Autophagy and metacaspase determine the mode of cell death in plants.

Although animals eliminate apoptotic cells using macrophages, plants use cell corpses throughout development and disassemble cells in a cell-autonomous manner by vacuolar cell death. During vacuolar cell death, lytic vacuoles gradually engulf and digest the cytoplasmic content. On the other hand, acute stress triggers an alternative cell death, necrosis, which is characterized by mitochondrial dysfunction, early rupture of the plasma membrane, and disordered cell disassembly. How both types of cell death are regulated remains obscure. In this paper, we show that vacuolar death in the embryo suspensor of Norway spruce requires autophagy. In turn, activation of autophagy lies downstream of metacaspase mcII-Pa, a key protease essential for suspensor cell death. Genetic suppression of the metacaspase­autophagy pathway induced a switch from vacuolar to necrotic death, resulting in failure of suspensor differentiation and embryonic arrest. Our results establish metacaspase-dependent autophagy as a bona fide mechanism that is responsible for cell disassembly during vacuolar cell death and for inhibition of necrosis.

Detection and measurement of necrosis in plants.

Necrosis plays a fundamental role in plant physiology and pathology. When plants or plant cell cultures are subjected to abiotic stress they initiate rapid cell death with necrotic morphology. Likewise, when plants are attacked by pathogens, they develop necrotic lesions, the reaction known as hypersensitive response. Great advances in the understanding of signaling pathways that lead to necrosis during plant-pathogen interaction have been made in the last two decades using Arabidopsis thaliana as a model plant. Further understanding of these signaling pathways, as well as those regulating the execution phase of necrotic cell death per se would require a robust set of readout assays to detect and measure necrosis in various plant model systems. Here we provide description of such assays, beginning from electron microscopy, as the "gold standard" to diagnose necrosis. This is followed by two groups of biochemical and cytochemical assays used by our group to detect and quantify mitochondrial dysfunction and the loss of protoplast integrity during necrosis in Arabidopsis plants and cell suspension cultures of both Arabidopsis and Norway spruce.

A newly described vancomycin-resistant ST412 Enterococcus faecium predominant in Greek hospitals.

A total of 359 vancomycin-resistant enterococci (344 Enterococcus faecium and 15 E. faecalis) collected during 2007 from eight tertiary-care hospitals in Greece were analysed for genotypic characteristics. Four common clones, ST412, ST203, ST16 and ST17, were identified among E. faecium and one clone, ST28, among E. faecalis strains.

In vitro activity of daptomycin against Gram-positive cocci: the first multicentre study in Greece.

A total of 10420 Gram-positive cocci (including staphylococci, enterococci and various groups of streptococci) collected from clinically significant specimens in ten Greek hospitals during 2006--2007 were tested for their susceptibility to daptomycin. The minimum inhibitory concentration (MIC) was determined by the broth microdilution method. Daptomycin demonstrated very high activity against Enterococcus faecalis (MIC at which 50% of the isolates were inhibited (MIC50) = 1mg/L and MIC at which 90% of the isolates were inhibited (MIC90) = 1.36 mg/L), Enterococcus faecium (MIC50 = 1.36 mg/L and MIC90 = 1.90 mg/L), Streptococcus pyogenes (MIC50 = 0.12 mg/L and MIC90 = 0.50mg/L), Streptococcus agalactiae (MIC50 = 0.09 mg/L and MIC90 = 0.12 mg/L), Streptococcus pneumoniae (MIC50 = 0.24 mg/L and MIC90 = 0.5 mg/L) and viridans group streptococci (MIC50 = 0.50 mg/L and MIC90 = 0.89 mg/L). Resistance to linezolid and vancomycin for enterococci and to penicillin for streptococci appears to be independent of reduced susceptibility to daptomycin. On the other hand, daptomycin was also active against meticillin-resistant Staphylococcus aureus (MIC50 = 0.44 mg/L and MIC90 = 0.78 mg/L) and meticillin-resistant coagulase-negative staphylococci (MIC50 = 0.24 mg/L and MIC90 = 0.44 mg/L); however, 0.9% of the staphylococci tested had an MIC > 1mg/L, which is the Clinical and Laboratory Standards Institute breakpoint proposed for susceptibility. For all tested organism groups, resistance to daptomycin was not associated with glycopeptide resistance.

