Autoubiquitination of the 26S proteasome on Rpn13 regulates breakdown of ubiquitin conjugates.

Degradation rates of most proteins in eukaryotic cells are determined by their rates of ubiquitination. However, possible regulation of the proteasome's capacity to degrade ubiquitinated proteins has received little attention, although proteasome inhibitors are widely used in research and cancer treatment. We show here that mammalian 26S proteasomes have five associated ubiquitin ligases and that multiple proteasome subunits are ubiquitinated in cells, especially the ubiquitin receptor subunit, Rpn13. When proteolysis is even partially inhibited in cells or purified 26S proteasomes with various inhibitors, Rpn13 becomes extensively and selectively poly-ubiquitinated by the proteasome-associated ubiquitin ligase, Ube3c/Hul5. This modification also occurs in cells during heat-shock or arsenite treatment, when poly-ubiquitinated proteins accumulate. Rpn13 ubiquitination strongly decreases the proteasome's ability to bind and degrade ubiquitin-conjugated proteins, but not its activity against peptide substrates. This autoinhibitory mechanism presumably evolved to prevent binding of ubiquitin conjugates to defective or stalled proteasomes, but this modification may also be useful as a biomarker indicating the presence of proteotoxic stress and reduced proteasomal capacity in cells or patients.

Effects of the Addition of Flaxseed and Amaranth on the Physicochemical and Functional Properties of Instant-Extruded Products.

The addition of flaxseed and amaranth on the physicochemical, functional, and microstructural changes of instant-extruded products was evaluated. Six mixtures with different proportions of amaranth (18.7-33.1%), flaxseed (6.6-9.3%), maize grits (55.6-67.3%) and minor ingredients (4.7%) were extruded in a twin-screw extruder. Insoluble and soluble fiber contents in extrudates increased as the proportions of amaranth and flaxseed increased. However, the highest flaxseed proportion had the highest soluble fiber content (1.9%). Extruded products with the highest proportion of flaxseed and amaranth resulted in the highest dietary fiber content and hardness values (5.2 N), which was correlated with the microstructural analysis where the crystallinity increased, resulting in larger, and more compact laminar structure. The extruded products with the highest maize grits proportion had the highest viscosity, expansion, and water absorption indexes, and the lowest water solubility index values. The mixtures with amaranth (18.7-22.9%), flaxseed (8.6-9.3%), and maize grits (63.8-67.3%) resulted in extruded products with acceptable physicochemical and functional properties.

The JAMM motif of human deubiquitinase Poh1 is essential for cell viability.

Poh1 deubiquitinase activity is required for proteolytic processing of polyubiquitinated substrates by the 26S proteasome, linking deubiquitination to complete substrate degradation. Poh1 RNA interference (RNAi) in HeLa cells resulted in a reduction in cell viability and an increase in polyubiquitinated protein levels, supporting the link between Poh1 and the ubiquitin proteasome pathway. To more specifically test for any requirement of the zinc metalloproteinase motif of Poh1 to support cell viability and proteasome function, we developed a RNAi complementation strategy. Effects on cell viability and proteasome activity were assessed in cells with RNAi of endogenous Poh1 and induced expression of wild-type Poh1 or a mutant form of Poh1, in which two conserved histidines of the proposed catalytic site were replaced with alanines. We show that an intact zinc metalloproteinase motif is essential for cell viability and 26S proteasome function. As a required enzymatic component of the proteasome, Poh1 is an intriguing therapeutic drug target for cancer.

Dificultad diagnóstica en masa retroperitoneal. Reporte de un caso / Diagnostic difficulty in retroperitoneal mass: A case report

El Schwannoma se origina de la vaina perineural de Schwannoma, se detecta con frecuencia incidentalmente en estudios imagenológicos siendo el principal método diagnóstico la Tomografía Computada. El tratamiento es la resección quirúrgica con márgenes libres. Se presenta una paciente femenina de 49 años, en control por oncología por enfermedad de base, cáncer de mama izquierda, se identifica por TAC y PECT/TC imagen voluminosa en retroperitoneo situación lateroaórtica izquierda de configuración no quística e hipermetabólica, solicita biopsia percutánea, ante la falta de ventana, se decide exeresis completa de masa. Diagnóstico definitivo patológico Schwannoma. Sin indicación de tratamiento adyuvante, cursa buena evolución postoperatoria sin recidiva.

