Isolation of two triterpenoids from Phlomis purpurea, one of them with anti-oomycete activity against Phytophthora cinnamomi, and insights into its biosynthetic pathway.

Phytophthora cinnamomi is an important plant pathogen responsible for dieback diseases in plant genera including Quercus, Fagus, Castanea, Eucalyptus, and Pinus, among others, all over the world. P. cinnamomi infection exerts tremendous ecological and economic losses. Several strategies have been developed to combat this pathogenic oomycete, including the search for novel anti-oomycete compounds. In this work, a Mediterranean vascular plant, Phlomis purpurea, has been screened for secondary bioactivity against this pathogen. The genus Phlomis includes a group of herbaceous plants and shrubs described as producers of many different bioactive compounds, including several triterpenoids. Triterpenoids are well-known molecules synthesized by plants and microorganisms with potent antioxidant, antitumoral, and antimicrobial activities. We have isolated by HPLC-DAD and characterized by HPLC-MS and NMR two nortriterpenoid compounds (phlomispentaol A and phlomispurtetraolone) from the root extracts of P. purpurea. One of them (phlomispentaol A) is active against the plant pathogenic oomycete P. cinnamomi (based on in vitro inhibition bioassays). Based on their chemical structure and their relationship to other plant triterpenoids, oleanolic acid is proposed to be the common precursor for these molecules. The anti-oomycete activity shown by phlomispentaol A represents a promising alternative to counteract the worldwide-scale damage caused to forest ecosystems by this pathogen.

Bioactive pectic polysaccharides from bay tree pruning waste: Sequential subcritical water extraction and application in active food packaging.

The potential isolation of bio-active polysaccharides from bay tree pruning waste was studied using sequential subcritical water extraction using different time-temperature combinations. The extracted polysaccharides were highly enriched in pectins while preserving their high molecular mass (10-100 kDa), presenting ideal properties for its application as additive in food packaging. Pectin-enriched chitosan films were prepared, improving the optical properties (≥95% UV-light barrier capacity), antioxidant capacity (˃95% radical scavenging activity) and water vapor permeability (≤14 g·Pa-1·s-1·m-1·10-7) in comparison with neat chitosan-based films. Furthermore, the antimicrobial activity of chitosan was maintained in the hybrid films. Addition of 10% of pectins improved mechanical properties, increasing the Young's modulus 12%, and the stress resistance in 51%. The application of pectin-rich fractions from bay tree pruning waste as an additive in active food packaging applications, with triple action as antioxidant, barrier, and antimicrobial has been demonstrated.

RanBP2-Mediated SUMOylation Promotes Human DNA Polymerase Lambda Nuclear Localization and DNA Repair.

Cellular DNA is under constant attack by a wide variety of agents, both endogenous and exogenous. To counteract DNA damage, human cells have a large collection of DNA repair factors. Among them, DNA polymerase lambda (Polλ) stands out for its versatility, as it participates in different DNA repair and damage tolerance pathways in which gap-filling DNA synthesis is required. In this work, we show that human Polλ is conjugated with Small Ubiquitin-like MOdifier (SUMO) proteins both in vitro and in vivo, with Lys27 being the main target of this covalent modification. Polλ SUMOylation takes place in the nuclear pore complex and is mediated by the E3 ligase RanBP2. This post-translational modification promotes Polλ entry into the nucleus, which is required for its recruitment to DNA lesions and stimulated by DNA damage induction. Our work represents an advance in the knowledge of molecular pathways that regulate cellular localization of human Polλ, which are essential to be able to perform its functions during repair of nuclear DNA, and that might constitute an important point for the modulation of its activity in human cells.

Isolation and characterization of lignins from wheat straw: Application as binder in lithium batteries.