Detection of programmed cell death in plant embryos.

Programmed cell death (PCD) is an integral part of embryogenesis. In plant embryos, PCD functions during terminal differentiation and elimination of the temporary organ, suspensor, as well as during establishment of provascular system. Embryo abortion is another example of embryonic PCD activated at pathological situations and in polyembryonic seeds. Recent studies identified the sequence of cytological events leading to cellular self-destruction in plant embryos. As in most if not all the developmental cell deaths in plants, embryonic PCD is hallmarked by autophagic degradation of the cytoplasm and nuclear disassembly that includes breakdown of the nuclear envelope and DNA fragmentation. The optimized setup of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) allows the routine in situ analysis of nuclear DNA fragmentation in plant embryos. This chapter provides step-by-step procedure of how to process embryos for TUNEL and how to combine TUNEL with immunolocalization of the protein of interest.

Cysteine protease mcII-Pa executes programmed cell death during plant embryogenesis.

Programmed cell death (PCD) is indispensable for eukaryotic development. In animals, PCD is executed by the caspase family of cysteine proteases. Plants do not have close homologues of caspases but possess a phylogenetically distant family of cysteine proteases named metacaspases. The cellular function of metacaspases in PCD is unknown. Here we show that during plant embryogenesis, metacaspase mcII-Pa translocates from the cytoplasm to nuclei in terminally differentiated cells that are destined for elimination, where it colocalizes with the nuclear pore complex and chromatin, causing nuclear envelope disassembly and DNA fragmentation. The cell-death function of mcII-Pa relies on its cysteine-dependent arginine-specific proteolytic activity. Accordingly, mutation of catalytic cysteine abrogates the proteolytic activity of mcII-Pa and blocks nuclear degradation. These results establish metacaspase as an executioner of PCD during embryo patterning and provide a functional link between PCD and embryogenesis in plants. Although mcII-Pa and metazoan caspases have different substrate specificity, they serve a common function during development, demonstrating the evolutionary parallelism of PCD pathways in plants and animals.

Programmed cell death in plant embryogenesis.

Successful embryonic development in plants, as in animals, requires a strict coordination of cell proliferation, cell differentiation, and cell-death programs. The role of cell death is especially critical for the establishment of polarity at early stages of plant embryogenesis, when the differentiation of the temporary structure, the suspensor, is followed by its programmed elimination. Here, we review the emerging knowledge of this and other functions of programmed cell death during plant embryogenesis, as revealed by developmental analyses of Arabidopsis embryo-specific mutants and gymnosperm (spruce and pine) model embryonic systems. Cell biological studies in these model systems have helped to identify and order the cellular processes occurring during self-destruction of the embryonic cells. While metazoan embryos can recruit both apoptotic and autophagic cell deaths, the ultimate choice depending on the developmental task and conditions, plant embryos use autophagic cell disassembly as a single universal cell-death pathway. Dysregulation of this pathway leads to aberrant or arrested embryo development. We address the role of distinct cellular components in the execution of the autophagic cell death, and outline an overall mechanistic view of how cells are eliminated during plant embryonic pattern formation. Finally, we discuss the possible roles of some of the candidate plant cell-death proteins in the regulation of developmental cell death.

Colistin for Klebsiella pneumoniae-associated sepsis.

Klebsiella pneumoniae that was resistant to all available antibiotics (minimum inhibitory concentration of imipenem, 32 microg/mL), including carbapenems, was isolated from blood samples obtained from a 48-year-old patient in the intensive care unit. The patient developed septic shock, which was successfully treated with colistin, the only antibiotic with activity against this multidrug-resistant strain.

Clonal dissemination of mupirocin-resistant staphylococci in Greek hospitals.

OBJECTIVES: To determine the rates of mupirocin resistance in staphylococci during a 4 year period (1999-2002) in Greece. MATERIALS: A total of 1200 Staphylococcus aureus and 2760 coagulase-negative staphylococci (CoNS), consecutively collected from four Greek hospitals located in different geographical areas, were tested for susceptibility to mupirocin using the Etest and a reference agar dilution method. RESULTS: Twenty-four S. aureus (2%) and 532 CoNS (19.2%) were found to be mupirocin-resistant during the study period. High-level mupirocin resistance was detected in 20 S. aureus (1.6%) and in 440 CoNS (15.9%), respectively. No variations in the rates of mupirocin-resistant S. aureus in relation to the year of collection were observed. In contrast, the rate of mupirocin-resistant CoNS increased dramatically from 9% in 1999, to 14% in 2000, 20% in 2001 and reached 33% in 2002. PFGE analysis revealed the presence of one main clone (A) among mupirocin-resistant S. aureus and two main clones (i and a) among Staphylococcus epidermidis isolates. CONCLUSIONS: In Greece, the rate of mupirocin-resistant S. aureus has remained low and steady since 1999. The high rate of mupirocin-resistant CoNS (33%) in 2002 was due mainly to clonal dissemination of epidemic hospital clones.

