Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor.

Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops.

Multilayer regulatory mechanisms control cleavage factor I proteins in filamentous fungi.

Cleavage factor I (CFI) proteins are core components of the polyadenylation machinery that can regulate several steps of mRNA life cycle, including alternative polyadenylation, splicing, export and decay. Here, we describe the regulatory mechanisms that control two fungal CFI protein classes in Magnaporthe oryzae: Rbp35/CfI25 complex and Hrp1. Using mutational, genetic and biochemical studies we demonstrate that cellular concentration of CFI mRNAs is a limited indicator of their protein abundance. Our results suggest that several post-transcriptional mechanisms regulate Rbp35/CfI25 complex and Hrp1 in the rice blast fungus, some of which are also conserved in other ascomycetes. With respect to Rbp35, these include C-terminal processing, RGG-dependent localization and cleavage, C-terminal autoregulatory domain and regulation by an upstream open reading frame of Rbp35-dependent TOR signalling pathway. Our proteomic analyses suggest that Rbp35 regulates the levels of proteins involved in melanin and phenylpropanoids synthesis, among others. The drastic reduction of fungal CFI proteins in carbon-starved cells suggests that the pre-mRNA processing pathway is altered. Our findings uncover broad and multilayer regulatory mechanisms controlling fungal polyadenylation factors, which have profound implications in pre-mRNA maturation. This area of research offers new avenues for fungicide design by targeting fungal-specific proteins that globally affect thousands of mRNAs.

Polyamine biosynthesis and control of the development of functional pollen in kiwifruit.

The role of polyamines (PAs) in plant reproduction, especially pollen development and germination has been demonstrated in several higher plants. The aim of the present research was to investigate PA involvement in pollen development and germination in dioecious kiwifruit (Actinidia deliciosa). Differences in PA content, level and gene expression for PA biosynthetic enzymes, and the effect of PA biosynthetic inhibitors were found during pollen development (or abortion in female flowers). Whereas PAs, especially spermidine (Spd), remained high throughout the development of functional pollen, the levels collapsed by the last stage of development of sterile pollen. Mature and functional pollen from male-fertile anthers showed S-adenosyl methionine decarboxylase activity (SAMDC; involved in Spd biosynthesis) throughout microgametogenesis, with high levels of soluble SAMDC found starting from the late uninucleate microspore stage. Soluble SAMDC was absent in male-sterile anthers. Arginine decarboxylase [ADC; for putrescine (Put) biosynthesis] showed little difference in functional vs sterile pollen; ornithine decarboxylase [ODC; also for putrescine (Put) biosynthesis] was present only in sterile pollen. Ultrastructural studies of aborted pollen grains in male-sterile flowers showed that cytoplasmic residues near the intine contain vesicles, extruding towards the pollen wall. Very high SAMDC activity was found in the wall residues of the aborted pollen. The combined application in planta of competitive inhibitors of S-adenosylmethionine decarboxylase (MGBG) and of spermidine synthase (CHA), or of D-arginine (inhibitor of Put synthesis), to male-fertile plants led to abnormal pollen grains with reduced viability. The importance of PAs during male-fertile pollen germination was also found. In fact, PA biosynthetic enzymes (ADC and, mainly, SAMDC) were active early during pollen hydration and germination in vitro. Two different SAMDC gene transcripts were expressed in germinating pollen together with a lower level of ADC transcript. Gene expression preceded PA enzyme activity. The application of PA inhibitors in planta drastically reduced pollen germination. Thus, low free Spd can lead either to degeneration or loss of functionality of kiwifruit pollen grains.

Polyamine metabolism and biosynthetic gene expression in Arabidopsis thaliana under salt stress.

In the present study we analysed polyamine metabolism in Arabidopsis thaliana (ecotype Columbia) rosette leaves collected at vegetative and reproductive stages from plants germinated and grown under increasing salt stress (0-75 mM NaCl) conditions. The expression level of the different isoforms of polyamine biosynthetic enzymes was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) and the polyamine biosynthetic enzyme activities were determined both in supernatant and pellet fractions. Free and perchloric acid (PCA)-conjugated (soluble and insoluble) polyamines, were measured. At vegetative stage, plants were able to adapt up to 50 mM NaCl, showing a significant growth inhibition only at 75 mM NaCl. At this growth stage and NaCl concentration there was an up-regulation of spermine biosynthesis. At reproductive stage, plants were able to flower up to 50 mM NaCl, even if with a delay of 7 days. On the contrary, at 75 mM NaCl two different phenotypes were isolated: 75/01 (salt sensitive) and 75/02 (salt tolerant). The sensitive plants (75/01) showed a severely stressed phenotype, compared to the tolerant ones (75/02), and the polyamine metabolism was up-regulated, with the increase of free putrescine and spermine.

