Synchrotron FTIR and Raman spectroscopy provide unique spectral fingerprints for Arabidopsis floral stem vascular tissues.

Cell walls are highly complex structures that are modified during plant growth and development. For example, the development of phloem and xylem vascular cells, which participate in the transport of sugars and water as well as providing support, can be influenced by cell-specific wall composition. Here, we used synchrotron radiation-based Fourier-transform infrared (SR-FTIR) and Raman spectroscopy to analyse the cell wall composition of floral stem vascular tissues of wild-type Arabidopsis and the double-mutant sweet11-1 sweet12-1, which has impaired sugar transport. The SR-FTIR spectra showed that in addition to modified xylem cell wall composition, phloem cell walls in the double-mutant line were characterized by modified hemicellulose composition. Combining Raman spectroscopy with a classification and regression tree (CART) method identified combinations of Raman shifts that could distinguish xylem vessels and fibers. In addition, the disruption of the SWEET11 and SWEET12 genes impacted on xylem wall composition in a cell-specific manner, with changes in hemicelluloses and cellulose observed at the xylem vessel interface. These results suggest that the facilitated transport of sugars by transporters that exist between vascular parenchyma cells and conducting cells is important in ensuring correct phloem and xylem cell wall composition.

Combined microscopy and molecular analyses show phloem occlusions and cell wall modifications in tomato leaves in response to 'Candidatus Phytoplasma solani'.

Callose deposition, phloem-protein conformational changes and cell wall thickening are calcium-mediated occlusions occurring in the plant sieve elements in response to different biotic and abiotic stresses. However, the significance of these structures in plant-phytoplasma interactions requires in-depth investigations. We adopted a novel integrated approach, based on the combined use of microscopic and molecular analyses, to investigate the structural modifications induced in tomato leaf tissues in presence of phytoplasmas, focusing on vascular bundles and on the occlusion structures. Phloem hyperplasia and string-like arrangement of xylem vessels were found in infected vascular tissue. The diverse occlusion structures were differentially modulated in the phloem in response to phytoplasma infection. Callose amount was higher in midribs from infected plants than in healthy ones. Callose was observed at sieve plates but not at pore-plasmodesma units. A putative callose synthase gene encoding a protein with high similarity to Arabidopsis CalS7, responsible for callose deposition at sieve plates, was upregulated in symptomatic leaves, indicating a modulation in the response to stolbur infection. P-proteins showed configuration changes in infected sieve elements, exhibiting condensation of the filaments. The transcripts for a putative P-protein 2 and a sieve element occlusion-related protein were localized in the phloem but only the first one was modulated in the infected tissues.

Phloem protein partners of Cucurbit aphid borne yellows virus: possible involvement of phloem proteins in virus transmission by aphids.

Poleroviruses are phytoviruses strictly transmitted by phloem-feeding aphids in a circulative and nonpropagative mode. During ingestion, aphids sample virions in sieve tubes along with sap. Therefore, any sap protein bound to virions will be acquired by the insects and could potentially be involved in the transmission process. By developing in vitro virus-overlay assays on sap proteins collected from cucumber, we observed that approximately 20 proteins were able to bind to purified particles of Cucurbit aphid borne yellows virus (CABYV). Among them, eight proteins were identified by mass spectrometry. The role of two candidates belonging to the PP2-like family (predominant lectins found in cucurbit sap) in aphid transmission was further pursued by using purified orthologous PP2 proteins from Arabidopsis. Addition of these proteins to the virus suspension in the aphid artificial diet greatly increased virus transmission rate. This shift was correlated with an increase in the number of viral genomes in insect cells and with an increase of virion stability in vitro. Surprisingly, increase of the virus transmission rate was also monitored after addition of unrelated proteins in the aphid diet, suggesting that any soluble protein at sufficiently high concentration in the diet and acquired together with virions could stimulate virus transmission.

Hypothermic in situ perfusion of the porcine liver using Celsior or Ringer-lactate solution.

