-induced cell death in primary neuronal cultures: role of calpain and caspase pathways.

Anandamide (arachidonoylethanolamide or AEA) is an endocannabinoid that acts at vanilloid (VR1) as well as at cannabinoid (CB1/CB2) and NMDA receptors. Here, we show that AEA, in a dose-dependent manner, causes cell death in cultured rat cortical neurons and cerebellar granule cells. Inhibition of CB1, CB2, VR1 or NMDA receptors by selective antagonists did not reduce AEA neurotoxicity. Anandamide-induced neuronal cell loss was associated with increased intracellular Ca(2+), nuclear condensation and fragmentation, decreases in mitochondrial membrane potential, translocation of cytochrome c, and upregulation of caspase-3-like activity. However, caspase-3, caspase-8 or caspase-9 inhibitors, or blockade of protein synthesis by cycloheximide did not alter anandamide-related cell death. Moreover, AEA caused cell death in caspase-3-deficient MCF-7 cell line and showed similar cytotoxic effects in caspase-9 dominant-negative, caspase-8 dominant-negative or mock-transfected SH-SY5Y neuroblastoma cells. Anandamide upregulated calpain activity in cortical neurons, as revealed by alpha-spectrin cleavage, which was attenuated by the calpain inhibitor calpastatin. Calpain inhibition significantly limited anandamide-induced neuronal loss and associated cytochrome c release. These data indicate that AEA neurotoxicity appears not to be mediated by CB1, CB2, VR1 or NMDA receptors and suggest that calpain activation, rather than intrinsic or extrinsic caspase pathways, may play a critical role in anandamide-induced cell death.

Mechanisms of JP-8 jet fuel toxicity. I. Induction of apoptosis in rat lung epithelial cells.

JP-8 is a kerosene-based fuel widely used by the U.S. military. Various models of human occupational and animal exposure to JP-8 have demonstrated the potential for local and systemic toxicity but the mechanisms involved are unknown. The purpose of our investigation was to study the molecular mechanisms of JP-8 toxicity by using an in vitro model. JP-8 exposure in a rat lung alveolar type II epithelial cell line (RLE-6TN) induces biochemical and morphological markers of apoptotic cell death: caspase-3 activation, poly(ADP-ribose) polymerase (PARP) cleavage, chromatin condensation, membrane blebbing, cytochrome c release from mitochondria, and genomic DNA cleavage into both oligonucleosomal (DNA ladder) and high-molecular-weight (HMW) fragments. The human histiocytic lymphoma cell line (U937) also responds to JP-8 with caspase-3 activation, cleavage of caspase substrates, including PARP, DNA-PK, and lamin B1, and degradation of genomic DNA with the production of HMW fragments. Caspase-3 activation and PARP cleavage also occur in the acute T-cell leukemia cell line (Jurkat) following treatment with JP-8. Furthermore, Jurkat cells stably transfected with a plasmid encoding the antiapoptotic protein Bcl-x(L) or pretreated with the pan-caspase inhibitor Boc-d-fmk, are relatively resistant to the cytotoxic effects of JP-8 compared to control cells. Finally, we demonstrate that PARP cleavage occurs in primary mouse thymocytes exposed to JP-8. In conclusion, our data support the hypothesis that apoptotic cell death is responsible at least partially for the cytotoxic effects of JP-8 and suggest that inhibition of the apoptotic cascade might reduce JP-8 toxicity.