The exocytotic trafficking of TC10 occurs through both classical and nonclassical secretory transport pathways in 3T3L1 adipocytes

To examine the structural determinants necessary for TC10 trafficking, localization, and function in adipocytes, we generated a series of point mutations in the carboxyl-terminal targeting domain of TC10. Wild-type TC10 (TC10/WT) localized to secretory membrane compartments and caveolin-positive lipid raft microdomains at the plasma membrane. Expression of a TC10/C206S point mutant resulted in a trafficking and localization pattern that was indistinguishable from that of TC10/WT. In contrast, although TC10/C209S or the double TC10/C206,209S mutant was plasma membrane localized, it was excluded from both the secretory membrane system and the lipid raft compartments. Surprisingly, inhibition of Golgi membrane transport with brefeldin A did not prevent plasma membrane localization of TC10 or H-Ras. Moreover, inhibition of trans-Golgi network exit with a 19 degrees C temperature block did not prevent the trafficking of TC10 or H-Ras to the plasma membrane. These data demonstrate that TC10 and H-Ras can both traffic to the plasma membrane by at least two distinct transport mechanisms in adipocytes, one dependent upon intracellular membrane transport and another independent of the classical secretory membrane system. Moreover, the transport through the secretory pathway is necessary for the localization of TC10 to lipid raft microdomains at the plasma membrane.     

Glucose-stimulated insulin secretion is coupled to the interaction of actin with the t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein) complex

The actin monomer sequestering agent latrunculin B depolymerized beta-cell cortical actin, which resulted in increased glucose-stimulated insulin secretion in both cultured MIN6 beta-cells and isolated rat islet cells. In perifused islets, latrunculin B treatment increased both first- and second-phase glucose-stimulated insulin secretion without any significant effect on total insulin content. This increase in secretion was independent of calcium regulation because latrunculin B also potentiated calcium-stimulated insulin secretion in permeabilized MIN6 cells. Confocal immunofluorescent microscopy revealed a redistribution of insulin granules to the cell periphery in response to glucose or latrunculin B, which correlated with a reduction in phalloidin staining of cortical actin. Moreover, the t-SNARE [target membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins Syntaxin 1 and SNAP-25 coimmunoprecipitated polymerized actin from unstimulated MIN6 cells. Glucose stimulation transiently decreased the amount of actin coimmunoprecipitated with Syntaxin 1 and SNAP-25, and latrunculin B treatment fully ablated the coimmunoprecipitation. In contrast, the actin stabilizing agent jasplakinolide increased the amount of actin coimmunoprecipitated with the t-SNARE complex and prevented its dissociation upon glucose stimulation. These data suggest a mechanism whereby glucose modulates beta-cell cortical actin organization and disrupts the interaction of polymerized actin with the plasma membrane t-SNARE complex at a distal regulatory step in the exocytosis of insulin granules.     

Organogenesis of heart-vascular system derived from mouse 2 cell stage embryos and from early embryonic stem cells in vitro

Regenerative medical treatment with embryonic stem cells (an ES cell) is a goal for organ transplantation. Structures that are tubular in nature (i.e. blood capillaries) were induced from early embryonic stem (EES) cells in vitro using embryotrophic factor (ETFs). In addition, cardiac muscle cells could be identified as well. However, differentiation of EES cells into a complete cardiovascular system was difficult because 3 germ layer primordial organs are directed embryologically in various ways and it is not possible to guide only cardiovascular organs. Thus, we introduced ETFs after the formation of an embryoid body and were successful in cloning cell clusters that beat, thus deriving only cardiovascular organs. The application of this to the treatment of various cardiovascular diseases is promising.     