VIM-1 Metallo-beta-lactamase-producing Klebsiella pneumoniae strains in Greek hospitals.

Seventeen Klebsiella pneumoniae clinical isolates carrying the bla(VIM-1) metallo-beta-lactamase gene were collected in the intensive care units of three hospitals in Athens, Greece, in 2002. They exhibited various carbapenem resistance levels (Etest MICs of imipenem ranged from 4 to 32 microg/ml). All isolates gave positive results by the imipenem-EDTA synergy Etest. The isolates were classified into four main types by pulsed-field gel electrophoresis; the majority of the isolates (5 and 10 isolates) belonged to two types. The bla(VIM-1) gene cassette was part of the variable region of a class 1 integron that also included aac6, dhfrI, and aadA. This structure was carried by transferable plasmids.

Re-organisation of the cytoskeleton during developmental programmed cell death in Picea abies embryos.

Cell and tissue patterning in plant embryo development is well documented. Moreover, it has recently been shown that successful embryogenesis is reliant on programmed cell death (PCD). The cytoskeleton governs cell morphogenesis. However, surprisingly little is known about the role of the cytoskeleton in plant embryogenesis and associated PCD. We have used the gymnosperm, Picea abies, somatic embryogenesis model system to address this question. Formation of the apical-basal embryonic pattern in P. abies proceeds through the establishment of three major cell types: the meristematic cells of the embryonal mass on one pole and the terminally differentiated suspensor cells on the other, separated by the embryonal tube cells. The organisation of microtubules and F-actin changes successively from the embryonal mass towards the distal end of the embryo suspensor. The microtubule arrays appear normal in the embryonal mass cells, but the microtubule network is partially disorganised in the embryonal tube cells and the microtubules disrupted in the suspensor cells. In the same embryos, the microtubule-associated protein, MAP-65, is bound only to organised microtubules. In contrast, in a developmentally arrested cell line, which is incapable of normal embryonic pattern formation, MAP-65 does not bind the cortical microtubules and we suggest that this is a criterion for proembryogenic masses (PEMs) to passage into early embryogeny. In embryos, the organisation of F-actin gradually changes from a fine network in the embryonal mass cells to thick cables in the suspensor cells in which the microtubule network is completely degraded. F-actin de-polymerisation drugs abolish normal embryonic pattern formation and associated PCD in the suspensor, strongly suggesting that the actin network is vital in this PCD pathway.

A key developmental switch during Norway spruce somatic embryogenesis is induced by withdrawal of growth regulators and is associated with cell death and extracellular acidification.

The biotechnology of somatic embryogenesis holds considerable promise for clonal propagation and breeding programs in forestry. To efficiently regulate the whole process of plant regeneration through somatic embryogenesis, it is of outmost importance to understand early developmental events when somatic embryos are just formed. In Norway spruce, somatic embryos transdifferentiate from proembryogenic masses (PEMs). This work describes the developmental dynamics (frequency distribution of PEMs and early somatic embryos) of the whole embryogenic suspension culture growing in the presence and absence of plant growth regulators (PGRs), auxin and cytokinin. The experiments have shown that PEM-to-somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos and ultimately plant production. This switch was induced by the withdrawal of PGRs in cell suspension leading to a rapid accumulation of early somatic embryos (to a maximum of 75% of the entire population of suspension culture) and concomitant degradation of PEMs. The latter was evident from increased level of cell death measured through spectrophotometric Evans blue staining assay. Proembryogenic mass-to-embryo transition and concomitant activation of cell death were mediated by strong extracellular acidification. Therefore, buffering PGR-free culture medium at high (pH 5.8) or low (pH 4.5) levels of pH inhibited both PEM-to-embryo transition and cell death. The yield of mature somatic embryos on abscisic acid (ABA)-containing medium was increased up to 10-fold if the suspension culture had been pretreated for 1 to 9 days in unbuffered PGR-free medium. In this case a large proportion (75%) of the total number of mature embryos was formed within a short, 5-week, contact with ABA. The latter is practically important because prolonged contact with ABA suppresses the growth of somatic embryo plants. Based on these results, an improved method for regulating somatic embryogenesis was set up and tested for nine genotypes of Norway spruce. Over 800 plants regenerated from all tested genotypes demonstrated a good performance in the greenhouse and they were transferred to the field.

