57, adjusted, P<0001): Culture supernatants from growth at 8, 15

57, adjusted, P<0.001): Culture supernatants from growth at 8, 15 and 37 °C were analysed in a Vero cell cytotoxicity assay (Table 1). The initially observed feature was the full cytotoxicity shared by both bacterial species after culturing at 15 °C. At 8 °C, two of the four B. weihenstephanensis strains showed high cytotoxicity, while the other two were negative or very low. After growth at 37 °C, no cytotoxic activity was seen from the four B. weihenstephanensis strains, while two of three B. cereus strains were TSA HDAC cost highly cytotoxic (80–100% range) and one strain (B. cereus NVH 1230-88)

was low (in the 20–50% range). The production of adequate levels of three-component protein cytotoxins Nhe and/or Hbl results in cytotoxicity in the Vero cell assay (Lund & Granum, 1996; Prüss et al., 1999; Dietrich et al., 2005). Components of those toxins were sought in the tested bacterial culture supernatants using commercially available antibody-based kits (Table 2). The L2 component of Hbl was detected at all three temperatures in all strains, except in this website B. cereus strain NVH 0075-95, which does not contain the operon encoding Hbl (Granum et al., 1996). On the other hand, the Nhe component NheA was detected

in all strains after growth at 15 °C, while when grown at 37 °C, NheA was only found in one of the four B. weihenstephanensis strains. One B. weihenstephanensis strain did not produce NheA at 8 °C. The boar spermatozoa assay was used to screen the Bacillus spp. strains for the production of cereulide (emetic B. cereus toxin) under standard conditions. Only one of the seven Bacillus strains (a B. cereus strain, NVH 1230-88) was weakly positive in this assay (results not

shown). However, screening by PCR for the genes encoding cereulide was negative for all the strains (results not shown). In vivo virulence results from experiments performed on G. mellonella larvae with the seven bacterial strains are presented in Fig. 1, which shows kinetics of induced mortality through the observation period following larval infection by two routes and at both temperatures. In order to give a general picture of virulence and because the exact dose repeats PAK5 were not possible to obtain, larval mortalities are shown as raw data, and B. cereus and B. weihenstephanensis strains are each grouped together. The results reveal variation in virulence level among strains (individual strain results not shown), even in the same group, but differences are particularly marked for mortality speed related to temperature between B. cereus and B. weihenstephanensis. Thus, using our linear regression model for in vivo experimental data, a significant difference was found in virulence between B. weihenstephanensis and B. cereus species (P<0.001), observed mainly at 37 °C (Fig. 1). Indeed, the psychrotolerant strains mostly showed negligible virulence both 24 and 90 h after infection, while B. cereus strains were generally highly virulent at 37 °C.

Lovastatin was termed monacolin K when isolated from Monascus pil

Lovastatin was termed monacolin K when isolated from Monascus pilosus (Staunton & Weissman, 2001). A structurally related compound named compactin was isolated from Penicillium citrinum (Abe et al., 2002). Our PKS1 protein showed 36% similarity to both MokA in the monacolin K biosynthesis pathway (Chen et al., 2008) and compactin nonaketide synthase (CNKS) in the compactin biosynthesis Rucaparib supplier pathway. The PKS1 protein also showed 37% sequence similarity to the PKS-NRPS hybrid equisetin synthetase (EqiS) in Fusarium heterosporum (Sims et al., 2005). LNKS contains a truncated NRPS module, and the biosynthesis of lovastatin and equisetin shares a common pathway up to the Diels–Alder

cyclization of hexaketide (Campbell & Vederas, 2010). Our PKS1 likely catalyzes a similar reaction, but the chain length of the polyketide cannot be predicted. The on-line software sbspks predicts that PKS1 accepts malonic or methylmalonic acid as a substrate, similar to LNKS and LDKS (Campbell & Vederas, 2010). There is a product template (PT) domain between the AT and ACP domains (Schuemann & Hertweck, 2009) controlling the chain length in non-reducing PKSs (Cox, 2007; Liu et al., 2011); however, the chain length determination in highly reduced PKSs, such as LNKS, LDKS and CNKS, is not well understood. The 760-bp fragment was located on an 11-kb hybrid pks-nrps gene (Fig. 3a). Hybrid gene clusters

