Pseudoautosomal abnormalities in terminal AZFb+c deletions are associated with isochromosomes Yp and may lead to abnormal growth and neuropsychiatric function.

STUDY QUESTION: Are copy number variations (CNVs) in the pseudoautosomal regions (PARs) frequent in subjects with Y-chromosome microdeletions and can they lead to abnormal stature and/or neuropsychiatric disorders? SUMMARY ANSWER: Only subjects diagnosed with azoospermia factor (AZF)b+c deletions spanning to the end of the Y chromosome (i.e. terminal deletions) harbor Y isochromosomes and/or cells 45,X that lead to pseudoautosomal gene CNVs, which were associated with abnormal stature and/or neuropsychiatric disorders. WHAT IS KNOWN ALREADY: The microdeletions in the long arm of the Y chromosome (Yq) that include the loss of one to three AZF regions, referred to as Yq microdeletions, constitute the most important known etiological factor for primary spermatogenic failure. Recently, controversy has arisen about whether Yq microdeletions are associated with gain or loss of PAR genes, which are implicated in skeletal development and neuropsychiatric function. STUDY DESIGN, SIZE, DURATION: We studied a cohort of 42 Chilean patients with complete AZF deletions (4 AZFa, 4 AZFb, 23 AZFc, 11 AZFb+c) from a university medical center, diagnosed over a period of 15 years. The subjects underwent complete medical examinations with special attention to their stature and neuropsychiatric function. PARTICIPANTS/MATERIALS, SETTING, METHODS: All subjects were characterized for Yq breakpoints by PCR, and for CNVs in PARs by multiplex ligation-dependent probe amplification (MLPA), followed by qPCR analysis for genes in PAR1 (SHOX and ZBED1), PAR2 (IL9R) and two single copy genes (SRY and DDX3Y, respectively located in Yp11.3 and AZFa). In addition, karyotypes revision and fluorescence in situ hybridization (FISH) for SRY and centromeric probes for X (DXZ1) and Y (DYZ3) chromosomes were performed in males affected with CNVs. MAIN RESULTS AND THE ROLE OF CHANCE: We did not detect CNVs in any of the 35 AZF-deleted men with interstitial deletions (AZFa, AZFb, AZFc or AZFb+c). However, six of the seven patients with terminal AZFb+c deletions showed CNVs: two patients showed a loss and four patients showed a gain of PAR1 genes, with the expected loss of VAMP-7 in PAR2. In these patients, the Yq breakpoints localized to the palindromes P8, P5 or P4. In the four cases with gain of PAR1, qPCR analysis showed duplicated signals for SRY and DDX3Y and one copy of IL9R, indicating isodicentric Yp chromosomes [idic(Y)] with breakpoint in Yq11.22. The two patients who had loss of PAR1, as shown by MLPA, had an additional reduction for SRY and DDX3Y, as shown by qPCR, associated with a high proportion of 45,X cells, as determined by FISH and karyotype. In agreement with the karyotype analysis, we detected DYZ3++ and DYZ3+ cells by FISH in the six patients, confirming idic(Y) and revealing additional monocentric Y chromosome [i(Y)]. Five patients had a history of major depressive disorders or bipolar disorder, and three had language impairment, whereas two patients showed severe short stature (Z score: -2.75 and -2.62), while a man with bipolar disorder was very tall (Z score: +2.56). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The number of males studied with Y-chromosome microdeletions and normozoospermic controls with normal karyotypes may not be enough to rule out an association between AZF deletions and PAR abnormalities. The prevalence of Y isochromosomes and/or 45,X cells detected in peripheral blood does not necessarily reflect the variations of PAR genes in target tissues. WIDER IMPLICATIONS OF THE FINDINGS: This study shows that CNVs in PARs were present exclusively in patients with terminal AZFb+c deletions associated with the presence of Y isochromosomes and 45,X cells, and may lead to neuropsychiatric and growth disorders. In contrast, we show that men with interstitial Yq microdeletions with normal karyotypes do not have an increased risk of PAR abnormalities and of phenotypical consequences. Moreover, our results highlight the importance of performing molecular studies, which are not considered in the usual screening for patients with Yq microdeletions. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the National Fund for Scientific and Technological Development of Chile (FONDECYT), grant no. 1120176 (A.C.). The authors declare that no conflicting interests exist.