SOCS1 deficiency results in accelerated mammary gland development and rescues lactation in prolactin receptor-deficient mice.

Prolactin is essential for proliferation and differentiation of the developing mammary gland. We have explored a role for Suppressor of Cytokine Signaling 1 (SOCS1) as a modulator of the prolactin response using mice deficient in SOCS1, which were rescued from neonatal death by deletion of the Interferon gamma (IFN gamma) gene. SOCS1(-/-)/IFN gamma(-/-) mice exhibited accelerated lobuloalveolar development in the mammary gland during late pregnancy and precocious lactation. Significantly, the lactogenic defect in prolactin receptor heterozygous females could be rescued by deletion of a single SOCS1 allele. These findings establish a role for SOCS1 as a negative regulator of prolactin signaling and suggest that SOCS1 is required for the prevention of lactation prior to parturition.

A Drosophila analogue of v-Cbl is a dominant-negative oncoprotein in vivo.

Cells rely on the ability to receive and interpret external signals to regulate growth, differentiation, and death. Positive transduction of these signals to the cytoplasm and nucleus has been extensively characterized, and genetic studies in Drosophila have made major contributions to the understanding of these pathways. Less well understood, but equally important, are the mechanisms underlying signal down-regulation. Here we report biochemical and genetic characterization of the Drosophila homologue of c-Cbl, a negative regulator of signal transduction with ubiquitin-protein ligase activity. A new isoform of D-Cbl, D-CblL, has been identified that contains SH3-binding and UBA domains previously reported to be absent. Genetic analysis demonstrates that Dv-cbl, analogous to the mammalian v-cbl oncogene, is a dominant negative mutation able to enhance signalling from the Drosophila Egfr and cooperate with activating mutations in the sevenless pathway to produce melanotic tumours. In addition, our data show genetic and biochemical links between D-Cbl and proteins involved in endocytosis and ubiquitination, suggesting that v-Cbl may exert its oncogenic effect by enhancing receptor signalling as a consequence of suppressing receptor endocytosis.

Isolation rate, T-serotyping and susceptibility to antibiotics of Group A Streptococcus from pediatric infections in Athens.

OBJECTIVE: The epidemiology of Group A Streptococcus (GAS) in Greece is not known. We have therefore conducted this prospective study to investigate the isolation rate of GAS from pediatric specimens, determine T-serotype frequency and examine the susceptibility of GAS to penicillin, erythromycin and clindamycin. METHODS: Over a 3-year study-period (1993-95) 11 597 clinical specimens obtained from sick children were inoculated on appropriate culture media. The isolation and identification of GAS strains were assessed by conventional methods. T-typing was performed by slide agglutination. Serum opacity factor (OF) was detected by microwell METHOD: The susceptibility of the strains was tested by the Kirby Bauer method. RESULTS: GAS were isolated from 1125 out of 11 597 (9.7%) clinical specimens, mostly from throat samples (15.6%). T-serotyping was performed in 652 GAS strains. A significant difference of the incidence of T-serotypes was observed within the 3 years studied (chi2 = 70.3, DF = 18, P < 0.001). The most dominant isolates were T-1 (25%), T-4 (20%) and T-12 (16%) during 1993, 1994 and 1995, respectively. Non-typeable (NT) strains were 4%. OF and hyaluronic acid were produced from 49.8% and 3% of the strains, respectively. All isolated strains were susceptible to penicillin and clindamycin. Resistance to erythromycin was 5.0-8.7% over the 3-year study period. CONCLUSIONS: There was a wide distribution of GAS T-serotypes in Athens and a significant change in their annual predominance. All strains were susceptible to penicillin and clindamycin, but a low level of erythromycin resistance was observed.