Structural features of N-glycans linked to glycoproteins from oil palm pollen, an allergenic pollen*

The pollen of oil palm (Elaeis guineensis Jacq.) is a strong allergen and causes severe pollinosis in Malaysia and Singapore. In the previous study (Biosci. Biotechnol. Biochem., 64, 820-827 (2002)), from the oil palm pollens, we purified an antigenic glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients. In this report, we describe the structural analysis of sugar chains linked to palm pollen glycoproteins to confirm the ubiquitous occurrence of antigenic N-glycans in the allergenic pollen. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine followed by purification with a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, electrospray ionization mass spectrometry (ESI-MS), and tandem MS analysis, as well as exoglycosidase digestions. The antigenic N-glycan bearing alpha1-3 fucose and/or beta1-2 xylose residues accounts for 36.9% of total N-glycans: GlcNAc2Man3Xyl1Fuc1GlcNAc2 (24.6%), GlcNAc2Man3Xyl1GlcNAc2 (4.4%), Man3Xyl1Fuc1-GlcNAc2 (1.1%), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (5.6%), and GlcNAc1Man3Xyl1GlcNAc2 (1.2%). The remaining 63.1% of the total N-glycans belong to the high-mannose type structure: Man9GlcNAc2 (5.8%), Man8GlcNAc2 (32.1%), Man7GlcNAc2 (19.9%), Man6GlcNAc2 (5.3%).     

Molecular Characterization of Photomixotrophic Tobacco Cells Resistant to Protoporphyrinogen Oxidase-Inhibiting Herbicides

Peroxidizing herbicides inhibit protoporphyrinogen oxidase (Protox), the last enzyme of the common branch of the chlorophyll- and heme-synthesis pathways. There are two isoenzymes of Protox, one of which is located in the plastid and the other in the mitochondria. Sequence analysis of the cloned Protox cDNAs showed that the deduced amino acid sequences of plastidial and mitochondrial Protox in wild-type cells and in herbicide-resistant YZI-1S cells are the same. The level of plastidial Protox mRNA was the same in both wild-type and YZI-1S cells, whereas the level of mitochondrial Protox mRNA YZI-1S cells was up to 10 times the level of wild-type cells. Wild-type cells were observed by fluorescence microscopy to emit strong autofluorescence from chlorophyll. Only a weak fluorescence signal was observed from chlorophyll in YZI-1S cells grown in the Protox inhibitor N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide. Staining with DiOC6 showed no visible difference in the number or strength of fluorescence between wild-type and YZI-1S mitochondria. Electron micrography of YZI-1S cells showed that, in contrast to wild-type cells, the chloroplasts of YZI-1S cells grown in the presence of N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide exhibited no grana stacking. These results suggest that the herbicide resistance of YZI-1S cells is due to the overproduction of mitochondrial Protox.     

RAD18 Activates the G2/M Checkpoint through DNA Damage Signaling to Maintain Genome Integrity after Ionizing Radiation Exposure

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure.     

Within-host Competition Does Not Select for Virulence in Malaria Parasites; Studies with Plasmodium yoelii

In endemic areas with high transmission intensities, malaria infections are very often composed of multiple genetically distinct strains of malaria parasites. It has been hypothesised that this leads to intra-host competition, in which parasite strains compete for resources such as space and nutrients. This competition may have repercussions for the host, the parasite, and the vector in terms of disease severity, vector fitness, and parasite transmission potential and fitness. It has also been argued that within-host competition could lead to selection for more virulent parasites. Here we use the rodent malaria parasite Plasmodium yoelii to assess the consequences of mixed strain infections on disease severity and parasite fitness. Three isogenic strains with dramatically different growth rates (and hence virulence) were maintained in mice in single infections or in mixed strain infections with a genetically distinct strain. We compared the virulence (defined as harm to the mammalian host) of mixed strain infections with that of single infections, and assessed whether competition impacted on parasite fitness, assessed by transmission potential. We found that mixed infections were associated with a higher degree of disease severity and a prolonged infection time. In the mixed infections, the strain with the slower growth rate was often responsible for the competitive exclusion of the faster growing strain, presumably through host immune-mediated mechanisms. Importantly, and in contrast to previous work conducted with Plasmodium chabaudi, we found no correlation between parasite virulence and transmission potential to mosquitoes, suggesting that within-host competition would not drive the evolution of parasite virulence in P. yoelii.     