Functionality predictors in acquired brain damage.

INTRODUCTION: Most individuals who have survived an acquired brain injury present consequences affecting the sensorimotor, cognitive, affective or behavioural components. These deficits affect the proper performance of daily living activities. The aim of this study is to identify functional differences between individuals with unilateral acquired brain injury using functional independence, capacity, and performance of daily activities. METHOD: Descriptive cross-sectional design with a sample of 58 people, with right-sided injury (n=14 TBI; n=15 stroke) or left-sided injury (n = 14 TBI, n = 15 stroke), right handed, and with a mean age of 47 years and time since onset of 4 ± 3.65 years. The functional assessment/functional independence measure (FIM/FAM) and the International Classification of Functioning (ICF) were used for the study. RESULTS: The data showed significant differences (P<.000), and a large size effect (dr=0.78) in the cross-sectional estimates, and point to fewer restrictions for patients with a lesion on their right side. The major differences were in the variables 'speaking' and 'receiving spoken messages' (ICF variables), and 'Expression', 'Writing' and 'intelligible speech' (FIM/FAM variables). In the linear regression analysis, the results showed that only 4 FIM/FAM variables, taken together, predict 44% of the ICF variance, which measures the ability of the individual, and up to 52% of the ICF, which measures the individual's performance. Gait alone predicts a 28% of the variance. CONCLUSIONS: It seems that individuals with acquired brain injury in the left hemisphere display important differences regarding functional and communication variables. The motor aspects are an important prognostic factor in functional rehabilitation.

[Evidence-based medicine: can we trust the results of well-designed randomized trials?]. / Medicina Basada en Evidencia: ¿podemos confiar en los resultados de los estudios clínicos aleatorizados bien diseñados?

Evidence based medicine assists in clinical decision-making by integrating critically appraised information with patient's values and preferences within an existing clinical context. A fundamental concept in this paradigm is the hierarchy of information. The randomized clinical trial is recognized as one of the designs that is less prone to bias and therefore of higher methodological quality. Clinical guidelines are one of the principal tools that evidence based medicine uses to transfer scientific information to clinical practice and many of their recommendations are based on these type of studies. In this review we present some of the limitations that the results can have, in even well designed and executed randomized clinical trials. We also discuss why valid results in these types of studies could not necessarily be extrapolated to the general population. Although the randomized clinical trial continues to be one of the best methodological designs, we suggest that the reader be careful when interpreting its results.

[Efficacy of nifurtimox for the treatment of chronic Chagas disease]. / Eficacia de nifurtimox para el tratamiento de pacientes con enfermedad de Chagas crónica.

BACKGROUND: Most Chagas patients belong to the chronic indeterminate stage, in which pharmacological treatment has an inconclusive outcome. OBJECTIVE: To evaluate the efficacy of nifurtimox treatment in chronic asymptomatic Trypanosoma cruzi infection. METHODS: We performed a systematic review and meta-analysis of electronically published literature, with no language, type of study, age or gender restrictions, until September 2010. Studies of chronic asymptomatic Chagas disease patients treated exclusively with nifurtimox were included in the analysis. Treatment efficacy was evaluated using parasitological or serological parameters. RESULTS: Of 463 identified studies, 7 were finally selected: 6 observational studies and 1 randomized clinical trial; 4 of the studies were in adults, 3 in children < 14 years. In 6 studies, outcomes were defined by serological techniques. Summary estimate (log odds) was 0.37 (CI9 -1.32 - 2.07). CONCLUSIONS: The analyzed studies gave discordant results. Those might be explained by differences in the populations studied, follow-up periods, diagnostic techniques, and sample size. More studies are necessary to obtain conclusive results about treatment efficacy of nifurtimox in this clinical phase of T. cruzi infection.

A mammalian H+ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1.

