Heteroresistance to Fosfomycin Is Predominant in Streptococcus pneumoniae and Depends on the murA1 Gene

Fosfomycin targets the first step of peptidoglycan biosynthesis in Streptococcus pneumoniae catalyzed by UDP-N-acetylglucosamine enolpyruvyltransferase (MurA1). We investigated whether heteroresistance to fosfomycin occurs in S. pneumoniae. We found that of 11 strains tested, all but 1 (Hungary(19A)) displayed heteroresistance and that deletion of murA1 abolished heteroresistance. Hungary(19A) differs from the other strains by a single amino acid substitution in MurA1 (Ala364Thr). To test whether this substitution is responsible for the lack of heteroresistance, it was introduced into strain D39. The heteroresistance phenotype of strain D39 was not changed. Furthermore, no relevant structural differences between the MurA1 crystal structures of heteroresistant strain D39 and nonheteroresistant strain Hungary(19A) were found. Our results reveal that heteroresistance to fosfomycin is the predominant phenotype of S. pneumoniae and that MurA1 is required for heteroresistance to fosfomycin but is not the only factor involved. The findings provide a caveat for any future use of fosfomycin in the treatment of pneumococcal infections.

Ontogeny of mRNA abundance of nuclear receptors and nuclear receptor target genes in young cattle

After birth the development of appropriate detoxification mechanisms is important. Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-alpha (PPARalpha), retinoid receptors (RAR, RXR), and NR target genes are involved in the detoxification of exogenous and endogenous substances. We quantified abundances of hepatic mRNA of NR and several NR target genes (cytochromes, CYP; cytochrome P450 reductase, CPR; UDP-glucuronosyl transferase, UDP) in calves at different ages. Gene expression was quantified by real-time RT-PCR. Abundance of mRNA of CAR and PXR increased from low levels at birth in pre-term calves (P0) and full-term calves (F0) to higher levels in 5-day-old calves (F5) and in 159-day-old veal calves (F159), whereas mRNA levels of PPARalpha did not exhibit significant ontogenetic changes. RARbeta mRNA levels were higher in F5 and F159 than in F0, whereas no age differences were observed for RARalpha levels. Levels of RXRalpha and RXRbeta mRNA were lower in F5 than in P0 and F0. Abundance of CYP2C8 and CYP3A4 increased from low levels in P0 and F0 to higher levels in F5 and to highest levels in F159. Abundance of CPR was transiently decreased in F0 and F5 calves. Levels of UGT1A1 mRNA increased from low levels in P0 and F0 to maximal level in F5 and F159. In conclusion, mRNA levels of NR and NR target genes exhibited ontogenetic changes that are likely of importance for handling of xeno- and endobiotics with increasing age.

Effects of somatosensory stimulation on use-dependent plasticity in chronic stroke

BACKGROUND AND PURPOSE: There is a need to develop strategies to enhance the beneficial effects of motor training, including use-dependent plasticity (UDP), in neurorehabilitation. Peripheral nerve stimulation (PNS) modulates motor cortical excitability in healthy humans and could influence training effects in stroke patients. METHODS: We compared the ability of PNS applied to the (1) arm, (2) leg, and (3) idle time to influence training effects in the paretic hand in 7 chronic stroke patients. The end point measure was the magnitude of UDP. RESULTS: UDP was more prominent with arm stimulation (increased by 22.8%) than with idle time (by 2.9%) or leg stimulation (by 6.4%). CONCLUSIONS: PNS applied to the paretic limb paired with motor training enhances training effects on cortical plasticity in stroke patients.