Three different lignin-rich fractions have been used as binder material for electrodes in rechargeable lithium batteries. Lignin samples were obtained through three different pulping processes; kraft, soda and organosolv pulping processes, using wheat straw as raw material. Physico-chemical characterization of three types of lignins was evaluated. Characterization has been performed using Fourier transform infrared spectroscopy (FTIR) and 31P NMR Spectroscopy to analyse the functional groups; gel permeation chromatography (GPC) for determining molar mass distribution (MWD), and thermogravimetric analysis (TGA) to follow the thermal behaviour. Electrodes containing lignin or poly vinylidene fluoride (PVDF) were tested electrochemically. The three different lignin samples exhibited excellent performance as binder, retaining the specific capacity after 50 cycles at a current density of 100mAg-1. These results show that lignin could be used as a low-cost and environmental binder, replacing the PVDF polymer in electrodes for energy storage applications.

Movimientos oculares anómalos del lactante como forma de inicio de una hemiplejía alternante de la infancia / Anomalous ocular movements of the infant as a form of onset of an alternating hemiplegia of childhood

La hemiplejía alternante de la infancia es un síndrome raro con manifestaciones tempranas. Su incidencia ha sido estimada en 1 de cada 1.000.000 nacimientos. Su patogenia es desconocida. Recientemente, se ha identificado la mutación ATP1A3 en la mayoría de estos pacientes. Por lo general, se suele presentar después de los 18 meses, produciéndose episodios repetidos de hemiplejía prolongada, así como eventos paroxísticos que se repiten, especialmente movimientos oculares, que pueden acompañar o apreciarse aisladamente en relación con la hemiplejía. La forma de presentación clínica consiste en movimientos oculares paroxísticos descubiertos en los 3 primeros meses de vida en más del 80% de los casos. Otro hallazgo es la desaparición de los síntomas con el sueño, que reaparecen sólo 10-20 minutos antes de despertar. Algunos niños manifiestan un retraso del desarrollo, así como alteraciones motoras adicionales, como ataxia, debilidad, distonía o espasticidad. El diagnóstico se establece mediante criterios clínicos. La flunarizina, un fármaco con efectos bloqueantes sobre los canales de calcio, disminuye la duración, la intensidad y/o la frecuencia de crisis en un gran número de pacientes, por lo que se ha convertido en el principal pilar del tratamiento (AU)

Alternating hemiplegia of childhood is a rare syndrome with early manifestations. The incidence has been estimated at 1 in 1,000,000 births. Its pathogenesis remains still unknown. Recently, the ATP1A3 mutation has been identified in the majority of these patients. Generally, it occurs after 18 months, with repeated episodes of prolonged hemiplegia, as well as recurring paroxysmal events, especially eye movements, which may accompany or be seen in isolation in relation to hemiplegia. The clinical presentation consists of paroxysmal ocular movements discovered in the first 3 months of life in more than 80% of cases. Another finding is the disappearance of symptoms with sleep, reappearing only 10-20 minutes before waking. In some children, developmental delays, as well as additional motor disturbances (such as ataxia, weakness, dystonia or spasticity) are described. The diagnosis is established by clinical criteria. Flunarizine, a drug with blocking effects on calcium channels, decreases duration, intensity and/or frequency of seizures in a large number of patients, and has become the main pillar of treatment (AU)

Análisis de los pacientes que reconsultaron a un servicio de emergencias pediátricas de un hospital materno infantil como indicador de calidad asistencial / Analysis of patients who consulted for a second time at a pediatric emergency department at a mother-and-child hospital as a marker of quality of care