Voltage-gated proton (H+) channels are found in many human and animal tissues and play an important role in cellular defense against acidic stress. However, a molecular identification of these unique ion conductances has so far not been achieved. A 191-amino acid protein is described that, upon heterologous expression, has properties indistinguishable from those of native H+ channels. This protein is generated through alternative splicing of messenger RNA derived from the gene NOH-1 (NADPH oxidase homolog 1, where NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate).

Evidence of involvement of neurone-glia/neurone-neurone communications via gap junctions in synchronised activity of KNDy neurones.

Pulsatile secretion of gonadotrophin-releasing hormone (GnRH)/luteinising hormone is indispensable for the onset of puberty and reproductive activities at adulthood in mammalian species. A cohort of neurones expressing three neuropeptides, namely kisspeptin, encoded by the Kiss1 gene, neurokinin B (NKB) and dynorphin A, localised in the hypothalamic arcuate nucleus (ARC), so-called KNDy neurones, comprises a putative intrinsic source of the GnRH pulse generator. Synchronous activity among KNDy neurones is considered to be required for pulsatile GnRH secretion. It has been reported that gap junctions play a key role in synchronising electrical activity in the central nervous system. Thus, we hypothesised that gap junctions are involved in the synchronised activities of KNDy neurones, which is induced by NKB-NK3R signalling. We determined the role of NKB-NK3R signalling in Ca2+ oscillation (an indicator of neuronal activities) of KNDy neurones and its synchronisation mechanism among KNDy neurones. Senktide, a selective agonist for NK3R, increased the frequency of Ca2+ oscillations in cultured Kiss1-GFP cells collected from the mediobasal hypothalamus of the foetal Kiss1-green fluorescent protein (GFP) mice. The senktide-induced Ca2+ oscillations were synchronised in the Kiss1-GFP and neighbouring glial cells. Confocal microscopy analysis of these cells, which have shown synchronised Ca2+ oscillations, revealed close contacts between Kiss1-GFP cells, as well as between Kiss1-GFP cells and glial cells. Dye coupling experiments suggest cell-to-cell communication through gap junctions between Kiss1-GFP cells and neighbouring glial cells. Connexin-26 and -37 mRNA were found in isolated ARC Kiss1 cells taken from adult female Kiss1-GFP transgenic mice. Furthermore, 18ß-glycyrrhetinic acids and mefloquine, which are gap junction inhibitors, attenuated senktide-induced Ca2+ oscillations in Kiss1-GFP cells. Taken together, these results suggest that NKB-NK3R signalling enhances synchronised activities among neighbouring KNDy neurones, and that both neurone-neurone and neurone-glia communications via gap junctions possibly contribute to synchronised activities among KNDy neurones.

Outcomes of a multicomponent intervention on occupational performance in persons with unilateral acquired brain injury.

Complications after unilateral acquired brain injury (ABI) can affect various areas of expertise causing (depending on the location of the lesion) impairment in occupational performance. The aim of this study was to analyze and compare the concepts of occupational performance and functional independence, both before and after a multicomponent intervention including occupational therapy, in persons with unilateral brain damage. This was a longitudinal quasi-experimental pretest post-test study in a sample of 58 patients with unilateral brain injury (28 with traumatic brain injury and 30 with ischemic stroke). The patients' level of independence was measured using the short version of the International Classification of Functioning, Disability and Health. We also measured quality of performance using the Assessment of Motor and Process Skills. The findings of this study showed that patients with injury in the right hemisphere improved more than those with left hemisphere damage (p<0.001). All the patients with ABI, especially those with right-sided injury, derived benefit from the multicomponent intervention, except in the area of motor skills. More research is needed on the specific techniques that might address such skills.

Amniotic fluid Clara cell protein concentration in normal pregnancy, a marker of fetal airway growth or fetal lung maturation?