are widely distributed in Ascomycetes (Collemare et al., 2008). The pks-nrps1

gene encodes a protein that displayed 36% similarity with three proteins: DmbS in the 2-pyridone MK 2206 desmethylbassianin (DMB) biosynthetic pathway (Heneghan et al., 2011), TenS in the tenellin biosynthetic pathway in B. bassiana (Eley et al., 2007), and FusS in the fusarin biosynthetic pathway in Fusarium moniliforme (teleomorph Gibberella moniliformis) (Song et al., 2004). sbspks predicts that malonic acid is the only accepted substrate for the AT domain of PKS-NRPS1. However, due to the highly variable signature sequences in the A domain binding pockets, we could not predict the substrates of all of the NRPSs reported here (Table S3). In the hybrid PKS-NRPS systems, the Dieckmann cyclase domain (also known as the R domain) often mediates product release (Halo et al., 2008; Du & Lou, 2010). Interestingly, the R domain of PKS-NRPS1 showed sequence similarity to the short-chain dehydrogenase/reductase OSBPL9 superfamily proteins in TenS, EqiS and DmbS (Halo et al., 2008; Sims & Schmidt, 2008; Heneghan et al., 2011) and therefore potentially mediates product release. Although PKS-NRPS1 contained an ER domain, it is likely to be inactive because there are three mutations in the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-binding motif (Fig. S1). Although the ER domains of LNKS, TenS and DmbS are inactive, reduction was catalyzed via the trans-acting ERs encoded by lovC, tenC and dmbC, respectively (Eley et al., 2007; Ma et al., 2009; Heneghan et al., 2011).

The RGS is designed to scale

The RGS is designed to scale Protease Inhibitor Library order task difficulty to the given performance level of the given subject or patient. Accordingly, we would expect similar activations as observed here whenever a patient works with the RGS, although recovery involved general motor abilities resulting from training with the RGS, as described after acute and chronic stroke (Cameirão et al., 2011, 2012). In conclusion, our results show that the VR-based RGS induces activation in brain regions associated with motor control,

including the SMA, the inferior frontal cortex, and the inferior parietal cortex. In agreement with our working hypothesis, these findings show the engagement of brain areas believed to represent the human mirror neuron system. As the RGS was shown to be an effective training tool for patients with acute and chronic

stroke (Cameirão et al., 2011, 2012), additional investigations are needed to address which brain areas become engaged when the RGS is applied to stroke patients. The study was supported by the consortium on the Rehabilitation Gaming System, AAL Joint Program 2008-1, AZD6244 chemical structure European Commission, and Microsystems 2004–2009, Bundesministerium für Bildung und Forschung, VDI-VDE, Germany. The authors thank Erika Rädisch for her assistance and support with the fMRI measurements. Our thanks also go to Albert Fabregat for his help with software programming, and to Klintsy Torres for translation. Abbreviations 3D three-dimensional ACC anterior cingulate cortex Tobramycin BOLD blood oxygenation level-dependent df degrees of freedom fMRI functional magnetic resonance imaging IFG inferior frontal gyrus

IPL inferior parietal lobule MRI magnetic resonance imaging RGS Rehabilitation Gaming System SD standard deviation SMA supplementary motor area VR virtual reality “
“During slow-wave sleep, the neocortex shows complex, self-organized spontaneous activity. Similar slow-wave oscillations are present under anesthesia where massive, persistent network activity (UP states) alternates with periods of generalized neural silence (DOWN states). To investigate the neuronal activity patterns occurring during UP states, we recorded simultaneously from populations of cells in neocortical layer V of ketamine/xylazine-anesthetized rats. UP states formed a diverse class. In particular, simultaneous-onset UP states were typically accompanied by sharp field potentials and 10–14 Hz modulation, and were often grouped in a 3 Hz (‘delta’) pattern. Longer, slow-onset UP states did not exhibit 10–14 Hz modulation, and showed a slow propagation across recording electrodes (‘traveling waves’). Despite this diversity, the temporal patterns of spiking activity were similar across different UP state types. Analysis of cross-correlograms revealed conserved temporal relationships among neurons, with each neuron having specific timing during UP states.