Objetivos: 1) Analizar las reconsultas (RC) de pacientes a las 72 horas de su primera consulta en el Servicio de Emergencias Pediatría (SEP) de un hospital materno infantil del conurbano bonaerense. 2) Comparar las reconsultas entre dos periodos, luego de implementar mejoras en los procesos asistenciales y de contrarreferencia. Materiales y Metodos: Diseño: retrospectivo, observacional y transversal. Criterios de inclusión: pacientes de 30 días a 15 años de edad, que reconsultaron en el SEP del 01/01 al 31/03 de los años 2010 y 2014. Resultados: Las consultas que se atendieron en el periodo de estudio fueron: 14.003 en 2010 y 13.011 en 2014. Los diagnósticos más frecuentes fueron: infección respiratoria aguda alta (19.2%), fiebre (13%) y diarrea/ gastroenteritis (13.3%). Hubo 7.11% (n=966) de RC en el 2010 y 12.21% (n=1589) en el 2014. La mediana de edad de los pacientes con RC fue 24 meses en ambos años. Los motivos de RC más frecuentes fueron: persistencia de los síntomas (41% y 53.67%); progresión de la enfermedad (25% y 11.43%); otros diagnósticos nuevos (14.07% y 19.5%) y control (14% y 8.3%). Las RC de los domiciliados en San Isidro fueron 72.97% (n=694) en el 2010 y 69.32% (n=1071) en el 2014 del total de RC. La disminución de las RC observada al comparar ambos períodos es estadísticamente no significativa. (Pearson chi2 2.4506, p = 0.117). Conclusiones: 1) Los principales motivos de consulta fueron fiebre, diarrea y patología de la vía aérea superior. 2) Las RC dentro de las primeras 72 horas fueron 7% en 2010 y 12.9% en 2014. 3) La mayoría de los niños que concurren por RC lo hacen por la persistencia de síntomas de procesos que revisten poca gravedad. 4) Hubo una leve disminución, estadísticamente no significativa, de la RC de los pacientes domiciliados en San Isidro (AU)

Aims: 1) To analyze second consultations (SC) in patients within 72 hours of the first consultation at the Pediatric Emergency Department (PED) of a mother-and-child hospital in Greater Buenos Aires. 2) To compare SC between two time periods, after implementing a program of care and counter-referral. Material and Methods: Study design: a retrospective, observational, and cross-sectional study was conducted. Inclusion criteria: patients between 30 days of life and 15 years of age, who consulted at the PED between 01/01 and 31/03 from 2010 to 2014. Results: Patients seen during the study period were: 14,003 in 2010 and 13,011 in 2014. The most common diagnoses were: Acute upper respiratory infection (19.2%), fever (13%) and diarrhea/gastroenteritis (13.3%). SC were 7.11% (n=966) in 2010 and 12.21% (n=1589) in 2014. Median age of patients with a SC was 24 months in both years. Most common reasons for SC were persistent symptoms (41% and 53.67%); disease progression (25% and 11.43%); new diagnosis (14.07% and 19.5%) and control (14% and 8.3%). Home visits for SC in San Isidro were 72.97% (n=694) in 2010 and 69.32% (n=1071) in 2014 of all SC. The decrease in SC when comparing both periods was not statistically significant (Pearson chi2 2.4506, p = 0.117). Conclusions: 1) The main reasons for consult were fever, diarrhea, and upper airway infections. 2) SC within 72 hours were 7% in 2010 and 12.9% in 2014. 3) The majority of childrenwho were seen in a SC had persistent symptoms that were not severe. 4) A slight decrease, that was not statistically significant, was observed in SC of patients seen in the area of San Isidro (AU)

Cytoplasmic interaction of the tumour suppressor protein hSNF5 with dynamin-2 controls endocytosis.

Human SNF5 (hSNF5; INI1, SMARCB1 or BAF47) is a component of the human SWI/SNF chromatin remodelling complex and a tumour suppressor mutated in rhabdoid tumours. It also associates with the integrase of the human immunodeficiency virus (HIV)-1. We show by fluorescence loss in photobleaching that hSNF5 is constantly shuttling between the nucleus and the cytoplasm, raising the question of what the role of hSNF5 is in the cytoplasm. Here, we demonstrate that hSNF5 directly interacts with the GTPase dynamin-2 (DNM2) in the cytoplasm. DNM2 is a large GTPase involved in endocytosis and vesicle dynamics, which has been related to HIV-1 internalization. We show that hSNF5 colocalizes with DNM2 in endocytic vesicles. Depletion of hSNF5, but not of other components of the SWI/SNF complex, destabilizes DNM2 and impairs DNM2-dependent endocytosis. Furthermore, we show that hSNF5 inhibits assembly-stimulated DNM2 GTPase activity but not basal GTPase activity in vitro. Altogether, these results indicate that hSNF5 affects both the stability and the activity of DNM2, uncovering an unexpected role of hSNF5 in modulating endocytosis, and open new perspectives in understanding the role of hSNF5 in tumour genesis.