Liver repopulation with bone marrow derived cells improves the metabolic disorder in the Gunn rat

BACKGROUND: Reversible ischaemia/reperfusion (I/R) liver injury has been used to induce engraftment and hepatic parenchymal differentiation of exogenous beta2-microglubulin(-)/Thy1(+) bone marrow derived cells. AIM: To test the ability of this method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, to correct the metabolic disorder in a rat model of congenital hyperbilirubinaemia. METHODS AND RESULTS: Analysis by confocal laser microscopy of fluorescence labelled cells and by immunohistochemistry for beta2-microglubulin, 72 hours after intraportal delivery, showed engraftment of infused cells in liver parenchyma of rats with I/R, but not in control animals with non-injured liver. Transplantation of bone marrow derived cells obtained from GFP-transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP positive hepatocytes in I/R liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP negative bone marrow derived cells into GFP positive transgenic rats resulted in the appearance of GFP negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow derived cells into hyperbilirubinaemic Gunn rats with deficient bilirubin conjugation after I/R damage resulted in 30% decrease in serum bilirubin, the appearance of bilirubin conjugates in bile, and the expression of normal UDP-glucuronyltransferase enzyme evaluated by polymerase chain reaction. CONCLUSIONS: I/R injury induced hepatic parenchymal engraftment and differentiation into hepatocyte-like cells of bone marrow derived cells. Transplantation of bone marrow derived cells from non-affected animals resulted in the partial correction of hyperbilirubinaemia in the Gunn rat.

Gilbert syndrome and the development of antiretroviral therapy-associated hyperbilirubinemia

BACKGROUND: Unconjugated hyperbilirubinemia results from Gilbert syndrome and from antiretroviral therapy (ART) containing protease inhibitors. An understanding of the interaction between genetic predisposition and ART may help to identify individuals at highest risk for developing jaundice. METHODS: We quantified the contribution of UGT1A1*28 and ART to hyperbilirubinemia by longitudinally modeling 1386 total bilirubin levels in 96 human immunodeficiency virus (HIV)-infected individuals during a median of 6 years. RESULTS: The estimated average bilirubin level was 8.8 micromol/L (0.51 mg/dL). Atazanavir increased bilirubin levels by 15 mu mol/L (0.87 mg/dL), and indinavir increased bilirubin levels by 8 micromol/L (0.46 mg/dL). Ritonavir, lopinavir, saquinavir, and nelfinavir had no or minimal effect on bilirubin levels. Homozygous UGT1A1*28 increased bilirubin levels by 5.2 micromol/L (0.3 mg/dL). As a consequence, 67% of individuals homozygous for UGT1A1*28 and receiving atazanavir or indinavir had > or =2 episodes of hyperbilirubinemia in the jaundice range (>43 micromol/L [>2.5 mg/dL]), versus 7% of those with the common allele and not receiving either of those protease inhibitors (P<.001). Efavirenz resulted in decreased bilirubin levels, which is consistent with the induction of UDP-glucuronosyltransferase 1A1. CONCLUSIONS: Genotyping for UGT1A1*28 before initiation of ART would identify HIV-infected individuals at risk for hyperbilirubinemia and decrease episodes of jaundice.

Hop-to-Hop Reliability in IP-Based Wireless Sensor Networks - A Cross-Layer Approach

To interconnect a wireless sensor network (WSN) to the Internet, we propose to use TCP/IP as the standard protocol for all network entities. We present a cross layer designed communication architecture, which contains a MAC protocol, IP, a new protocol called Hop-to-Hop Reliability (H2HR) protocol, and the TCP Support for Sensor Nodes (TSS) protocol. The MAC protocol implements the MAC layer of beacon-less personal area networks (PANs) as defined in IEEE 802.15.4. H2HR implements hop-to-hop reliability mechanisms. Two acknowledgment mechanisms, explicit and implicit ACK are supported. TSS optimizes using TCP in WSNs by implementing local retransmission of TCP data packets, local TCP ACK regeneration, aggressive TCP ACK recovery, congestion and flow control algorithms. We show that H2HR increases the performance of UDP, TCP, and RMST in WSNs significantly. The throughput is increased and the packet loss ratio is decreased. As a result, WSNs can be operated and managed using TCP/IP.