OBJECTIVE: To evaluate the relationship of Clara cell protein (CCP) in amniotic fluid (AF) with the lecithin/sphingomyelin (L/S) ratio, and the concentrations of saturated phosphatidylcholine (Sat PC) and surfactant protein A (SP-A). STUDY DESIGN: AF samples were obtained by amniocentesis from 98 pregnancies without conditions known to influence fetal lung maturation between 25 and 41 weeks of gestation. These samples were used for determinations of CCP, L/S ratio, Sat PC, and SP-A. Simple and multiple linear regressions were used to analyze the data. RESULTS: CCP in AF increased logarithmically with gestational age (R(2)=0.51, p=0.006). The L/S ratio (R(2)=0.41, p<0.001), and the concentrations of Sat PC (R(2)=0.26, p<0.001) and SP-A (R(2)=0.52, p<0.001) also increased with advancing gestation. Weak correlations of CCP with the L/S ratio (R(2)=0.22, p=0.009) and Sat PC (R(2)=0.12, p=0.004), but not with SP-A (R(2)=0.07, p=0.10), were found. Using multiple linear regressions, gestational age was the only predictor of CCP (F=10.9, R(2)=0.13, p=0.015). Conversely, gestational age, Sat PC, and SP-A accounted for most of the variation of the L/S ratio (F=34.7, R(2)=0.61, p=0.0001). CONCLUSION: CCP correlated very poorly with known and widely accepted indices of fetal lung maturation. The increasing concentration of CCP in AF throughout gestation probably reflects growth and development of the fetal airways.

[Intramuscular epithelioid hemangioma of the parapharyngeal space]. / Hemangioma epitelioide intramuscular del espacio parafaríngeo.

A clinical case of epithelioid hemangioma of the scalene muscle that occupied the parapharyngeal space is reported. The patient was a 34 year old man with a 2-month history of sensation of pharyngeal foreign body and mild dysphagia. The exploration revealed a tumor of the posterior and lateral wall of the oropharynx that extended from the rhinopharynx to the hypopharynx. The diagnostic sequence included CT, MRI, Doppler echography, and arteriography, which identified a right post-styloid tumor located behind and medial to the jugular vein, internal carotid artery, and vagal nerve, but did not affect arterial blood flow. The patient underwent surgical treatment consisting of lateral cervicotomy, tumor excision, and histological study.

[Benign esophagobronchial fistula in an adult]. / Fístula esofagobronquial benigna en un adulto.

The case of a senile dementia patient 86 year old, and a supposed clinical history of swallowing a foreign body is presented. After a clinical study (esophagogram, flexible and rigid esophagoscopy and bronchoscopy) and finally the autopsy of the patient, we found and idiopathic esophago-bronchial fistula.

[Comparative study of the use of manual and mechanical suturing in the closure of the mucosal defect in total laryngectomy]. / Estudio comparativo entre el empleo de la sutura manual y la sutura mecánica en el cierre del defecto mucoso en la laringectomía total.

With the present study we try to achieve an objective confirmation of the efficacy given by the mechanical suture in the closing of the pharyngeal defect following a total laryngectomy against the conventional manual suture with absorbable material. Therefore we have studied three random groups of patients: in the first one we include those who had a manual suture (n = 50), in the second one those who had performed a mechanical suture with closed technique (n = 38) and in the third one those who had a mechanical suture with an open technique (n = 12). We have checked that mechanical suture with a closed technique improves significantly the surgical length (212 minutes), the beginning of the oral feeding (10.73 days) and the time of in-hospital stay (13.5 days). Mechanical sutures with a closed technique also reduce the rate of complications such as fistulae (5.26%), infection (0%) and haemorrhage (0%).

[Evaluation of the community integration of persons with lateralised post-acute acquired brain injury]. / Valoracion de la integracion en la comunidad de las personas con daño cerebral adquirido postagudo lateralizado.

INTRODUCTION. Hemispheric specialization is a topic of interest that has motivated an enormous amount of research in recent decades. After a unilateral brain injury, the consequences can affect various areas of specialization, leading, depending on the location of the injury, impairment in quality of life and community integration. PATIENTS AND METHODS. Cross-sectional study with a sample of 58 patients, 28 traumatic brain injury (TBI) and 30 cerebrovascular accidents, both lateralized. The level of integration in the community is measured by the Community Integration Questionnaire. RESULTS. There were three groups analyzed by considering unilateral injury (full sample, stroke sample, and TBI sample). Results showed a significantly high community integration of people with right hemisphere injury. However, to measure the level of community integration between TBI and stroke, the results showed no significant differences. CONCLUSION. According to the results of the study people with brain injury in the right hemisphere have a better community integration than people with lesions in the left hemisphere regardless of the origin of the lesions (vascular or traumatic). We discussed the reasons that may motivate the differences and clinical implications.