The chromatin remodeling factor CHD8 interacts with elongating RNA polymerase II and controls expression of the cyclin E2 gene.

CHD8 is a chromatin remodeling ATPase of the SNF2 family. We found that depletion of CHD8 impairs cell proliferation. In order to identify CHD8 target genes, we performed a transcriptomic analysis of CHD8-depleted cells, finding out that CHD8 controls the expression of cyclin E2 (CCNE2) and thymidylate synthetase (TYMS), two genes expressed in the G1/S transition of the cell cycle. CHD8 was also able to co-activate the CCNE2 promoter in transient transfection experiments. Chromatin immunoprecipitation experiments demonstrated that CHD8 binds directly to the 5' region of both CCNE2 and TYMS genes. Interestingly, both RNA polymerase II (RNAPII) and CHD8 bind constitutively to the 5' promoter-proximal region of CCNE2, regardless of the cell-cycle phase and, therefore, of the expression of CCNE2. The tandem chromodomains of CHD8 bind in vitro specifically to histone H3 di-methylated at lysine 4. However, CHD8 depletion does not affect the methylation levels of this residue. We also show that CHD8 associates with the elongating form of RNAPII, which is phosphorylated in its carboxy-terminal domain (CTD). Furthermore, CHD8-depleted cells are hypersensitive to drugs that inhibit RNAPII phosphorylation at serine 2, suggesting that CHD8 is required for an early step of the RNAPII transcription cycle.

Comportamiento de los parámetros glucémicosdurante el primer trimestre del embarazo en mujeres con diabetes mellitus tipo 1 / The behaviour of glycemic parameters during the first trimester of pregnancy in women with type 1 diabetes mellitus

Introducción: Se sabe que las mujeres con diabetes mellitus tipo 1tienen una frecuencia aumentada de hipoglucemia durante el primertrimestre del embarazo. Pacientes y métodos: Realizamos un estudiolongitudinal, retrospectivo, en 64 mujeres embarazadas conDM1. Se delimitaron 4 periodos: preconcepcional (28 días previosa la concepción), primer, segundo y tercer mes de gestación. Secompararon distintos parámetros glucémicos entre estos 4 periodos.Resultados: La glucemia media fue similar en el periodo preconcepcionaly en el primer mes. A continuación, disminuyó progresivamentedurante el segundo y el tercer mes. La hemoglobina glucosiladatuvo un comportamiento paralelo a éste. Tanto la hipoglucemiano grave como la hipoglucemia nocturna aumentaron durante el primertrimestre. No hubo cambios significativos en el número diario demediciones de la glucemia ni en la dosis media diaria de insulina.Conclusión: Junto con un mejor control glucémico, se corroborauna tendencia hacia la hipoglucemia, tanto diurna como nocturna,durante el primer trimestre del embarazo en mujeres con DM1, queno parece atribuible a cambios en la dosis de insulina (AU)

Introduction: It is known that there is a higher frequency of hypoglycemiaduring the fi rst trimester of pregnancy in women with type 1diabetes mellitus. Patients and methods: We undertook a retrospectivelongitudinal study of 64 pregnant women with type 1 diabetesmellitus. Four periods were defi ned: preconception (28 days prior toconception), fi rst, second and third month of pregnancy. Different glycemicparameters between those 4 periods were compared. Results:Mean blood glucose level was similar in the preconception period andin the fi rst month. Afterwards, it progressively decreased during thesecond and third months. HbA1c showed a similar tendency as the meanblood glucose. Both non severe hypoglycemia as well as nocturnalhypoglycemia increased throughout the fi rst trimester. There were nosignifi cant changes either in the number of daily blood glucose measurementsor in the average daily dose of insulin. Conclusion: Togetherwith an improved glycemic control, a tendency towards hypoglycemia–daytime as well as nocturnal– during the fi rst trimester ofpregnancy in women with type 1 diabetes is corroborated, and doesnot seem to be attributable to the dose of insulin(AU)

Young man with asthma and infertility.