Activity of the glycosylating enzyme, core 2 GlcNAc (beta1,6) transferase, is higher in polymorphonuclear leukocytes from diabetic patients compared with age-matched control subjects: relevance to capillary occlusion in diabetic retinopathy

The exact mechanism for capillary occlusion in diabetic retinopathy is still unclear, but increased leukocyte-endothelial cell adhesion has been implicated. We examined the possibility that posttranslational modification of surface O-glycans by increased activity of core 2 transferase (UDP-Glc:Galbeta1-3GalNAcalphaRbeta-N-acetylglucoaminyltr ansferase) is responsible for increased adhesion of leukocytes to vascular endothelium in diabetes. The mean activity of core 2 transferase in polymorphonuclear leukocytes isolated from type 1 and type 2 diabetic patients was higher compared with age-matched control subjects (1,638 +/- 91 [n = 42] vs. 249 +/- 35 pmol x h(-1) x mg(-1) protein [n = 24], P = 0.00013; 1,459 +/- 194 [n = 58] vs. 334 +/- 86 [n = 11], P = 0.01). As a group, diabetic patients with retinopathy had significantly higher mean activity of core 2 transferase compared with individuals with no retinopathy. There was a significant association between enzyme activity and severity of retinopathy in type 1 and type 2 diabetic patients. There was a strong correlation between activity of core 2 transferase and extent of leukocyte adhesion to cultured retinal capillary endothelial cells for diabetic patients but not for age-matched control subjects. Results from transfection experiments using human myelocytic cell line (U937) demonstrated a direct relationship between increased activity of core 2 transferase and increased binding to cultured endothelial cells. There was no relationship between activity of core 2 transferase and HbA(1c) (P = 0.8314), serum advanced glycation end product levels (P = 0.4159), age of the patient (P = 0.7896), and duration of diabetes (P = 0.3307). On the basis that branched O-glycans formed by the action of core 2 transferase participate in leukocyte adhesion, the present data suggest the involvement of this enzyme in increased leukocyte-endothelial cell adhesion and the pathogenesis of capillary occlusion in diabetic retinopathy.

Enterprise Integration of Smart Objects using Semantic Service Descriptions

Integrating physical objects (smart objects) and enterprise IT systems is still a labor intensive, mainly manual task done by domain experts. On one hand, enterprise IT backend systems are based on service oriented architectures (SOA) and driven by business rule engines or business process execution engines. Smart objects on the other hand are often programmed at very low levels. In this paper we describe an approach that makes the integration of smart objects with such backends systems easier. We introduce semantic endpoint descriptions based on Linked USDL. Furthermore, we show how different communication patterns can be integrated into these endpoint descriptions. The strength of our endpoint descriptions is that they can be used to automatically create REST or SOAP endpoints for enterprise systems, even if which they are not able to talk to the smart objects directly. We evaluate our proposed solution with CoAP, UDP and 6LoWPAN, as we anticipate the industry converge towards these standards. Nonetheless, our approach also allows easy integration with backend systems, even if no standardized protocol is used.