In contrast to lactoferrin, desferrioxamine and deferiprone, DIBI

In contrast to lactoferrin, desferrioxamine and deferiprone, DIBI provided almost complete inhibition of the growth of both C. albicans and C. vini over a 4-day incubation period. Candida albicans has been reported to use iron from the ferriproteins haemin, haemoglobin and myoglobin (Han, 2005), and to acquire iron from transferrin (Knight et al., 2005). However, the slight increase of the maximum specific

growth yields observed in the presence of some chelators in this study was not significant enough to support chelator-assisted iron acquisition. In a long-term study with reduced, subinhibitory concentrations (0.17 g L−1), DIBI did allow delayed and gradual growth of both yeasts, which was comparable to inhibition by EDTA for C. albicans and to BPS in C. vini. In contrast to EDTA and BPS, which are known to readily chelate other transition metals (Ueno et al., 1992), DIBI was shown to be iron-selective and its inhibitory activity was shown to be Dasatinib supplier Fe reversible. Accordingly, DIBI appeared to be a more potent iron scavenger than any of the other clinically

relevant chelators examined. This work presents the first evidence of the iron requirements of C. vini, a nonpathogenic food spoilage organism, and the inhibition of www.selleckchem.com/products/PF-2341066.html C. vini and the opportunistic pathogen C. albicans by several strong chelators. The differences observed with respect to the ability of C. vini and C. albicans to grow under iron-restricted conditions were consistent with the respective environmental niches and pathogenicity. Protein kinase N1 The present work provides a foundation for future studies that may investigate the possible synergistic effects of iron withdrawal in combination with

antifungal preservative addition. The authors thank Chelation Partners for supplying the FEC-1 chelating adsorbent and the DIBI chelator. “
“Sinorhizobium meliloti associates with Medicago and Melilotus species to develop nitrogen-fixing symbioses. The agricultural relevance of these associations, the worldwide distribution of acid soils, and the remarkable acid sensitivity of the microsymbiont have all stimulated research on the responses of the symbionts to acid environments. We show here that an adaptive acid-tolerance response (ATR) can be induced in S. meliloti, as shown previously for Sinorhizobium medicae, when the bacteria are grown in batch cultures at the slightly acid pH of 6.1. In marked contrast, no increased tolerance to hydrogen ions is obtained if rhizobia are grown in a chemostat under continuous cultivation at the same pH. The adaptive ATR appears as a complex process triggered by an increased hydrogen-ion concentration, but operative only if other – as yet unknown – concomitant factors that depend on the culture conditions are present (although not provided under continuous cultivation). Although the stability of the ATR and its influence on acid tolerance has been characterized in rhizobia, no data have been available on the effect of the adapted state on symbiosis.

[34] We identified two different groups of clinical trials based<

[34] We identified two different groups of clinical trials based

on their recruitment method and found that this classification was useful in describing other important aspects of trial design and outcome. Eight of the clinical trials recruited trekkers as they ascended and then aimed to assess the same trekkers later on their expedition.[27, 29, 30, 33, 34, 36, 37, 43] We designated Selleck EX527 this type of trial “location-based.” The other nine trials, including the trial which was excluded from quantitative analysis, recruited people to the trial prior to embarking on an organized expedition(s) and we designated this type of trial “expedition-based.”[28, 31, 32, 35, 38-42] There are a number of key differences between the two different types of trial summarized in Table 2. Most importantly, location-based trials tended to be larger (median 160.5 vs 35) but have a higher dropout rate (median 52 vs 0.5). Expedition-based trials had a higher rate of ascent (mean 450 vs 2,800 m/d). All of the

studies used questionnaires to assess outcome. These were either administered by blinded researchers or self-administered. A number of assessment tools were used as shown in Table 1. The most commonly used assessment score was the Lake Louise Symptom buy Belinostat score (LLS),[44] which was used in 10 studies (63%). Four studies (25%) used variations of the Acute Mountain Sickness score cerebral and respiratory domains (AMS-C and AMS-R) which are derived from the modified Environmental Systems Questionnaire.[45] Of the remaining clinical trials, one used the General High Altitude Questionnaire (GHAQ)[46] and one used a score developed for the clinical trial.[43] All of the scores were similar in that they were combined interval scores incorporating several symptom

domains and the diagnosis of AMS was made if a specific score was reached (often with the presence of headache mandatory). It is likely from the individual trial HA-1077 supplier reports that timing of assessment after arrival at altitude varied; however, they generally did not contain enough information on this factor to allow analysis. None of the study protocols were available for review. It was generally not possible to ascertain whether sequence generation, allocation, or blinding were satisfactory from the trial report since they were usually described briefly. However, no cause for concern about bias in any of these domains was found. All trials were therefore found to have low or unclear risk of bias in these domains. The main source of bias was found in the outcome data domain. As discussed above, studies which relied on location-based recruitment had a high dropout rate. We decided to perform a worst-case analysis of the missing data and exclude studies in which the worst-case analysis resulted in a change of result.