TITLE: Valoracion de la integracion en la comunidad de las personas con daño cerebral adquirido postagudo lateralizado.Introduccion. La especializacion hemisferica es un tema de interes que ha motivado un gran volumen de investigacion en las ultimas decadas. Tras una lesion cerebral lateralizada, las secuelas pueden afectar a diversas areas de especializacion, provocando, en funcion de la localizacion de la lesion, una afectacion en la calidad de vida y en la integracion comunitaria. Pacientes y metodos. Estudio transversal con una muestra de 58 pacientes, 28 con traumatismos craneoencefalicos (TCE) y 30 con accidentes cerebrovasculares (ACV), ambos lateralizados. El nivel de integracion en la comunidad se midio con el cuestionario de integracion comunitaria (CIQ). Resultados. De los grupos analizados teniendo en cuenta la lesion lateralizada (muestra completa, muestra de ACV y muestra de TCE), los resultados mostraron una significativamente mayor integracion comunitaria de las personas con lesion en el hemisferio derecho. Sin embargo, al medir el nivel de integracion comunitaria entre TCE y ACV, los resultados no mostraron diferencias significativas. Conclusion. Segun los resultados del estudio, las personas con lesion cerebral en el hemisferio derecho presentan una mejor integracion comunitaria que las personas con lesion en el hemisferio izquierdo, con independencia del origen de las lesiones (vascular o traumatica). Se discuten las razones que pueden motivar las diferencias y las implicaciones clinicas.

[Energy-saving strategies in the treatment of fatigue in patients with multiple sclerosis. A pilot study]. / Estrategias de conservación de la energía en el tratamiento de la fatiga en pacientes con esclerosis múltiple. Estudio piloto.

INTRODUCTION: Multiple sclerosis (MS) is the third most common cause of neurological disability in adults, and 75-95% of subjects suffer from fatigue, which is the symptom that interferes most with activities of daily living and is the most frequent reason why patients have to stop working. AIM: To evaluate the adherence to and effectiveness of a course on energy-saving as a way to diminish fatigue and to increase the quality of life in people with MS. PATIENTS AND METHODS: This pilot study was almost random (although allocation to the control and intervention groups was randomised, the researchers chose the number of patients assigned to each group), with a control group of 14 patients with MS who showed signs of fatigue. Two 2-hour sessions of the course on energy-saving techniques were given by an occupational therapist in a group of nine patients. Measures were made of the self-efficiency to perform the energy-saving strategies assessment, the Fatigue Impact Scale (FIS), the Short-Form health questionnaire (SF-36) and the open questionnaire on improvements in the course. RESULTS: Adherence to treatment by participants was high (with a mean increase of 2.17) and improvements were obtained on the FIS and SF-36 scales, although they were not statistically significant. CONCLUSIONS: The degree of adherence to a course on energy-saving strategies by patients with MS is high. Although no statistically significant improvements were found, patients did report clinical improvements in both fatigue and quality of life.

Eficacia de nifurtimox para el tratamiento de pacientes con enfermedad de Chagas crónica / Efficacy of nifurtimox for the treatment of chronic Chagas disease

Background: Most Chagas patients belong to the chronic indeterminate stage, in which pharmacological treatment has an inconclusive outcome. Objective: To evaluate the efficacy of nifurtimox treatment in chronic asymptomatic Trypanosoma cruzi infection. Methods: We performed a systematic review and meta-analysis of electronically published literature, with no language, type of study, age or gender restrictions, until September 2010. Studies of chronic asymptomatic Chagas disease patients treated exclusively with nifurtimox were included in the analysis. Treatment efficacy was evaluated using parasitological or serological parameters. Results: Of 463 identified studies, 7 were finally selected: 6 observational studies and 1 randomized clinical trial; 4 of the studies were in adults, 3 in children < 14 years. In 6 studies, outcomes were defined by serological techniques. Summary estimate (log odds) was 0.37 (CI9 -1.32 - 2.07). Conclusions: The analyzed studies gave discordant results. Those might be explained by differences in the populations studied, follow-up periods, diagnostic techniques, and sample size. More studies are necessary to obtain conclusive results about treatment efficacy of nifurtimox in this clinical phase of T. cruzi infection.