BACKGROUND: Hypothermic perfusion (HP) of the liver is applied during total vascular exclusion (TVE) to reduce ischemic injury during liver resection. No studies have been performed comparing different perfusion solutions for HP. The aim of this experimental study was to compare Ringer-lactate solution (RL) with Celsior solution (Cs) for HP in a pig model of 60-min TVE. METHOD: Twenty pigs underwent 60-min TVE of the liver. Groups were TVE without HP (no-HP, n = 9), TVE with HP using RL (n = 6), and TVE with HP using Cs (n = 5). Blood and liver tissue samples were taken before TVE and during 24-h reperfusion. RESULTS: In the no-HP group, plasma aspartate aminotransferase values were significantly increased during reperfusion (p < 0.05), while liver tissue pO(2) levels (p < 0.01) were decreased when compared to the HP groups. After 24-h reperfusion, bile production and liver tissue glutathione content were significantly higher (p < 0.05) in the Cs group (42.0 +/- 1.7 mL/h and 44.9 +/- 2.2 nmol/mg, respectively) as compared to the RL group (31.5 +/- 3.5 mL/h and 19.6 +/- 1.8 nmol/mg, respectively). CONCLUSION: The protective effect of HP during TVE was confirmed in this study. HP with Cs was more effective in reducing ischemic injury as compared to HP with RL.

Original article: New surgical approaches to the Klatskin tumour.

BACKGROUND: Surgical treatment of hilar cholangiocarcinoma (Klatskin tumours) is difficult because of its central location in the liver hilum. Recent developments in surgical techniques have improved the outcome after resection. AIM: To describe the surgical approaches currently applied in our centre and the impact of these strategies on outcome and criteria for resection. METHODS: From 1988 to 2003, 99 consecutive patients underwent resection for hilar cholangiocarcinoma. Patients were analysed for rate of R0 resections in relation with Bismuth classification. Morbidity, mortality and survival were assessed. RESULTS: The rate of hilar resections in combination with (extended) liver resections for type III and IV tumours increased from 24% to 95% in the last 5 years of the study period. Eight patients (8%) had Bismuth type IV tumours. Four of these patients underwent palliative local excisions of the hepatic duct confluence whereas the other four patients underwent hilar resection in combination with partial liver resection, resulting in microscopically radical resections. There was no mortality in this group. Overall postoperative morbidity and mortality were 68% and 10%, respectively. CONCLUSIONS: An aggressive surgical approach consisting of hilar resections combined with partial liver resections including segments 1 and 4, resulted in a higher rate of R0 resections. Even Bismuth type IV tumours may be resectable depending on the biliary anatomy of the hepatic duct confluence.

Machine perfusion preservation of the pig liver using a new preservation solution, polysol.

INTRODUCTION: The current gold standard for donor liver preservation is cold storage in a preservation solution (4 degrees C), such as Celsior or the University of Wisconsin solution (UW). Recent studies have suggested the benefits of machine perfusion (MP) over cold storage. To improve the results of MP, an enriched preservation solution (named Polysol) was developed, which in a rat liver preservation model proved to be superior to the UW-gluconate solution. The aim of this study was to assess Polysol in a pig liver preservation model. MATERIALS AND METHODS: Female pigs (35 to 40 kg) were used as liver donors. After heparinization, the liver was washed out using Ringer's lactate, followed by the preservation solution (4 degrees C). The liver was preserved for 24 hours by either cold storage using Celsior (n=5) or MP using Polysol (n=5). For analysis of liver damage and function, livers were reperfused for 60 minutes using oxygenated Krebs-Henseleit buffer. RESULTS: CS-Celsior caused significantly more damage compared with MP-Polysol (t=60, AST: 622+/-215 versus 222+/-55; ALT: 17+/-6 versus 5+/-1). Intravascular resistance during reperfusion was significantly higher after CS-Celsior compared with MP-Polysol (t=0, 0.20+/-0.01 and 0.11+/-0.02 mm Hg/mL/min, respectively). No differences were seen regarding ammonia clearance and urea production. In both groups, no bile was produced during reperfusion. CONCLUSIONS: In an ex vivo pig liver preservation model significantly less damage was observed after machine perfusion preservation using Polysol, in comparison to cold storage using Celsior.

Alkaline phosphatase reduces hepatic and pulmonary injury in liver ischaemia -- reperfusion combined with partial resection.

BACKGROUND: Lipopolysaccharides mediate inflammation in liver ischaemia-reperfusion (I/R) and partial liver resection (PHX). Bovine intestinal alkaline phosphatase (BIAP) detoxifies lipopolysaccharides by dephosphorylation and reduces inflammation in models of sepsis. This study examined the protective effects of BIAP administration in models of partial (70 per cent) liver I/R with or without partial resection of all non-ischaemic lobes during ischaemia (30 per cent). METHODS: Male Wistar rats were divided into six groups: I/R + BIAP, I/R + saline, I/R + PHX + BIAP and I/R + PHX + saline, PHX only or sham laparotomy only. A single dose of BIAP (0.5 units/g) or vehicle (saline) was administered 5 min before reperfusion. Inflammatory response, and hepatic and pulmonary injury were assessed during 24 h of reperfusion. RESULTS: I/R, with or without PHX, increased all markers of inflammation, and hepatic and pulmonary damage (P < 0.050 versus sham operation). I/R + PHX significantly increased release of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and hepatic neutrophil influx compared with I/R only (P < 0.050). BIAP treatment decreased hepatic wet/dry ratios, neutrophil influx and histopathological damage after I/R with or without PHX (P < 0.050), and also AST, ALT and interleukin (IL)-6 production after I/R + PHX (P < 0.050). BIAP treatment reduced the neutrophil influx after I/R, and pulmonary histopathological injury was decreased after I/R with or without PHX. CONCLUSION: BIAP attenuates hepatic and pulmonary injury after partial liver I/R and PHX.

