A Brain-specific Grb2-associated Regulator of Extracellular Signal-regulated Kinase (Erk)/Mitogen-activated Protein Kinase (MAPK) (GAREM) Subtype,GAREM2, Contributes to Neurite Outgrowth of Neuroblastoma Cells by Regulating Erk Signaling

Grb2-associated regulator of Erk/MAPK1 (GAREM) is an adaptor molecule in the EGF-mediated signaling pathway. GAREM is expressed ubiquitously in human organs and cultured cells. Two GAREM homologues are encoded by the human genome. Therefore, previously identified GAREM is named GAREM1. Here we characterized a new subtype of GAREM, GAREM2, that is specifically expressed in the mouse, rat, and human brain. Three GAREM2 tyrosines (Tyr-102, Tyr-429, and Tyr-551) are phosphorylated upon EGF stimulation and are necessary for binding to Grb2. Furthermore, GAREM2 and Shp2 regulate Erk activity in EGF-stimulated cells. These characteristics are similar to those of GAREM1. GAREM2 is expressed in some neuroblastoma cell lines and is also tyrosine-phosphorylated and bound to Grb2 after treatment with EGF. Eventually, GAREM2 regulates Erk activation in the presence of EGF or insulin like growth factor 1. GAREM2 also regulates insulin-like growth factor 1-induced neuronal differentiation of the SH-SY5Y neuroblastoma cell line. Although the structure and function of both GAREM subtypes are similar, GAREM1 is recruited into the nucleus and GAREM2 is not. Nuclear localization of GAREM1 might be controlled by a GAREM1-specific nuclear localization sequence and 14-3-3ϵ binding. The N-terminal 20 amino acids of GAREM1 make up its nuclear localization sequence that is also a 14-3-3ϵ binding site. The GAREM family is a new class of adaptor molecules with subtype-specific biological functions.     

M-CSF-mediated macrophage differentiation but not proliferation is correlated with increased and prolonged ERK activation

M-CSF is a cytokine essential for both the proliferation and differentiation of monocytes/macrophages. In this study, we established a new M-CSF-mediated differentiation-inducing system, and examined how the level and duration of the activation of ERK preceded M-CSF-mediated differentiation. TF-1-fms human leukemia cells rapidly proliferated in response to M-CSF. However, in the presence of a phorbol ester, TPA, TF-1-fms cells definitely switched their responsiveness to M-CSF from proliferation to differentiation, as evidenced by a more drastic morphological change and the appearance of cells with a higher level of phagocytic activity. In TF-1-fms cells expressing HIV-1 Nef protein in a conditionally active-manner, both M-CSF-mediated proliferation and M-CSF/TPA-mediated differentiation were inhibited by the activation of Nef. The Nef-active cells showed perturbed patterns of ERK activation. Under the proliferation-inducing conditions (TPA-free), parental or Nef-inactive cells showed modest ERK activation following M-CSF stimulation, whereas Nef-active cells showed an earlier and transient ERK activation, despite a decrease in their proliferation rate. Under the differentiation-inducing conditions, parental or Nef-inactive cells showed increased and prolonged ERK activation following M-CSF stimulation, whereas Nef-active cells showed transient ERK activation. These results supported the idea that the increased and prolonged ERK activation led to M-CSF-mediated macrophage differentiation but not to proliferation.     

Regulation of Vibrio parahaemolyticus T3SS2 gene expression and function of T3SS2 effectors that modulate actin cytoskeleton

Vibrio parahaemolyticus is a leading causative agent of seafood‐borne gastroenteritis worldwide. Most clinical isolates from patients with diarrhoea possess two sets of genes for the type III secretion system (T3SS) on each chromosome (T3SS1 and T3SS2). T3SS is a protein secretion system that delivers effector proteins directly into eukaryotic cells. The injected effectors modify the normal cell functions by altering or disrupting the normal cell signalling pathways. Of the two sets of T3SS genes present in V. parahaemolyticus, T3SS2 is essential for enterotoxicity in several animal models. Recent studies have elucidated the biological activities of several T3SS2 effectors and their roles in virulence. This review focuses on the regulation of T3SS2 gene expression and T3SS2 effectors that specifically target the actin cytoskeleton.     

Parental death in childhood and pathways to increased mortality across the life course in Stockholm,Sweden: A cohort study

BackgroundPrevious studies have shown that the experience of parental death during childhood is associated with increased mortality risk. However, few studies have examined potential pathways that may explain these findings. The aim of this study is to examine whether familial and behavioural factors during adolescence and socioeconomic disadvantages in early adulthood mediate the association between loss of a parent at age 0 to 12 and all-cause mortality by the age of 63.Methods and findingsA cohort study was conducted using data from the Stockholm Birth Cohort Multigenerational Study for 12,615 children born in 1953, with information covering 1953 to 2016. Familial and behavioural factors at age 13 to 19 included psychiatric and alcohol problems in the surviving parent, receipt of social assistance, and delinquent behaviour in the offspring. Socioeconomic disadvantage in early adulthood included educational attainment, occupational social class, and income at age 27 to 37. We used Cox proportional hazard regression models, combined with a multimediator analysis, to separate direct and indirect effects of parental death on all-cause mortality.Among the 12,582 offspring in the study (men 51%; women 49%), about 3% experienced the death of a parent in childhood. During follow-up from the age of 38 to 63, there were 935 deaths among offspring. Parental death was associated with an elevated risk of mortality after adjusting for demographic and household socioeconomic characteristics at birth (hazard ratio [HR]: 1.52 [95% confidence interval: 1.10 to 2.08, p-value = 0.010]). Delinquent behaviour in adolescence and income during early adulthood were the most influential mediators, and the indirect associations through these variables were HR 1.03 (1.00 to 1.06, 0.029) and HR 1.04 (1.01 to 1.07, 0.029), respectively. After accounting for these indirect paths, the direct path was attenuated to HR 1.35 (0.98 to 1.85, 0.066). The limitations of the study include that the associations may be partly due to genetic, social, and behavioural residual confounding, that statistical power was low in some of the subgroup analyses, and that there might be other relevant paths that were not investigated in the present study.ConclusionsOur findings from this cohort study suggest that childhood parental death is associated with increased mortality and that the association was mediated through a chain of disadvantages over the life course including delinquency in adolescence and lower income during early adulthood. Professionals working with bereaved children should take the higher mortality risk in bereaved offspring into account and consider its lifelong consequences. When planning and providing support to bereaved children, it may be particularly important to be aware of their increased susceptibility to delinquency and socioeconomic vulnerability that eventually lead to higher mortality.

In this cohort study, Ayako Hiyoshi and colleagues show associations between parental death in a child’s life and mortality risk later in that child’s life.     

A Mixture of Paraphenylenediamine and Imidazole: Its Effect on the Extraction of Lipid Droplets during Electron Microscopy Staining

To prevent extraction of lipids during a double staining procedure for electron microscopy, the tissue slices, double fixed with glutaraldehyde and osmium tetroxide to preserve microvesicular lipid droplets in the cytoplasm, were immersed for 2 hr in veronal buffer (pH 9.0) containing 0.5% p-phenylenediamine and 0.5% imidazole immediately after postfixation. The stained sections of the immersed tissue slice showed blackened, well circumscribed lipid droplets similar to those in corresponding unstained sections. