DivIVA is required for polar growth in the MreB-lacking rod-shaped actinomycete Corynebacterium glutamicum

The actinomycete Corynebacterium glutamicum grows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVA protein (DivIVA(Cg)) resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA from Streptomyces or Mycobacterium localized to the cell poles of DivIVA(Cg)-depleted C. glutamicum and restored polar peptidoglycan synthesis, in contrast to DivIVA proteins from Bacillus subtilis or Streptococcus pneumoniae, which localized at the septum of C. glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVA(Cg) localized at the septum after cell wall synthesis had started and the nucleoids had already segregated, suggesting that in C. glutamicum DivIVA is not involved in cell division or chromosome segregation.     

High Mutability of the Tumor Suppressor Genes RASSF1 and RBSP3 (CTDSPL) in Cancer

Background

Many different genetic alterations are observed in cancer cells. Individual cancer genes display point mutations such as base changes, insertions and deletions that initiate and promote cancer growth and spread. Somatic hypermutation is a powerful mechanism for generation of different mutations. It was shown previously that somatic hypermutability of proto-oncogenes can induce development of lymphomas.

Methodology/Principal Findings

We found an exceptionally high incidence of single-base mutations in the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) both located in 3p21.3 regions, LUCA and AP20 respectively. These regions contain clusters of tumor suppressor genes involved in multiple cancer types such as lung, kidney, breast, cervical, head and neck, nasopharyngeal, prostate and other carcinomas. Altogether in 144 sequenced RASSF1A clones (exons 1–2), 129 mutations were detected (mutation frequency, MF = 0.23 per 100 bp) and in 98 clones of exons 3–5 we found 146 mutations (MF = 0.29). In 85 sequenced RBSP3 clones, 89 mutations were found (MF = 0.10). The mutations were not cytidine-specific, as would be expected from alterations generated by AID/APOBEC family enzymes, and appeared de novo during cell proliferation. They diminished the ability of corresponding transgenes to suppress cell and tumor growth implying a loss of function. These high levels of somatic mutations were found both in cancer biopsies and cancer cell lines.

Conclusions/Significance

This is the first report of high frequencies of somatic mutations in RASSF1 and RBSP3 in different cancers suggesting it may underlay the mutator phenotype of cancer. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread.     

The landscape of human mutually exclusive splicing

Mutually exclusive splicing of exons is a mechanism of functional gene and protein diversification with pivotal roles in organismal development and diseases such as Timothy syndrome, cardiomyopathy and cancer in humans. In order to obtain a first genomewide estimate of the extent and biological role of mutually exclusive splicing in humans, we predicted and subsequently validated mutually exclusive exons (MXEs) using 515 publically available RNA‐Seq datasets. Here, we provide evidence for the expression of over 855 MXEs, 42% of which represent novel exons, increasing the annotated human mutually exclusive exome more than fivefold. The data provide strong evidence for the existence of large and multi‐cluster MXEs in higher vertebrates and offer new insights into MXE evolution. More than 82% of the MXE clusters are conserved in mammals, and five clusters have homologous clusters in Drosophila. Finally, MXEs are significantly enriched in pathogenic mutations and their spatio‐temporal expression might predict human disease pathology.     

Regulation of autophagy by cytoplasmic p53

Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53(-/-) cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.     

Food and secretagogue stimulation decrease the digestive enzyme content remaining in the rat pancreas

The aim of the study reported here was to investigate changes in the digestive enzyme content in the pancreas after food and secretagogue stimulation. Rats from which food had been withheld overnight were either fed (between 6 and 8 a.m.) or not before euthanasia and pancreatic excision (at 8 a.m.: 21 not fed and 21 fed) and at 4 (12 p.m.: six not fed and six fed) and 8 h later (4 p.m.: six not fed and six fed). Another 16 rats were anesthetized, fitted with jugular vein and pancreatic duct catheters, and infused with the secretagogues, CCK-33 and secretin, during 1.5 h of pancreatic juice collection before euthanasia and pancreatic excision. The pancreata were homogenized, and total soluble protein and individual enzyme (trypsin and amylase) tissue contents were analyzed. Results indicated lower amounts of protein and enzymes remaining in the pancreata of the fed, compared with non-fed rats. Enzyme values indicated recovery within four hours in fed rats, but non-fed rats also had increased values during daytime. High enzyme secretion during the high dose of hormonal stimulation was reflected in lower enzyme values remaining in the pancreas, compared with that in response to low-dose stimulation. Results indicated that stimulation of the pancreas, either by food ingestion or exogenous secretagogues, lowers the amounts of digestive enzymes remaining in the pancreas, and imply that stimulation and circadian rhythms influence the pancreatic enzyme content at euthanasia. This finding should be borne in mind in interpretation of data from pancreatic studies.     

Identification of functional p53-binding motifs in the mouse wig-1 promoter

With selective excitation around BChl-B800 and BChl-B850 absorption bands, we observed the evolution of excited-state dynamics in LH2 from Rhodobacter sphaeroides 601. The dynamical traces demonstrate a dominant excited-state absorption (ESA) followed concomitantly by an ultrafast transmission increase and decay with pulse-width limited time scale at 818 nm and 828 nm excitation. The ESA occurring prior to excitonic thermalization or ground-state bleach was observed at 840 nm as well. These experimental results indicate the competition between the transition from excitonic states to higher-lying excited states and interexciton relaxation, which are of physical significance for understanding excitation transfer and related mechanisms in LH2.     

Mitochondria and ischemic reperfusion damage in the adult and in the developing brain

The developing and the adult brain respond in similar ways to ischemia, but also display clear differences. For example, the relative contributions of necrosis and apoptosis to neuronal death may be different, such that apoptotic mechanisms would be more prevalent in the developing brain. During normal development, more than half of the neurons in some brain regions are removed through apoptosis, and effectors like caspase-3 are highly upregulated in the immature brain. Mitochondria are pivotal regulators of cell death through their role in energy production and calcium homeostasis, their capacity to release apoptogenic proteins and to produce reactive oxygen species. This review will summarize some of the current studies dealing with mitochondria-related mechanisms of ischemic brain damage, with special reference to developmental aspects.     

Effects of human cytomegalovirus infection on ligands for the activating NKG2D receptor of NK cells: up-regulation of UL16-binding protein (ULBP)1 and ULBP2 is counteracted by the viral UL16 protein

Human CMV (HCMV) interferes with NK cell functions at various levels. The HCMV glycoprotein UL16 binds some of the ligands recognized by the NK-activating receptor NKG2D, namely UL16-binding proteins (ULBP) 1 and 2 and MHC class I-related chain B, possibly representing another mechanism of viral immune escape. This study addressed the expression and function of these proteins in infected cells. HCMV induced the expression of all three ULBPs, which were predominantly localized in the endoplasmic reticulum of infected fibroblasts together with UL16. However, while at a lower viral dose ULBP1 and 2 surface expression was completely inhibited compared to ULBP3, at a higher viral dose cell surface expression of ULBP1 and ULBP2 was delayed. The induction of ULBPs correlated with an increased dependency on NKG2D for recognition; however, the overall NK sensitivity did not change (suggesting that additional viral mechanisms interfere with NKG2D-independent pathways for recognition). Infection with a UL16 deletion mutant virus resulted in a different pattern compared to the wild type: all three ULBP molecules were induced with similar kinetics at the cell surface, accompanied by a pronounced, entirely NKG2D-dependent increase in NK sensitivity. Together our findings show that upon infection with HCMV, the host cell responds by expression of ULBPs and increased susceptibility to the NKG2D-mediated component of NK cell recognition, but UL16 limits these effects by interfering with the surface expression of ULBP1 and ULBP2.     

Determination of aldehydes in human breath by on-fibre derivatization, solid-phase microextraction and GC-MS

A GC-MS method for the simultaneous determination of hexanal, heptanal, octanal, nonanal and decanal in exhaled breath was established and validated. The aldehydes were derivatized on PDMS/DVB fibres using O-2,2,4,5,6-(pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA) as the headspace derivatization reagent. The resultant oximes were quantified by GC-MS in selected ion monitoring (SIM) mode. The method provides detection limits of 0.01-0.03 nM for the aldehydes, with a linear response in the concentration range 0.002-20 nM. Within-day precision values for the five aldehydes at 0.02-0.04 nM and 0.2-0.4 nM were in the ranges: 3-9% and 3-8%, respectively; the corresponding between-day precision values were 11-22% and 10-24%. Exhaled breath samples could be stored at -20 degrees C for 48 h.     

Pathological apoptosis in the developing brain

More than half of the initially-formed neurons are deleted in certain brain regions during normal development. This process, whereby cells are discretely removed without interfering with the further development of remaining cells, is called programmed cell death (PCD). The term apoptosis is used to describe certain morphological manifestations of PCD. Many of the effectors of this developmental cell death program are highly expressed in the developing brain, making it more susceptible to accidental activation of the death machinery, e.g. following hypoxia-ischemia or irradiation. Recent evidence suggests, however, that activation and regulation of cell death mechanisms under pathological conditions do not exactly mirror physiological, developmentally regulated PCD. It may be argued that the conditions after e.g. ischemia are not even compatible with the execution of PCD as we know it. Under pathological conditions cells are exposed to various stressors, including energy failure, oxidative stress and unbalanced ion fluxes. This results in parallel triggering and potential overshooting of several different cell death pathways, which then interact with one another and result in complex patterns of biochemical manifestations and cellular morphological features. These types of cell death are here called "pathological apoptosis," where classical hallmarks of PCD, like pyknosis, nuclear condensation and caspase-3 activation, are combined with non-PCD features of cell death. Here we review our current knowledge of the mechanisms involved, with special focus on the potential for therapeutic intervention tailored to the needs of the developing brain.