Asthma and male infertility are common diseases that can occur in the same patient. In some cases they could have patho-physiological changes common to both diseases. Our patient was seen as a result of having an irritating cough with wheezing, mainly at night, for more than a month. Asthma was diagnosed, and he responded favourably to the treatment given. Upon being informed that he had been examined for infertility for 5 years, alpha-1 antitrypsin (AAT) levels were requested. These confirmed that he had a phenotype SZ AAT deficiency. These findings, together with some evidence published recently, suggested that there is a need to rule out AAT deficiency in males with asthma and infertility.

[Liver transplantation. Nursing in hospital-based home care]. / Trasplante hepático. Enfermería en la hospitalización a domicilio.

At home hospital treatment nursing by the "Hospital de Cruces" staff provides integrated treatment for liver transplant patients in their home environments, their own houses, with the assistance of full hospital care and resources. The number of patients attended to since the start of this program in February 1996 until November 2001 has reached 164 patients in 205 episodes. The continuity of at home nursing treatment in a liver transplant patient's home is an effective tool which shortens the duration of a patient's hospital stay and the period of a patient's integration into his/her habitual environment with its beneficial consequences in aspects so important as the quality of their lives.

Trasplante hepático / Liver Transplant

La enfermería de hospitalización a domicilio del Hospital de Cruces está realizando cuidados de carácter integral a enfermos trasplantados hepáticos en el entorno familiar, el domicilio, con la ayuda de todos los recursos hospitalarios. El número de enfermos atendidos desde el comienzo del programa, en febrero de 1996, hasta noviembre de 2001, ha ascendido a 164 en 205 episodios. La continuidad de cuidados de enfermería en el domicilio del enfermo trasplantado hepático es una herramienta eficaz para acortar el tiempo de estancia hospitalaria y la integración en su medio habitual con las consecuencias beneficiosas en aspectos tan importantes como la calidad de vida (AU)

Control of the migratory pathway of facial branchiomotor neurones.

Facial branchiomotor (fbm) neurones undergo a complex migration in the segmented mouse hindbrain. They are born in the basal plate of rhombomere (r) 4, migrate caudally through r5, and then dorsally and radially in r6. To study how migrating cells adapt to their changing environment and control their pathway, we have analysed this stereotyped migration in wild-type and mutant backgrounds. We show that during their migration, fbm neurones regulate the expression of genes encoding the cell membrane proteins TAG-1, Ret and cadherin 8. Specific combinations of these markers are associated with each migratory phase in r4, r5 and r6. In Krox20 and kreisler mutant mouse embryos, both of which lack r5, fbm neurones migrate dorsally into the anteriorly positioned r6 and adopt an r6-specific expression pattern. In embryos deficient for Ebf1, a gene normally expressed in fbm neurones, part of the fbm neurones migrate dorsally within r5. Accordingly, fbm neurones prematurely express a combination of markers characteristic of an r6 location. These data suggest that fbm neurones adapt to their changing environment by switching on and off specific genes, and that Ebf1 is involved in the control of these responses. In addition, they establish a close correlation between the expression pattern of fbm neurones and their migratory behaviour, suggesting that modifications in gene expression participate in the selection of the local migratory pathway.

NtcA represses transcription of gifA and gifB, genes that encode inhibitors of glutamine synthetase type I from Synechocystis sp. PCC 6803.

Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) activity is controlled by direct interaction with two inactivating factors (IF7 and IF17). IF7 and IF17 are homologous polypeptides encoded by the gifA and gifB genes respectively. We investigated the transcriptional regulation of these genes. Expression of both genes is maximum in the presence of ammonium, when GS is inactivated. Nitrogen starvation attenuates the ammonium-mediated induction of gifA and gifB as well as the ammonium-mediated inactivation of GS. Putative binding sites for the transcription factor NtcA were identified at -7.5 and -30.5 bp upstream of gifB and gifA transcription start points respectively. Synechocystis NtcA protein binding to both promoters was demonstrated by gel electrophoresis mobility shift assays. Constitutive high expression levels of both genes were found in a Synechocystis NtcA non-segregated mutant (SNC1), which showed a fourfold reduction in the ntcA expression. These experiments indicate a repressive role for NtcA on the transcription of gifA and gifB genes. Our results demonstrate that NtcA plays a central role in GS regulation in cyanobacteria, stimulating transcription of the glnA gene (GS structural gene) and suppressing transcription of the GS inactivating factor genes gifA and gifB.

A gene cluster involved in metal homeostasis in the cyanobacterium Synechocystis sp. strain PCC 6803.

A gene cluster composed of nine open reading frames (ORFs) involved in Ni(2+), Co(2+), and Zn(2+) sensing and tolerance in the cyanobacterium Synechocystis sp. strain PCC 6803 has been identified. The cluster includes an Ni(2+) response operon and a Co(2+) response system, as well as a Zn(2+) response system previously described. Expression of the Ni(2+) response operon (nrs) was induced in the presence of Ni(2+) and Co(2+). Reduced Ni(2+) tolerance was observed following disruption of two ORFs of the operon (nrsA and nrsD). We also show that the nrsD gene encodes a putative Ni(2+) permease whose carboxy-terminal region is a metal binding domain. The Co(2+) response system is composed of two divergently transcribed genes, corR and corT, mutants of which showed decreased Co(2+) tolerance. Additionally, corR mutants showed an absence of Co(2+)-dependent induction of corT, indicating that CorR is a transcriptional activator of corT. To our knowledge, CorR is the first Co(2+)-sensing transcription factor described. Our data suggest that this region of the Synechocystis sp. strain PCC 6803 genome is involved in sensing and homeostasis of Ni(2+), Co(2+), and Zn(2+).

Light-dependent regulation of cyanobacterial phytochrome expression.

A histidine kinase protein (Cph1) with sequence homology and spectral characteristics very similar to those of the plant phytochrome has been recently identified in the cyanobacterium Synechocystis sp. strain PCC 6803. Cph1 together with Rcp1 (a protein homologue to the response regulator CheY) forms a light-regulated two-component system whose function is presently unknown. Levels of cph1 rcp1 mRNA increase in the dark and decrease upon reillumination. A dark-mediated increase in cph1 rcp1 mRNA levels was inhibited by the presence of glucose, but not by inhibition of the photosynthetic electron flow. The half-life of cph1 rcp1 transcript in the light was about fourfold shorter than in the dark, indicating that control of cph1 rcp1 transcript stability is one of the mechanisms by which light regulates expression of the cyanobacterial phytochrome. After 15 min of darkness, 3-min pulses of red, blue, green, and far-red light were equally efficient in decreasing the cph1 rcp1 mRNA levels. Red light downregulation was not reversed by far-red light, suggesting that cph1 rcp1 mRNA levels are not controlled by a phytochrome-like photoreceptor. Furthermore, a Synechocystis strain containing an H538R Cph1 point mutation, unable to phosphorylate Rcp1, shows normal light-dark regulation of the cph1 rcp1 transcript levels. Our data suggest a role of cyanobacterial phytochrome in the control of processes required for adaptation in light-dark and dark-light transitions.

Glutamine synthetase inactivation by protein-protein interaction.