Medical Decision-Making Incapacity among Newly Diagnosed Older Patients with Hematological Malignancy Receiving First Line Chemotherapy: A Cross-Sectional Study of Patients and Physicians

Background

Decision-making capacity to provide informed consent regarding treatment is essential among cancer patients. The purpose of this study was to identify the frequency of decision-making incapacity among newly diagnosed older patients with hematological malignancy receiving first-line chemotherapy, to examine factors associated with incapacity and assess physicians’ perceptions of patients’ decision-making incapacity.

Methods

Consecutive patients aged 65 years or over with a primary diagnosis of malignant lymphoma or multiple myeloma were recruited. Decision-making capacity was assessed using the Structured Interview for Competency and Incompetency Assessment Testing and Ranking Inventory-Revised (SICIATRI-R). Cognitive impairment, depressive condition and other possible associated factors were also evaluated.

Results

Among 139 eligible patients registered for this study, 114 completed the survey. Of these, 28 (25%, 95% confidence interval [CI]: 17%-32%) were judged as having some extent of decision-making incompetency according to SICIATRI-R. Higher levels of cognitive impairment and increasing age were significantly associated with decision-making incapacity. Physicians experienced difficulty performing competency assessment (Cohen’s kappa -0.54).

Conclusions

Decision-making incapacity was found to be a common and under-recognized problem in older patients with cancer. Age and assessment of cognitive impairment may provide the opportunity to find patients that are at a high risk of showing decision-making incapacity.     

Protracted Administration of L-Asparaginase in Maintenance Phase Is the Risk Factor for Hyperglycemia in Older Patients with Pediatric Acute Lymphoblastic Leukemia

Although L-asparaginase related hyperglycemia is well known adverse event, it is not studied whether the profile of this adverse event is affected by intensification of L-asparaginase administration. Here, we analyzed the profile of L-asparaginase related hyperglycemia in a 1,176 patients with pediatric acute lymphoblastic leukemia treated according to the Japan Association of Childhood Leukemia Study ALL-02 protocol using protracted L-asparaginase administration in maintenance phase. We determined that a total of 75 L-asparaginase related hyperglycemia events occurred in 69 patients. Although 17 events (17/1176, 1.4%) developed in induction phase, which was lower incidence than those (10–15%) in previous reports, 45 events developed during the maintenance phase with protracted L-asparaginase administration. Multivariate analysis showed that older age at onset (≥10 years) was a sole independent risk factor for L-asparaginase-related hyperglycemia (P<0.01), especially in maintenance phase. Contrary to the previous reports, obesity was not associated with L-asparaginase-related hyperglycemia. These findings suggest that protracted administration of L-asparaginase is the risk factor for hyperglycemia when treating adolescent and young adult acute lymphoblastic leukemia patients.     

A power-law distribution of inter-spike intervals in renal sympathetic nerve activity in salt-sensitive hypertension-induced chronic heart failure

To assess sympathetic variability in chronic heart failure (CHF), we evaluated a distribution of inter-spike intervals (ISIs) in renal sympathetic nerve activity (RSNA) in salt-sensitive hypertension-induced CHF (DSSH-CHF) rats. Dahl salt-sensitive rats were fed an 8% NaCl diet for 9 weeks to induce salt-sensitive hypertension-induced CHF. ISIs in RSNA were obtained from chronically instrumented conscious rats, and counts (frequency) and ranks of ISIs in RSNA were plotted with a histogram. We found that ISIs in RSNA followed a power-law distribution in rats, and the power-law distribution of ISIs for RSNA in DSSH-CHF rats was significantly different from that in normal rats. These results indicated that sympathetic variability may be significantly different between salt-sensitive hypertension-induced CHF and healthy individuals, which suggests that sympathetic variability may be used to predict abnormality of the sympathetic regulatory system.