Energy-Efficient Management Of Heterogeneous Wireless Sensor Networks

Various applications for the purposes of event detection, localization, and monitoring can benefit from the use of wireless sensor networks (WSNs). Wireless sensor networks are generally easy to deploy, with flexible topology and can support diversity of tasks thanks to the large variety of sensors that can be attached to the wireless sensor nodes. To guarantee the efficient operation of such a heterogeneous wireless sensor networks during its lifetime an appropriate management is necessary. Typically, there are three management tasks, namely monitoring, (re) configuration, and code updating. On the one hand, status information, such as battery state and node connectivity, of both the wireless sensor network and the sensor nodes has to be monitored. And on the other hand, sensor nodes have to be (re)configured, e.g., setting the sensing interval. Most importantly, new applications have to be deployed as well as bug fixes have to be applied during the network lifetime. All management tasks have to be performed in a reliable, time- and energy-efficient manner. The ability to disseminate data from one sender to multiple receivers in a reliable, time- and energy-efficient manner is critical for the execution of the management tasks, especially for code updating. Using multicast communication in wireless sensor networks is an efficient way to handle such traffic pattern. Due to the nature of code updates a multicast protocol has to support bulky traffic and endto-end reliability. Further, the limited resources of wireless sensor nodes demand an energy-efficient operation of the multicast protocol. Current data dissemination schemes do not fulfil all of the above requirements. In order to close the gap, we designed the Sensor Node Overlay Multicast (SNOMC) protocol such that to support a reliable, time-efficient and energy-efficient dissemination of data from one sender node to multiple receivers. In contrast to other multicast transport protocols, which do not support reliability mechanisms, SNOMC supports end-to-end reliability using a NACK-based reliability mechanism. The mechanism is simple and easy to implement and can significantly reduce the number of transmissions. It is complemented by a data acknowledgement after successful reception of all data fragments by the receiver nodes. In SNOMC three different caching strategies are integrated for an efficient handling of necessary retransmissions, namely, caching on each intermediate node, caching on branching nodes, or caching only on the sender node. Moreover, an option was included to pro-actively request missing fragments. SNOMC was evaluated both in the OMNeT++ simulator and in our in-house real-world testbed and compared to a number of common data dissemination protocols, such as Flooding, MPR, TinyCubus, PSFQ, and both UDP and TCP. The results showed that SNOMC outperforms the selected protocols in terms of transmission time, number of transmitted packets, and energy-consumption. Moreover, we showed that SNOMC performs well with different underlying MAC protocols, which support different levels of reliability and energy-efficiency. Thus, SNOMC can offer a robust, high-performing solution for the efficient distribution of code updates and management information in a wireless sensor network. To address the three management tasks, in this thesis we developed the Management Architecture for Wireless Sensor Networks (MARWIS). MARWIS is specifically designed for the management of heterogeneous wireless sensor networks. A distinguished feature of its design is the use of wireless mesh nodes as backbone, which enables diverse communication platforms and offloading functionality from the sensor nodes to the mesh nodes. This hierarchical architecture allows for efficient operation of the management tasks, due to the organisation of the sensor nodes into small sub-networks each managed by a mesh node. Furthermore, we developed a intuitive -based graphical user interface, which allows non-expert users to easily perform management tasks in the network. In contrast to other management frameworks, such as Mate, MANNA, TinyCubus, or code dissemination protocols, such as Impala, Trickle, and Deluge, MARWIS offers an integrated solution monitoring, configuration and code updating of sensor nodes. Integration of SNOMC into MARWIS further increases performance efficiency of the management tasks. To our knowledge, our approach is the first one, which offers a combination of a management architecture with an efficient overlay multicast transport protocol. This combination of SNOMC and MARWIS supports reliably, time- and energy-efficient operation of a heterogeneous wireless sensor network.

Deletion of exon 8 from the EXT1 gene causes multiple osteochondromas (MO) in a family with three affected members.

Multiple osteochondromas (also called hereditary multiple exostoses) is an autosomal dominant disorder characterized by multiple cartilaginous tumors, which are caused by mutations in the genes for exostosin-1 (EXT1) and exostosin-2 (EXT2). The goal of this study was to elucidate the genetic alterations in a family with three affected members. Isolation of RNA from the patients' blood followed by reverse transcription and PCR amplification of selected fragments showed that the three patients lack a specific region of 90 bp from their EXT1 mRNA. This region corresponds to the sequence of exon 8 from the EXT1 gene. No splice site mutation was found around exon 8. However, long-range PCR amplification of the region from intron 7 to intron 8 indicated that the three patients contain a deletion of 4318 bp, which includes exon 8 and part of the flanking introns. There is evidence that the deletion was caused by non-homologous end joining because the breakpoints are not located within a repetitive element, but contain multiple copies of the deletion hotspot sequence TGRRKM. Exon 8 encodes part of the active site of the EXT1 enzyme, including the DXD signature of all UDP-sugar glycosyltransferases. It is conceivable that the mutant protein exerts a dominant negative effect on the activity of the EXT glycosyltransferase since it might interact with normal copies of the enzyme to form an inactive hetero-oligomeric complex. We suggest that sequencing of RNA might be superior to exome sequencing to detect short deletions of a single exon.