Glutamine synthetase (GS; EC 6.3.1.2) is the pivotal enzyme of nitrogen metabolism in prokaryotes. Control of bacterial GS activity by reversible adenylylation has provided one of the classical paradigms of signal transduction by cyclic cascades. By contrast, in the present work we show that cyanobacterial GS is controlled by a different mechanism that involves the interaction of two inhibitory polypeptides with the enzyme. Both inactivating factors (IFs), named IF7 and IF17, are required in vivo for complete GS inactivation. Inactive GS-IF7 and GS-IF17 complexes were reconstituted in vitro by using Escherichia coli-expressed purified proteins. Our data suggest that control of GS activity is exerted by regulating the levels of IF7 and IF17.

Nitrogen control of the glnN gene that codes for GS type III, the only glutamine synthetase in the cyanobacterium Pseudanabaena sp. PCC 6903.

Pseudanabaena sp. strain PCC 6903 is the first cyanobacteria lacking the typical prokaryotic glutamine synthetase type I encoded by the glnA gene. The glnN gene product, glutamine synthetase type III, is the only glutamine synthetase activity present in this cyanobacterium. Analysis of glnN expression clearly indicated a nitrogen-dependent regulation. Pseudanabaena glnN gene expression and GSIII activity were upregulated under nitrogen starvation or using nitrate as a nitrogen source, while low levels of transcript and activity were found in ammonium-containing medium. Primer extension analysis showed that the glnN gene promoter structure resembled that of the NtcA-related promoters. Mobility shift assays demonstrated that Synechocystis sp. PCC 6803 NtcA protein, expressed and purified from Escherichia coli, bound to the promoter of the Pseudanabaena 6903 glnN gene. The NtcA control of the glnN gene in this cyanobacterium suggested that, in the absence of a glnA gene, NtcA took control of the only glutamine synthetase gene in a fashion similar to the way the glnA gene is governed in those cyanobacteria harbouring a glnA gene.

One-stage immediate reconstruction of partial auricular amputation by dog bite.

A case of one-stage immediate reconstruction of partial auricular amputation secondary to dog bite is presented. Primary reconstruction was performed using the cartilage of the avulsed portion of the ear nourished by means of a pedicled temporofascial flap and split-thickness skin graft. The perioperative antimicrobial protocol is also detailed.

Purification and characterization of a new type of glutamine synthetase from cyanobacteria.

The cyanobacterium Synechocystis sp. PCC 6803 contains two genes encoding two different types of glutamine synthetases (GS), glnA and glnN. The first codes for a typical prokaryotic GS type I and the second one codes for a GS type III, different in amino acid sequence to the prokaryotic GSI and the eukaryotic GSII. The glnN gene has been expressed in Escherichia coli and the corresponding protein purified almost to homogeneity (92%). The native enzyme (500 kDa) was composed of six identical subunits with an apparent molecular mass of 80 kDa. The protein was strongly stabilized in the presence of Mn2+ but not with other divalent cations. Biosynthetic activity of GSIII required the same substrates and cofactors as GSI and GSII enzymes. Apparent Km values for ATP, glutamate and ammonium were 0.43 mM, 0.9 mM and 0.19 mM, respectively. The enzyme was weakly inhibited by several amino acids and strongly inhibited by ADP. Synechocystis GSIII was also inhibited by L-methionine sulfoximine and DL-phosphinotricin, two transition-state analogs of the GS reaction mechanism. GSIII has also been purified from nitrogen-starved Synechocystis 6803 glnA mutant cells, demonstrating that the GS activity, strongly induced under nitrogen starvation in these cells, corresponds to the glnN gene product. In addition, a Synechocystis 6803 glnN mutant lacks the corresponding 80-kDa protein (GSIII). Polyclonal antibodies specific for GSIII cross-react with GSIII from other cyanobacteria. In all the strains analysed, levels of GSIII protein increased under nitrogen deficiency. These data suggest that GSIII is specifically required under conditions of nitrogen starvation.