Introducción: La mayoría de los pacientes con enfermedad de Chagas se encuentran en fase crónica indeterminada donde los resultados de tratamiento farmacológico no han sido concluyentes. Objetivo: Evaluar la evidencia que apoya la eicacia del tratamiento con nifurtimox en la infección crónica por Trypanosoma cruzi asintomática. Método: Revisión sistemática y meta-análisis de literatura publicada en forma electrónica, sin restricción de lenguaje, tipo de estudio, edad y género, hasta septiembre de 2010. Se incluyeron estudios de pacientes con enfermedad de Chagas crónica asintomáticos que recibieron tratamiento exclusivo con nifurtimox. La eicacia del tratamiento fue evaluada mediante métodos parasitológicos o serológicos. Resultados: Se identiicaron 463 estudios primarios seleccionando inalmente siete: seis observacionales y un ensayo clínico randomizado; cuatro en pacientes adultos y tres en niños bajo14 años de edad. En seis estudios los resultados se midieron mediantes técnicas serológicas. La medida resumen (log de la chance) fue de 0,37 (IC95% -1,32 -2,07). Conclusiones: Los resultados son discordantes. La incertidumbre se maniiesta por las diferencias en las poblaciones estudiadas, periodos de seguimiento, técnicas diagnósticas y tamaño de las muestras. Es necesario realizar nuevos estudios que consideren las fuentes de incertidumbre para obtener resultados concluyentes sobre la eicacia del nifurtimox en esta fase clínica de la infección por T. cruzi.

Attitudes of general practitioners in Bizkaia, Spain, towards the terminally ill patient.

This paper reports the results of a survey of general practitioners in Biscay which investigated the types of narcotics used in the treatment of chronic malignant pain, the degree of their use and the difficulties encountered both in the use of narcotics and the general care of the terminally ill patient. We found that (1) 50% of the doctors surveyed used narcotics in the treatment of at least 60% of their patients with terminal disease; (2) morphine is the most frequently used narcotic, usually being given orally, and (3) those who completed the questionnaire showed themselves to be highly motivated when treating terminally ill patients. However, they considered that their motivation exceeded their level of competence and, according to the majority, collaboration between the different health services and the formation of interdisciplinary teams would be the best way to facilitate an increase in competence.

Duality of the voltage-dependent calcium influx in adrenal glomerulosa cells.

Both T- and L-type calcium channels are expressed in bovine adrenal glomerulosa cells and both channels are sensitive to moderate depolarizations of the cell membrane induced by angiotensin II (AngII) or physiological concentrations of extracellular K+. These channels present distinct pharmacology, L-type channels being more sensitive to dihydropyridines, whereas T channels are inhibited by lower concentrations of mibefradil, a new type of calcium antagonist currently used for treating hypertension. The activity of these channels is also differently modulated by AngII, which inhibits T channels through activation of protein kinase C and L channels through a Pertussis toxin-sensitive G protein. Finally, whereas the activity of L-type channels is directly reflected on the levels of the cytosolic calcium concentration ([Ca2+]c), T-type channels are more closely related to the control of steroidogenesis, possibly through a kind of "calcium pipeline" linking the plasma membrane to the mitochondria. In conclusion, two types of calcium channels, with distinct functions and differential modulation by AngII, are activated by agonists of aldosterone biosynthesis in adrenal glomerulosa cells. Most importantly, these channels have distinct sensitivities to currently used antihypertensive therapeutic drugs.

Microbial removal of chlorinated phenols during aerobic treatment of effluents from radiata pine kraft pulps bleached with chlorine-based chemicals, with or without hemicellulases.