Transmisión bipolar: la difusión de la arquitectura moderna latinoamericana en España (1949-1968)

En las décadas de 1950 y 1960, pese a las aparentes dificultades de comunicación con el exterior, la arquitectura española miró hacia fuera constantemente. La potencia de la arquitectura moderna latinoamericana, junto a las diferencias instrumentales y geográficas, la hacían difícilmente importable a un contexto materialmente escaso y excesivamente tradicional. Sin embargo, dicha producción interesó y se difundió notablemente durante ambas décadas. Este artículo pretende varias cosas a la vez. Por una parte, proporcionar una panorámica global sobre difusión de la arquitectura latinoamericana fuera de su continente. Para ello nos serviremos básicamente del que probablemente fue el medio más potente con el que contaron: las revistas especializadas de arquitectura. Pero también pretende ser una llamada de atención hacia las publicaciones periódicas españolas, mucho menos conocidas que otras europeas pero, sin embargo, repletas de buena arquitectura.

Desarrollo de una aplicación iOS para el manejo remoto de un sistema de control

En los países desarrollados el Internet de las Cosas (IoT) ya es una realidad. El mundo físico y el digital cada vez están más unidos, gracias a la reducción del tamaño, el descenso del coste de los sensores, la posibilidad de disponer de una conexión a Internet en todo momento y al desarrollo de las aplicaciones, que ponen en uso la gran cantidad de información generada por todos los objetos conectados. Los campos de aplicación del IoT son muy variados, lo que otorga grandes oportunidades a los fabricantes de cada uno de los diferentes sectores, y desafíos, en particular, a los desarrolladores de software de tecnologías móviles. Los Smartphone serán los ojos y los oídos de las aplicaciones y estarán comunicados con el resto de las cosas. Pero con la evolución de las aplicaciones y dispositivos ya no sólo se verá y escuchará la información enviada por los sensores conectados a internet, sino que además, también existirá una comunicación completa entre los dispositivos y el SmartPhone. Este proyecto tiene por objetivo la realización de una aplicación móvil, para el sistema operativo móvil iOS, que cubra la posibilidad de comunicarse, controlar e interaccionar con un sistema de control para aumentar así la calidad y el bienestar del usuario. Para ello, se parte de una situación determinada en la que ya existe un modelado de dispositivos que incorporan la tecnología necesaria para recibir órdenes y contestar, y de un servidor que dispone de una comunicación directa con estos dispositivos y que a su vez gestiona un sistema de licencias con el cual se controla qué usuario tiene acceso a qué dispositivo. El futuro de la aplicación pasa por la posibilidad de comunicarse con el dispositivo directamente mediante una red WiFi propia, generada por él mismo, o bien, mediante bluetooh, o llamada perdida si el dispositivo incorporara una tarjeta SIM. La comunicación del SmartPhone con el servidor será mediante protocolo UDP. Mientras que la comunicación directa entre el SmartPhone y el dispositivo sería mediante TCP, siendo similar a la que ya existe entre el servidor y el dispositivo. La aplicación incorporará tres grandes bloques a nivel de control que se desarrollan a lo largo del trabajo. Un bloque de comunicación, un bloque de protocolos de estado y un bloque de modelaje de los mensajes que la aplicación intercambia con el servidor. Para dotar de una mayor seguridad a la aplicación, se hará que los mensajes que se intercambian con el servidor vayan cifrados y firmados de forma digital, lo que permitirá al receptor determinar el origen del mensaje (autenticación de origen y no repudio), y confirmar que dicho mensaje no haya sufrido alteraciones desde que fue firmado (integridad y confidencialidad)