Two of the most frequent sources of malaria education reported du

Two of the most frequent sources of malaria education reported during buy GSK J4 this investigation were “word of mouth” and “casual conversation.” These methods can be beneficial if a trusted person was passing along correct information, but detrimental if the information or advice from a trusted person was incorrect. In order to ensure crew members receive correct and consistent information, education should be provided in an appropriate learning environment,

which may be different between pilots and FA. Additionally, there should be ample opportunities to ask questions from a knowledgeable health care professional. Both occupational groups reported a strong preference to hear about the experiences of fellow crew members who were recently ill with malaria. This practice should be pursued with a crew member trained to serve in this role and assist in raising crew members’ awareness of their occupational risk for malaria. Training can be re-emphasized with educational material in airport lounges, such as posters and the FAQ sheets. As scheduling work trips can occur months in advance, sending text and e-mail messages 2 to 3 days prior to travel to a malaria-intense destination would remind crew members to prepare their preventive measures before departure. This investigation was subject to at least five limitations. The low participation

rate, which was ICG-001 price not unexpected for

an Internet survey, makes generalizability to all crew members difficult. Selection bias was introduced as FA whose travel included West Africa in the previous year were actively solicited by a company e-mail to participate in the Palmatine survey. Their responses may be different from other FA eligible for international travel. Also, selection bias by the participants may have occurred, as those who completed the survey may have been different from nonparticipants. The assessment of malaria knowledge may have been biased if participants sought assistance while completing the questions. Finally, reporting bias could be present, as participants may under or over report the frequencies of their practices knowing that their employer would receive the cumulative information, participants were free to skip questions, and without personal identification information or IP addresses, there was no control to avoid duplicate questionnaire submissions from the same participant. Despite a sound basic knowledge of malaria transmission and preventive measures, both the FA and pilot populations had a low perception of their occupational risks for malaria. Many participants practiced risky, but some unavoidable, activities that may have increased their malaria exposure and rarely used all the recommended preventive measures during layovers at malaria-intense destinations.

While 10mmol/L is the upper limit of normal BG levels, this may i

While 10mmol/L is the upper limit of normal BG levels, this may in practice indicate

that levels are much higher. Together, this information about glucose control reveals that, while convenient, pump therapy might be less effective than reported, although not necessarily less effective than MDI therapy. It may be that an anonymised survey elicits information that differs from other sources for a variety of reasons that relate to surveys in general as well as to diabetes. It also implies that despite being on a reliably constant basal dose of insulin and with boosts conveniently selected for delivery to a tailored pattern coupled with features such as electronic memory and safety lockout features, respondents were commonly above the target BG range. An increase in BG with CSII may result from an occlusion of the BYL719 infusion line or cannula, although more commonly problems arise from human SGI-1776 chemical structure error, for example inaccurate carbohydrate estimation, inaccurate insulin carbohydrate

ratios, insulin sensitivity factors, as well as lifestyle factors such as exercise and stress. Whether the postprandial BG peak would be detected would depend on the user testing at the relevant times. The positive attitude towards an artificial pancreas such as INSmart focused on the control of BG and user independence as well as improved quality of life. Negative responses were perceptions about relying on an automated system that could possibly fail or not be reliable. The concept of an implantable device rather than an external (and therefore easily-removable) pump

was clearly worrying to some. There were comments about the need for comfort, the safety of implantation and maintenance including refill which would all need to be demonstrated for an INSmart type device to secure approval from the Medical Devices Directive in the UK25 (FDA in the USA). The behaviour, PIK3C2G attitude and use of existing external pump users from the open ended questions from this survey provided some useful feedback toward a redesign of the existing device which has now successfully been implanted into diabetic pigs. It is apparent that current external pumps have shortcomings which an implantable INSmart device could overcome: Automated delivery of insulin to real time changing glucose levels by the fast uptake of glucose in the peritoneum. No changing of infusion lines, rotation of sites and not visible. No moving parts or electronic power requirements. No need to regularly check BG levels. No need to bolus for meal times. However, an implantable INSmart device would still need to overcome risks such as leakage of insulin or smart gel, infection and surgery. The general consensus from the survey was that most respondents felt that an implantable artificial pancreas would be a close match to a functioning healthy pancreas and therefore appealing.