Rapid and massive expansion of cord blood-derived cytokine-induced killer cells: an innovative proposal for the treatment of leukemia relapse after cord blood transplantation.

We have used a standardized 21-day expansion protocol to produce cytokine-induced killer (CIK) cells starting from very small amounts of nucleated cells (approximately 15 x 10(6) cells) isolated from cord blood. Mononuclear cells are stimulated with anti CD3 (OKT3) and IFNgamma and then expanded with IL-2. Moreover, we show that washouts of cord blood units bags (at the end of the infusion) may be sufficient to yield almost 500 x 10(6) CIK by the same expansion protocol. CIK cells show strong cytotoxic activity against a variety of tumor target cell lines including B and T lymphomas and myeloid leukemias. More importantly, expanded cord blood-derived CIK cells are cytotoxic against fresh leukemic blasts and express perforin, granzyme and NKG2D molecule at high levels. The same in vitro protocol has already been used to expand CIK cells from peripheral blood of adult donors under GMP conditions and therefore these observations open up the possibility of imagining a future clinical application of leukemia relapse following cord blood transplantation with CIK cells obtained from the same cord blood unit.

Translational regulation of the plant S-adenosylmethionine decarboxylase.

It is becoming apparent that control of protein synthesis by metabolites is more common than previously thought. Much of that control is exerted at the level of initiation of mRNA translation, orchestrated by upstream open reading frames (uORFs) and RNA secondary structure. S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in polyamine biosynthesis and both mammalian and plant AdoMetDCs are translationally regulated by uORFs in response to polyamine levels by distinct mechanisms.

Comparison of the standard laryngeal mask airway and the ProSeal laryngeal mask airway in obese patients.

BACKGROUND: The ProSeal laryngeal mask airway (PLMA) may have advantages over the laryngeal mask airway (LMA) in obese patients. We tested this hypothesis in a clinical setting. METHODS: Sixty obese patients (BMI >30) were randomized to receive mechanical ventilation (tidal volume 7 ml kg(-1), PEEP 10 cm H(2)O), through either the PLMA or the LMA. A gastric tube was used in all patients. Cuff pressure was set at 60 cm H(2)O and increased progressively until excessive leak occurred. The incidence of sore throat was assessed at recovery and after 1 week. RESULTS: The mean leak fraction was 6.1 (SD 2.9)% with the LMA and 6.4 (3.5)% with the PLMA (P=0.721). With the PLMA, with no sign of ventilation problems, the drainage tube was not patent in three patients. The cuff pressure was >100 cm H(2)O in 38% of the LMA group and 7% of the PLMA group (P=0.05). The incidence of sore throat was similar in both groups and it was similarly scored in the recovery room and 1 week after surgery. CONCLUSIONS: Both the PLMA and the LMA can be used for mechanical ventilation of obese patients. The patency of the PLMA drainage tube needs to be checked constantly even when an optimal airtight seal is present. In obese patients the LMA requires a greater cuff pressure than the PLMA, but sore throat is not related to the cuff pressure. Sore throat assessment in the recovery room appears as reliable as assessment later.

Impact of laryngeal mask airway and tracheal tube on pulmonary function during the early postoperative period.

BACKGROUND: The tracheal tube (TT) produces reversible bronchoconstriction and increases pulmonary airway resistance compared to the laryngeal mask airway (LMA). The possible persistence of this effect in the postoperative period has not been studied. The aim of this study was to compare the early postoperative pulmonary function in healthy patients undergoing minor surgical procedures with the LMA or with the TT. METHODS: Sixty patients scheduled for saphenous vein stripping under general anaesthesia were randomised to receive the LMA or the TT. Before anaesthesia and 20 min after LMA or TT removal, pulse oxymetry values (SpO(2)) were recorded and patients performed forced spirometry in the supine position. RESULTS: Preoperative pulmonary function was normal in both groups. There were no differences between groups in the preoperative respiratory function test and SpO(2). Following surgery SpO(2), forced expiratory volume in the first second (FEV1), forced vital capacity (FVC) and peak expiratory flow (PEF) decreased in both groups. The FEV1/FVC did not change in either of the groups. In the TT group, compared to patients using the LMA, there was a greater relative decrease of SpO(2) (2.7 +/- 2.7% vs. 1.3 +/- 2.2%, P=0.017), FEV1 (17.6 +/- 12.2% vs. 8 +/- 17.4%, P=0.008), FVC (15.8 +/- 12.4% vs. 9 +/- 13.4%, P=0.023) and PEF (20.6% +/- 15.3% vs. 8.1 +/- 33.3%, P=0.033). CONCLUSIONS: This study demonstrates greater early postoperative respiratory restrictive syndrome and lower arterial oxygen saturation following tracheal intubation compared to LMA use in patients without respiratory disease.