The removal of chlorophenolic compounds from kraft mill effluents bleached with chlorine (cBKME) or chlorine plus hemicellulases (bBKME) was studied in reactors of aerobic treatment lagoons. In these laboratory models, a stable microbial population removed biochemical oxygen demand at similar rates of the mill lagoon. Complete removal of nine chlorophenols and chloroguaiacols during microbial treatment of these effluents was detected by gas chromatography. Abiotic removal was only observed with 2,4-dichlorophenol and 2,4,5-trichlorophenol. There were no significant differences in degradative ability between microorganisms acclimated to grow in reactors fed with cBKME or bBKME. The latter had a lower content of adsorbable organic halogen and chlorophenols than cBKME. Microorganisms acclimated to cBKME or bBKME were only able to grow on phenol or guaiacol as sole carbon source. However, these microorganisms removed (0.1-0.5 mM) 4-chlorophenol, 2,4-dichlorophenol and 2,4-dichlorophenoxyacetate with BKME as primary carbon source. Under these conditions, 2,4,6- and 2,4,5-trichlorophenol, 4,5-dichloroguaiacol, 4,5,6-trichloroguaiacol and tetrachloroguaiacol were not removed. These results suggest that the microbial removal of bleaching chlorophenols and chloroguaiacols during aerobic treatment, probably takes place only because of their very low concentration (1-200 ppb) in BKME.

Angiotensin II negatively modulates L-type calcium channels through a pertussis toxin-sensitive G protein in adrenal glomerulosa cells.

In bovine adrenal glomerulosa cells, angiotensin II and extracellular K+ stimulate aldosterone secretion in a calcium-dependent manner. In these cells, physiological concentrations of extracellular potassium activate both T-type (low threshold) and L-type (high threshold) voltage-operated calcium channels. Paradoxically, the cytosolic calcium response to 9 mM K+ is inhibited by angiotensin II. Because K+-induced calcium changes observed in the cytosol are almost exclusively due to L-type channel activity, we therefore studied the mechanisms of L-type channel regulation by angiotensin II. Using the patch-clamp method in its perforated patch configuration, we observed a marked inhibition (by 63%) of L-type barium currents in response to angiotensin II. This effect of the hormone was completely prevented by losartan, a specific antagonist of the AT1 receptor subtype. Moreover, this inhibition was strongly reduced when the cells were previously treated for 1 night with pertussis toxin. An effect of pertussis toxin was also observed on the modulation by angiotensin II of the K+ (9 mM)-induced cytosolic calcium response in fura-2-loaded cells, as well as on the angiotensin II-induced aldosterone secretion, at both low (3 mM) and high (9 mM) K+ concentrations. Finally, the expression of both Go and Gi proteins in bovine glomerulosa cells was detected by immunoblotting. Altogether, these results strongly suggest that in bovine glomerulosa cells, a pertussis toxin-sensitive G protein is involved in the inhibition of L-type channel activity induced by angiotensin II.

Heme histidine ligands within gp91(phox) modulate proton conduction by the phagocyte NADPH oxidase.

The membrane subunit of the phagocyte NADPH oxidase, gp91(phox), possesses a H(+) channel motif formed by membrane-spanning histidines postulated to coordinate the two heme groups forming the redox center of the flavocytochrome. To study the role of heme-binding histidines on proton conduction, we stably expressed the gp91(phox) cytochrome in human embryonic kidney 293 cells and measured proton currents with the patch clamp technique. Similar to its shorter homologue, NADPH oxidase homologue 1, which is predicted not to bind heme, gp91(phox) generated voltage-activated, pH-dependent, H(+)-selective currents that were reversibly blocked by Zn(2+). The gp91(phox) currents, however, activated faster, deactivated more slowly, and were markedly affected by the inhibition of heme synthesis. Upon heme removal, the currents had larger amplitude, activated faster and at lower voltages, and became sensitive to the histidine reagent diethylpyrocarbonate. Mutation of the His-115 residue to leucine abolished both the gp91(phox) characteristic 558-nm absorbance peak and voltage-activated currents, indicating that His-115 is involved in both heme ligation and proton conduction. These results indicate that the gp91(phox) proton channel is activated upon release of heme from its His-115 ligand. During activation of the oxidase complex, changes in heme coordination within the cytochrome might increase the mobility of histidine ligands, thereby coupling electron and proton transport.

A Ca(2+)-activated NADPH oxidase in testis, spleen, and lymph nodes.

Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.