Clasificación de tráfico de Internet mediante análisis de datos

Poder clasificar de manera precisa la aplicación o programa del que provienen los flujos que conforman el tráfico de uso de Internet dentro de una red permite tanto a empresas como a organismos una útil herramienta de gestión de los recursos de sus redes, así como la posibilidad de establecer políticas de prohibición o priorización de tráfico específico. La proliferación de nuevas aplicaciones y de nuevas técnicas han dificultado el uso de valores conocidos (well-known) en puertos de aplicaciones proporcionados por la IANA (Internet Assigned Numbers Authority) para la detección de dichas aplicaciones. Las redes P2P (Peer to Peer), el uso de puertos no conocidos o aleatorios, y el enmascaramiento de tráfico de muchas aplicaciones en tráfico HTTP y HTTPS con el fin de atravesar firewalls y NATs (Network Address Translation), entre otros, crea la necesidad de nuevos métodos de detección de tráfico. El objetivo de este estudio es desarrollar una serie de prácticas que permitan realizar dicha tarea a través de técnicas que están más allá de la observación de puertos y otros valores conocidos. Existen una serie de metodologías como Deep Packet Inspection (DPI) que se basa en la búsqueda de firmas, signatures, en base a patrones creados por el contenido de los paquetes, incluido el payload, que caracterizan cada aplicación. Otras basadas en el aprendizaje automático de parámetros de los flujos, Machine Learning, que permite determinar mediante análisis estadísticos a qué aplicación pueden pertenecer dichos flujos y, por último, técnicas de carácter más heurístico basadas en la intuición o el conocimiento propio sobre tráfico de red. En concreto, se propone el uso de alguna de las técnicas anteriormente comentadas en conjunto con técnicas de minería de datos como son el Análisis de Componentes Principales (PCA por sus siglas en inglés) y Clustering de estadísticos extraídos de los flujos procedentes de ficheros de tráfico de red. Esto implicará la configuración de diversos parámetros que precisarán de un proceso iterativo de prueba y error que permita dar con una clasificación del tráfico fiable. El resultado ideal sería aquel en el que se pudiera identificar cada aplicación presente en el tráfico en un clúster distinto, o en clusters que agrupen grupos de aplicaciones de similar naturaleza. Para ello, se crearán capturas de tráfico dentro de un entorno controlado e identificando cada tráfico con su aplicación correspondiente, a continuación se extraerán los flujos de dichas capturas. Tras esto, parámetros determinados de los paquetes pertenecientes a dichos flujos serán obtenidos, como por ejemplo la fecha y hora de llagada o la longitud en octetos del paquete IP. Estos parámetros serán cargados en una base de datos MySQL y serán usados para obtener estadísticos que ayuden, en un siguiente paso, a realizar una clasificación de los flujos mediante minería de datos. Concretamente, se usarán las técnicas de PCA y clustering haciendo uso del software RapidMiner. Por último, los resultados obtenidos serán plasmados en una matriz de confusión que nos permitirá que sean valorados correctamente. ABSTRACT. Being able to classify the applications that generate the traffic flows in an Internet network allows companies and organisms to implement efficient resource management policies such as prohibition of specific applications or prioritization of certain application traffic, looking for an optimization of the available bandwidth. The proliferation of new applications and new technics in the last years has made it more difficult to use well-known values assigned by the IANA (Internet Assigned Numbers Authority), like UDP and TCP ports, to identify the traffic. Also, P2P networks and data encapsulation over HTTP and HTTPS traffic has increased the necessity to improve these traffic analysis technics. The aim of this project is to develop a number of techniques that make us able to classify the traffic with more than the simple observation of the well-known ports. There are some proposals that have been created to cover this necessity; Deep Packet Inspection (DPI) tries to find signatures in the packets reading the information contained in them, the payload, looking for patterns that can be used to characterize the applications to which that traffic belongs; Machine Learning procedures work with statistical analysis of the flows, trying to generate an automatic process that learns from those statistical parameters and calculate the likelihood of a flow pertaining to a certain application; Heuristic Techniques, finally, are based in the intuition or the knowledge of the researcher himself about the traffic being analyzed that can help him to characterize the traffic. Specifically, the use of some of the techniques previously mentioned in combination with data mining technics such as Principal Component Analysis (PCA) and Clustering (grouping) of the flows extracted from network traffic captures are proposed. An iterative process based in success and failure will be needed to configure these data mining techniques looking for a reliable traffic classification. The perfect result would be the one in which the traffic flows of each application is grouped correctly in each cluster or in clusters that contain group of applications of similar nature. To do this, network traffic captures will be created in a controlled environment in which every capture is classified and known to pertain to a specific application. Then, for each capture, all the flows will be extracted. These flows will be used to extract from them information such as date and arrival time or the IP length of the packets inside them. This information will be then loaded to a MySQL database where all the packets defining a flow will be classified and also, each flow will be assigned to its specific application. All the information obtained from the packets will be used to generate statistical parameters in order to describe each flow in the best possible way. After that, data mining techniques previously mentioned (PCA and Clustering) will be used on these parameters making use of the software RapidMiner. Finally, the results obtained from the data mining will be compared with the real classification of the flows that can be obtained from the database. A Confusion Matrix will be used for the comparison, letting us measure the veracity of the developed classification process.