Characterization of monocot and dicot plant S-adenosyl-l-methionine decarboxylase gene families including identification in the mRNA of a highly conserved pair of upstream overlapping open reading frames.

S-Adenosyl-L-methionine decarboxylase (AdoMetDC; EC 4.1.1.50) is one of the key regulatory enzymes in the biosynthesis of polyamines. Isolation of genomic and cDNA sequences from rice and Arabidopsis had indicated that this enzyme is encoded by a small multigene family in monocot and dicot plants. Analysis of rice, maize and Arabidopsis AdoMetDC cDNA species revealed that the monocot enzyme possesses an extended C-terminus relative to dicot and human enzymes. Interestingly, we discovered that all expressed plant AdoMetDC mRNA 5' leader sequences contain a highly conserved pair of overlapping upstream open reading frames (uORFs) that overlap by one base. The 5' tiny uORF consists of two or three codons and the 3' small uORF encodes 50-54 residues. Sequences of the small uORFs are highly conserved between monocot, dicot and gymnosperm AdoMetDC mRNA species and the C-terminus of the plant small uORFs is conserved with the C-terminus of nematode AdoMetDC uORFs; such a conserved arrangement is strongly suggestive of a translational regulatory mechanism. No introns were found in the main AdoMetDC proenzyme ORF from any of the plant genes encoding AdoMetDC, whereas introns were found in conserved positions flanking the overlapping uORFs. The absence of the furthest 3' intron from the Arabidopsis gene encoding AdoMetDC2 suggests that this intron was lost recently. Reverse-transcriptase-mediated PCR analysis of the two Arabidopsis genes for AdoMetDC indicated that AdoMetDC1 is abundant and ubiquitous, whereas the gene for AdoMetDC2 is expressed preferentially in leaves and inflorescences. Investigation of recently released Arabidopsis genome sequences has revealed that in addition to the two genes encoding AdoMetDC isolated as part of the present work, four additional genes are present in Arabidopsis but they are probably not expressed.

[Salmonella C infection of aortic abdominal aneurysm]. / Infezione di aneurisma dell'aorta addominale da salmonella di gruppo C.

The infected aneurysms of the aorta represent the 0.65% of all the aneurysms and they are associated with high morbidity and mortality. The vascular infections from Salmonella are not particularly frequent, even if in the last years their reports are more numerous. The authors report their experience in the surgical treatment of one case of aneurysm of the abdominal aorta infected by group C Salmonella, making a review of the Literature on this matter.

[True aneurysm of the profunda femoris artery. Case report and literature review]. / Aneurisma vero dell'arteria femorale profonda. Caso clinico e revisione della letteratura.

Aneurysms of the profunda femoris artery rarely occur as an isolated lesion because of anatomical position and characteristics of wall tissue. The case of a 79-year-old man admitted to the hospital because of acute pain and swelling of the right leg with pulsating tumor in the groin is reported. Duplex scanning and angiography, performed in order to establish the diagnosis and to plan the operation, demonstrated a right large aneurysm of the deep femoral artery (8 x 12 cm). At operation, the aneurysm was resected associated to a PTFE Stretch graft replacement because the autogenous saphenous vein was not available. A review of the literature on the clinical manifestation and management in emergency or elective surgery of the isolated true atherosclerotic aneurysm of the deep femoral artery, is presented.

[Aneurysm of the celiac trunk. Presentation of a clinical case and review of the literature]. / Aneurisma del tripode celiaco. Presentazione di un caso clinico e revisione della letteratura.

The aneurysms of the celiac trunk are the rarest aneurysms of the visceral arteries. From 1958 only 69 cases have been reported in the international literature. They are frequently asymptomatic and their discovery is more often occasional. They can rupture in 15-20% of the cases with a mortality approaching 80% of the cases. This explains the need of a surgical treatment even in the asymptomatic cases. Personal experience in the surgical treatment of a case of aneurysm of the celiac trunk is reported and a survey of the literature on this matter is made.