This work was supported by grants from Fundación para la Investig

This work was supported by grants from Fundación para la Investigación Sanitaria (FIS) del Ministerio de Sanidad y Consumo (FIS PI07/0236) and from Fundación para la Investigación y la prevención del SIDA en España (FIPSE 36644/07 and 36650/07). SR received grants from Fondo de Investigación Sanitaria (FIS) del Ministerio de Ciencia e Innovación (PI07/90201; PI08/0738), Instituto de Salud Carlos III (UIPY 1467/07) and Fundación para la Investigación y la Prevención del SIDA en España (FIPSE) (36650/07). 12 Octubre Hospital: Ivacaftor mouse M. I. González-Tomé and P. Rojo; Gregorio Marañón

Hospital: S. Resino, B. Larrú, R. Resino, J. M. Bellón, M. D. Gurbindo, M. L. Navarro, J. Saavedra and M. A. Muñoz-Fernández; La Paz Hospital: M. I. Isabel de José; Carlos III Hospital: P. Martín-Fontelos and M. J. Mellado; Niño Jesús Hospital: J. Martínez; Getafe Hospital: J. T. Ramos, S. Guillén, L. Prieto, B. Rubio and L. García San Miguel; Móstoles Hospital: M. A. Roa; Principe de Asturias Hospital: J. Beceiro; Leganés Hospital: C. Calvo. “
“After structured treatment interruption

(STI) of treatment for HIV-1, a fraction of patients maintain suppressed viral loads. Prospective identification of such patients might improve HIV-1 treatment, if selected patients are offered STI. We analysed the effect of previously identified genetic modulators of HIV-1 disease progression on patients’ ability to suppress viral replication after STI. Polymorphisms in the genes killer cell immunoglobulin-like receptor 3DLI (KIR3DL1)/KIR3DS1, human leucocyte antigen B (HLA-B) and HLA Complex P5 (HCP5), and this website a polymorphism affecting HLA-C surface expression were analysed in 130 Swiss HIV Cohort Study patients undergoing STI. Genotypes were correlated with viral load levels after STI. We observed a statistically Protein kinase N1 significant reduction in viral load

after STI in carriers of HLA-B alleles containing either the Bw480Thr or the Bw480Ile epitope (mean adjusted effect on post-STI viral load: −0.82 log HIV-1 RNA copies/ml, P < 0.001; and −1.12 log copies/ml, P < 0.001, respectively). No significant effects were detected for the other polymorphisms analysed. The likelihood of being able to control HIV-1 replication using a prespecified cut-off (viral load increase < 1000 copies/ml) increased from 39% in Bw4-negative patients to 53% in patients carrying Bw4-80Thr, and to 65% in patients carrying Bw4-80Ile (P = 0.02). These data establish a significant impact of HLA-Bw4 on the control of viral replication after STI. Antiretroviral therapy (ART) enables long-term control of HIV-1 infection through suppression of viral replication in the majority of treated individuals. This leads to substantial immune reconstitution, significantly delays morbidity and mortality, and transforms HIV infection into a chronic disease [1]. However, ART is not curative and life-long pharmacological treatment is required, which can lead to numerous adverse effects.

Both KCC2-FL and KCC2-ΔNTD can interact with the actin cytoskelet

Both KCC2-FL and KCC2-ΔNTD can interact with the actin cytoskeleton by direct structural interaction of the intracellular C-terminus with the actin-binding protein 4.1N (Li et al., 2007). We found aberrant actin and 4.1N

patterns in the neural tube of transgenic embryos. The cells of the neural tube had diffuse cytoplasmic levels of actin and 4.1N. Similar results were obtained in the neural cell line C17.2. The cytoplasmic staining in KCC2-overexpressing cells points to a redistribution of the 4.1N protein within the cell, perhaps leading to a defective formation of F-actin. KCC2-C568A did not produce similar effects on the actin cytoskeleton, indicating that the point mutation rendered KCC2 less effective in binding to GSK458 clinical trial Epacadostat 4.1N. Indeed, immunoprecipitation of the three variants of the KCC2 protein demonstrated a significantly lower binding of KCC2-C568A to 4.1N (Fig. 8). Previous studies have employed KCC2-C568A as a control for KCC2-FL overexpression (Cancedda et al., 2007; Reynolds et al., 2008). The lack of effects of KCC2-C568A was