Towards deciphering phloem: a transcriptome analysis of the phloem of Apium graveolens.

Events occurring in the phloem tissue are key to understanding a wide range of developmental and physiological processes in vascular plants. While a considerable amount of molecular information on phloem proteins has emerged in the past decade, a unified picture of the molecular mechanisms involved in phloem differentiation and function is still lacking. New models to increase our understanding of this complex tissue can be created by the development of global approaches such as genomic analysis. In order to obtain a comprehensive overview of the molecular biology of the phloem tissue, we developed a genomic approach using Apium graveolens as a model. cDNA libraries were constructed from mRNAs extracted from isolated phloem of petioles. Expression data obtained from the analysis of 989 expressed sequence tags (ESTs) and the transcript profile deduced from a cDNA macroarray of 1326 clones were combined to identify genes showing distinct expression patterns in the vascular tissues. Comparisons of expression profiles obtained from the phloem, xylem and storage parenchyma tissues uncovered tissue-specific differential expression patterns for given sets of genes. The major classes of mRNAs predominantly found in the phloem encode proteins related to phloem structure, metal homeostasis or distribution, stress responses and degradation or turnover of proteins. Of great interest for future studies are the genes we found to be specifically expressed in the phloem but for which the function is still unknown, and also those genes described in previous reports to be up or downregulated by specific interactions. From a broader prospective, our results also clearly demonstrate that cDNA macroarray technology can be used to identify the key genes involved in various physiological and developmental processes in the phloem.

In vitro function of porcine carotid arteries preserved in UW, HTK and Celsior solutions.

We compared the efficacy of histidine-tryptophan-ketoglutarate (HTK) and University of Wisconsin (UW) solution with Celsior solution using hypothermically-preserved porcine carotid arteries and studied the importance of different components of these solutions by preserving carotid arteries in modified HTK solutions. Excised carotid arteries were stored at 4 degrees C in 0.9% (w/v) NaCl, UW, HTK, Celsior, or a modified HTK solution for up to 14 days. Preservation-induced changes in smooth muscle cell and endothelial cell function were determined using an organ bath for isometric tension recording. Short-term preservation (1-3 days) in UW, HTK and Celsior did not significantly alter contractile and relaxation responses of arterial segments when compared to freshly-excised segments, but significantly impaired these responses in arterial segments stored in 0.9% (w/v) NaCl solution. Long-term hypothermic preservation of arterial segments (7 and 14 days) in 0.9% (w/v) NaCl and HTK solution almost completely abolished all responses, but only slightly reduced the responses of arterial segments stored in UW solution. Intermediate results were obtained for Celsior. Modifying HTK by replacement of chloride for sulfate and phosphate resulted in improved contractile and relaxation responses after long-term preservation. With respect to smooth muscle and endothelial function, UW is superior to HTK and Celsior and the absence of chloride or presence of sulfate and phosphate plays a relevant role in this in vitro model of hypothermic preservation of porcine carotid arteries.

Plasmodesmata and plant cytoskeleton.

Plant cell-to-cell communication is achieved by membranous conduits called plasmodesmata, which bridge the cytoplasm of neighboring cells. A growing body of immunolocalization data shows an association of the cytoskeleton machinery with plasmodesmata. The role of the cytoskeleton in the plasmodesmata-mediated transport has been well documented for virus movement. Because viruses are known to exploit existing host pathways and because the cytoskeleton is involved in intracellular trafficking, the cytoskeleton is thought to drive and target macromolecules to plasmodesmata. It is this link between plasmodesmata and the cytoskeleton that will be described here.

Synthesis of (-)-strand RNA from the 3' untranslated region of plant viral genomes expressed in transgenic plants upon infection with related viruses.