Bacterial resistance to vancomycin: Overproduction, purification, and characterization of VanC2 from Enterococcus casseliflavus as a d-Ala-d-Ser ligase

The VanC phenotype for clinical resistance of enterococci to vancomycin is exhibited by Enterococcus gallinarum and Enterococcus casseliflavus. Based on the detection of the cell precursor UDP-N-acetylmuramic acid pentapeptide intermediate terminating in d-Ala-d-Ser instead of d-Ala-d-Ala, it has been predicted that the VanC ligase would be a d-Ala-d-Ser rather than a d-Ala-d-Ala ligase. Overproduction of the E. casseliflavus ATCC 25788 vanC2 gene in Escherichia coli and its purification to homogeneity allowed demonstration of ATP-dependent d-Ala-d-Ser ligase activity. The kcat/Km2 (Km2 = Km for d-Ser or C-terminal d-Ala) ratio for d-Ala-d-Ser/d-Ala-d-Ala dipeptide formation is 270/0.69 for a 400-fold selection against d-Ala in the C-terminal position. VanC2 also has substantial d-Ala-d-Asn ligase activity (kcat/Km2 = 74 mM−1min−1).

A reversibly glycosylated polypeptide (RGP1) possibly involved in plant cell wall synthesis: Purification, gene cloning, and trans-Golgi localization

We purified from pea (Pisum sativum) tissue an ≈40 kDa reversibly glycosylated polypeptide (RGP1) that can be glycosylated by UDP-Glc, UDP-Xyl, or UDP-Gal, and isolated a cDNA encoding it, apparently derived from a single-copy gene (Rgp1). Its predicted translation product has 364 aminoacyl residues and molecular mass of 41.5 kDa. RGP1 appears to be a membrane-peripheral protein. Immunogold labeling localizes it specifically to trans-Golgi dictyosomal cisternae. Along with other evidence, this suggests that RGP1 is involved in synthesis of xyloglucan and possibly other hemicelluloses. Corn (Zea mays) contains a biochemically similar and structurally homologous RGP1, which has been thought (it now seems mistakenly) to function in starch synthesis. The expressed sequence database also reveals close homologs of pea Rgp1 in Arabidopsis and rice (Oryza sativa). Rice possesses, in addition, a distinct but homologous sequence (Rgp2). RGP1 provides a polypeptide marker for Golgi membranes that should be useful in plant membrane studies.