Schwannoma, a benign tumor that arises from Schwann cells of the perineural nerve sheath, is often incidentally detected in imaging tests and mainly diagnosed by CT scan. Treatment consists of surgical resection with clear margins. We present the case of a 49-year-old female patient subject to Oncology Department follow-up due to an underlying disease, left breast cancer. A large, hypermetabolic, noncystic mass in the retroperitoneal region is identified by CT and PECT/CT scan in the left lateral aortic area. A percutaneous biopsy is requested. Due to the limited acoustic window, complete resection of the mass is decided. Final histopathology diagnosis of Schwannoma. No adjuvant treatment indication; undergoing favorable postoperative progress, without recurrence

Assessment of the effects of D-003, a new antiplatelet and lipid-lowering compound, in healthy volunteers. A phase I clinical study.

OBJECTIVE: To investigate the effects of D-003 on the bleeding time (BT) and lipid profile of healthy human volunteers. METHODS: This single-blind, randomised, placebo-controlled, parallel-group study was conducted in healthy volunteers. Step 1 investigated the effects of single doses of D-003 5, 25 or 50 mg on BT in comparison with placebo. Step 2 investigated the effects of 30 days of D-003 5, 25 or 50 mg/day compared with placebo on lipid profile with an interim assessment at 14 days. BT, lipid profile, physical and haematological safety indicators were measured and adverse events (AEs) recorded. Both steps were followed by a 14- or 30-day washout period. RESULTS: Step 1: D-003 25 and 50 mg significantly increased mean BT 2 hours after administration compared with baseline, but a significant difference versus placebo occurred only with the 50 mg dose. Individual values from participants taking this dose, however, remained within normal limits. This effect was reversible. BT values obtained 2 hours after drug administration showed a moderate dose-dependent relationship. No drug-related changes in safety indicators were found with D-003. Step 2: After 7 days on D-003 50 mg/day, BT was significantly increased compared with baseline and placebo up to the end of the active treatment period. However, all individual values for participants taking this dosage remained within the normal range. This effect was reversible by the end of the washout period. After 30 days, D-003 (5, 25 and 50 mg/day) significantly reduced serum TC (by 13.3 to 17.4%) and LDL-C (by 11.6 to 22.6%) levels, and raised HDL-C levels (by 14.6 to 29.7%), but did not affect triglyceride levels. The significant increase in HDL-C was observed after 14 days on treatment. The effects on the lipid profile were reversible by the end of the 30-day washout period, although after 14 days of washout the effects on HDL-C and LDL-C still remained significant, revealing a certain persistence of effect. Eight participants (four receiving placebo and four receiving D-003 5, 25 or 50 mg/day) reported a total of nine AEs, none of which were drug-related. Of these patients, only two treated with D-003 25 and 50 mg/day discontinued treatment. CONCLUSIONS: D-003 in single or repeated doses (50 mg) induced significant and reversible increases in BT. In addition, repeated doses (5, 25 and 50 mg/day) significantly and reversibly lowered serum LDL-C and TC levels and significantly raised serum HDL-C levels. These effects were reversible by 30 days after the end of treatment.

Continuous venovenous hemodiafiltration in patients with multiple organ dysfunction syndrome in an intensive care unit.

INTRODUCTION: Continuous venovenous hemodiafiltration, generally used in patients with acute renal failure, enables elimination of humoral mediators of systemic inflammatory response and sepsis from blood. This effect should improve treatment results in patients with multiple organ dysfunction, but evidence of improved survival is insufficient. OBJECTIVES: Describe the effect of continuous venovenous hemodiaflitration on patients with multiple organ dysfunction syndrome in terms of systemic and brain hemodynamics, oxygenation, metabolism and status on ICU separation. METHODS: An observational case series was done of 18 patients (11 men and 7 women) aged 24-78 years with multiple organ dysfunction syndrome treated with continuous venovenous hemodiafiltration in the Medical-Surgical Research Center's ICU in Havana. General, systemic and brain hemodynamic, oxygenation and metabolic variables were assessed immediately before and 12 hours after starting the procedure; vital status on separation from intensive care was recorded. For analysis, patients were grouped by whether cause of multiple organ dysfunction syndrome was septic or nonseptic. Variable means before and after treatment were compared using the Wilcoxon matched pairs test. Standardized mortality ratios were calculated for both groups, with survival efficacy defined by a ratio of <0.9. RESULTS: After 12 hours continuous venovenous hemodiafitration, the septic group showed clinical improvement, with statistically significant improvement in all variables except mean arterial pressure and brain hemodynamics. Survival to discharge from ICU was 64%, with a standardized mortality ratio of 0.66. In the nonseptic group, survival was 0% and ratio was 2.13; temperature was the only variable found to improve significantly. CONCLUSIONS: Continuous venovenous hemodiafltration improved clinical parameters and survival in patients with multiple organ dysfunction of septic origin. Further studies are needed with larger numbers of patients to corroborate these results.