suggested to be due to inactivation of the ion transport function. However, this interpretation does not exclude a structural effect of KCC2, as our data suggest. It is not clear whether the C568A mutation interferes with the folding or intracellular trafficking Beta adrenergic receptor kinase of the protein or resides in an important 4.1N-binding structure. However, the mutation lies within a central domain of the KCC2 protein, and the 4.1N-binding domain has been localized to the C-terminus (Li et al., 2007). As we have detected expression of KCC2-C568A at the protein level, we propose that the mutation has a major influence on the tertiary structure of KCC2, yielding a protein inactive both as an ion transporter and as an interacting partner of 4.1N. Taken together,

our results indicate that KCC2 regulates early neuronal differentiation and migration by effects mediated through direct structural interaction with 4.1N and the actin cytoskeleton. This interaction may be essential for neural tube development. We wish to thank Ruth Detlofsson, Panagiotis Papachristou, Maria Lindqvist and the Karolinska Center for Transgene Technologies for technical support, and Evan Y. Snyder for the C17.2 cells. This study was supported by grants from the Swedish Research Council, Stockholm County Council, M&M Wallenberg, Sällskapet Barnavård, Swedish Heart and Lung Foundations (E.H.), the Academy of Finland and the Sigrid Jusélius Foundation (K.K.). Z.H. is supported by the League of European Research Universities (LERU). K.K. is a member of the Finnish Center of Excellence in Molecular and Integrative Neuroscience Research.

The aqueous phase was added to a 07 volume of dPEG solution (20%

The aqueous phase was added to a 0.7 volume of dPEG solution (20% polyethylene glycol 6000, 2.5 M NaCl) on

ice for 10 min. Genomic DNA was finally recovered by centrifugation at 10 000 g at 4 °C for 10 min, washed in 70% ethanol, and resuspended in 50 μL of TE buffer. The concentrations of each genomic DNA were measured by NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE). Each 1.5 μg of genomic DNA was electrophoresed on 1.0% agarose gel in TAE buffer. We observed buy LY294002 hyphal contact in the compatible and incompatible combinations (Fig. 1). In the compatible pairing (V18 vs. V18), almost 70% of hyphal contact zones were anastomosed (Fig. 2). Various types of hyphal anastomosis (Rayner et al., 1994) were observed: tip to tip, tip to side, side to side, and side to side extending hyphal neogenesis (data not shown). In the incompatible pairing, the frequency of hyphal anastomosis was reduced and most of contacting hyphae crossed over each other (Fig. 2). We also evaluated the effects of the growing medium (i.e. water Ipilimumab agar and 1/10-strength oatmeal agar media), with or without activated charcoal. In the compatible pairing, supplementation with activated charcoal significantly decreased the frequency of fusion but significantly increased the frequencies of cross-over. In the incompatible combination, supplementation with activated charcoal increased

the frequency of hyphal fusion and cross-over but the changes were not dramatic (Fig. 2). TEM observation in the compatible combination (V18 vs. V18) revealed that hyphal anastomosis, cell wall fusion and reconstruction of septa had occurred (Fig. 3b). We also recognized a healthy plasma membrane, tonoplast, mitochondria, and nuclear membrane. In the incompatible combination (V18 vs. V670), we observed the disconnection of membranes (plasma membrane, tonoplast, nuclear membrane, and mitochondria membrane) and collapse of cell contents (cytoplasm, nuclei and nucleolus, mitochondria, and vacuole) (Fig. 3c–f). When we noticed that some

cell structures had collapsed at the hyphal contact zones, we rated the parts of the cell Meloxicam collapse by each cell component. We calculated the percentage of collapsed organella from 50 hyphae in which the following were observed: disconnection of tonoplast (10%), disconnection of tonoplast and plasma membrane (22%), disconnection of tonoplast, plasma membrane, and nuclear membrane (36%), collapse of most cell contents except mitochondria (28%), and collapse of all cell contents (4%). We found that the collapse of plasma membrane, nuclear membrane, and mitochondria never preceded the collapse of tonoplast. We estimated that PCD occurred in the following order: (1) collapse of the tonoplast, (2) collapse of the plasma membrane and nuclear membrane, (3) reduction of the electron density of nuclei and nucleolus, and (4) collapse of mitochondria.