When expressed in transgenic tobacco plants, transgene mRNA that includes the 3' untranslated region (3' UTR) of Lettuce mosaic virus served as template for synthesis of complementary (-)-strand RNA following an infection by Tobacco etch virus, Tobacco vein mottle virus or Pepper mottle virus, but not when infected with Cucumber mosaic virus. Deletion of the 3' UTR from the transgene abolished the synthesis of (-)-strand transcripts. Similar results were obtained in transgenic tobacco plants expressing mRNA that includes the RNA3 3' UTR of Cucumber mosaic virus when infected with Tomato aspermy virus. These results show that the viral RNA-dependent RNA polymerase of several potyviruses and Tomato aspermy virus have the ability to recognize heterologous 3' UTRs when included in transgene mRNAs, and to use them as transcription promoters.

Relationship of the pelargonium flower break carmovirus (PFBV) coat protein gene with that of other carmoviruses.

The 3'-terminal 1500 nucleotides of the genome of pelargonium flower break carmovirus (PFBV) were sequenced from RT-PCR amplification products. One large ORF was found, encoding a 345 amino acid protein of Mr 37 kDa, which corresponds to the coat protein, as confirmed by immunoprecipitation of products of in vitro transcription and translation. The sequence also included the putative promoter of the coat protein gene subgenomic RNA, as well as its 5' and 3' untranslated regions. The PFBV coat protein was more similar to that of saguaro cactus virus and carnation mottle virus than to that of other carmoviruses. Despite the lower level of similarity of CP gene sequences compared to the RNA dependent RNA polymerase (RdRp) gene sequences of small icosahedral viruses used in taxonomic studies, PFBV CP sequence comparisons and alignments confirmed that PFBV is related to carmoviruses, tombusviruses and a dianthovirus, as previously concluded from the analysis of a PFBV RdRp gene fragment.

Bromovirus RNA replication and transcription require compatibility between the polymerase- and helicase-like viral RNA synthesis proteins.

The positive-strand RNA bromoviruses encode two nonstructural proteins, 1a and 2a, involved in RNA-dependent RNA replication. These proteins have extensive sequence similarities with methyltransferase, helicase, and polymerase proteins of other plant and animal viruses. 1a and 2a can also form a complex in vitro. To explore whether 1a-2a interaction is required for RNA replication in vivo, we reassorted the 1a and 2a genes from two different bromoviruses, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV). 1a and 2a were expressed independently of viral replication by using RNA- or DNA-based transient expression, and their in vivo RNA replication activities were tested in protoplasts with BMV and CCMV RNA3 templates. RNA-based transient expression confirmed prior indications that bromovirus RNA replication is more sensitive to reductions in 1a expression than to reductions in 2a expression. DNA-based expression of the homologous combinations of 1a and 2a supported high levels of RNA synthesis, but both 1a-2a heterologous combinations exhibited RNA synthesis defects. The combination of CCMV 1a and BMV 2a did not support detectable synthesis of negative-strand, positive-strand, or subgenomic RNA. The converse combination of BMV 1a and CCMV 2a was preferentially defective in positive-strand and subgenomic RNA accumulation, showing that 1a-2a interaction is involved in these processes in ways distinct from negative-strand RNA synthesis, which was only slightly affected. These results indicate that at least some functions of 1a and 2a operate in a mutually dependent manner in vivo and that the mechanisms of positive- and negative-strand RNA synthesis are differentiated in part by features of such interactions.

Nucleotide sequence of the 3' terminal region of lettuce mosaic potyvirus RNA shows a Gln/Val dipeptide at the cleavage site between the polymerase and the coat protein.

DNA complementary to the 3' terminal 1651 nucleotides of the genome of the common strain of lettuce mosaic virus (LMV-O) has been cloned and sequenced. Microsequencing of the N-terminus enabled localization of the coat protein gene in this sequence. It showed also that the LMV coat protein coding region is at the 3' end of the genome, and that the coat protein is processed from a larger protein by cleavage at an unusual Q/V dipeptide between the polymerase and the coat protein. This is the first report of such a site for cleavage of a potyvirus polyprotein, where only Q/A, Q/S, and Q/G cleavage sites have been reported. The LMV coat protein gene encodes a 278 amino acid polypeptide with a calculated Mr of 31,171 and is flanked by a region which has a high degree of homology with the putative polymerase and a 3' untranslated region of 211 nucleotides in length. Percentage of homology with the coat protein of other potyviruses confirms that LMV is a distinct member of this group. Moreover, amino acid homologies noticed with the coat protein of potexvirus, bymovirus, and carlavirus elongated plant viruses suggest a functional significance for the conserved domains.