La importancia de garantizar la calidad y minimizar los riesgos de contaminación en el análisis genético forense / The importance of ensuring quality and minimising the risks of contamination in genetics forensic analysis

La alta sensibilidad conseguida en los análisis genéticos de interés forense permite obtener perfiles genéticos procedentes de mínimas trazas de material biológico depositado sobre los indicios, antes o incluso después de los hechos investigados. La contaminación de los indicios biológicos de manera accidental y la interpretación errónea de los resultados genéticos tienen importantes consecuencias con la consiguiente repercusión en el proceso judicial. Minimizar y detectar la presencia de contaminaciones accidentales que se pueden generar durante algunas de las fases de recogida o análisis genético es una prioridad para los laboratorios de análisis genéticos. El presente artículo revisa el marco normativo, así como los estándares de calidad nacionales e internacionales aplicables al ámbito de la genética forense que tienen como objetivo garantizar la calidad de la prueba pericial y la fiabilidad de las conclusiones emitidas en los informes periciales (AU)

The high sensitivity achieved in genetic analysis of forensic interest allows to obtain genetic profiles from minimal traces of biological material deposited on the evidence before or even subsequent to the events under investigation. Accidental contamination of biological evidence and erroneous interpretation of genetic findings have important implications with the consequent impact on the judicial process. Minimising and detecting the presence of accidental contamination that may be generated during some phases of collection of samples or genetic analysis are a priority for forensic genetics laboratories. This article reviews legislation and national and international standards applicable to field of forensic genetics, which aim to ensure the quality of expert evidence and the reliability of the conclusions reached in the expert reports (AU)

Femoral central venous catheter-associated deep venous thrombosis in children with diabetic ketoacidosis.

OBJECTIVE: To describe the incidence of clinical deep venous thrombosis associated with femoral central venous catheters (CVC-DVT) in children with diabetic ketoacidosis (DKA). DESIGN: Retrospective case-matched control series. SETTING: Pediatric intensive care units of two university-affiliated hospitals. PATIENTS: All eight pediatric DKA patients with femoral central venous catheters between 1998 and 2001, and 16 age-matched control patients with femoral central venous catheters and circulatory shock. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The records of all children with DKA and the control patients were reviewed. CVC-DVT was defined as persistent ipsilateral leg swelling after removal of a femoral central venous catheter. Control patients with coagulopathies, thrombocytopenia, cancer, and hyperglycemia were excluded. Four of eight patients with DKA developed CVC-DVT compared with none of the 16 control patients (p = .007, Fisher's exact test). All four patients with DKA and CVC-DVT were <3 yrs old. Doppler ultrasound examination was performed on three of the four patients with clinical CVC-DVT, confirming the diagnosis in each case. CONCLUSIONS: This study suggests that young children with DKA have an increased incidence of clinical DVT associated with the placement of femoral central venous catheters.

Characterization of Staphylococcus aureus enterotoxin L.

Staphylococcus aureus causes a wide variety of diseases. Major virulence factors of this organism include enterotoxins (SEs) that cause both food poisoning and toxic shock syndrome. Recently, a novel SE, tentatively designated SEL, was identified in a pathogenicity island from a bovine mastitis isolate. The toxin had a molecular weight of 26,000 and an isoelectric point of 8.5. Recombinant SEL shared many biological activities with SEs, including superantigenicity, pyrogenicity, enhancement of endotoxin shock, and lethality in rabbits when administered in subcutaneous miniosmotic pumps, but the protein lacked emetic activity. T cells bearing the T-cell receptor beta chain variable regions 5.1, 5.2, 6.7, 16, and 22 were significantly stimulated by recombinant SEL.

Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress.

The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, to tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of S. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS17, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology to the ylxM-ffh operon of Bacillus subtilis. The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an S. mutans genomic library. Sequencing of the complementing clone identified the intact ylxM and ffh genes as well as a partial ORF with homology to the proUlopuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat-1::Tn917 mutation on ffh expression. Upon acid shock (pH 5), wild-type ffh mRNA levels were found to increase approximately four- to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptive/tolerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acid-shocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable to ferment sorbitol although it was able to grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans to changing environmental conditions.

Enfermedad de Cowden: presentación de un caso y revisión de la bibliografía / Cowden disease: report of a case and review of the bibliography

La Enfermedad de Cowden (EC), también conocida como 'Síndrome de Neoplasias-Hamartomas Múltiples', es una rara e infradiagnosticada entidad hereditaria autosómica dominante caracterizada por la presencia de múltiples hamartomas de origen mesodérmico, endodérmico y ectodérmico , así como por una alta incidencia de presentación de tumores malignos. Actualmente se clasifica como un síndrome preneoplásico hereditario. Describimos los hallazgos en un paciente con EC con especial énfasis sobre los aspectos de imagen, revisando las principales manifestaciones de esta entidad (AU)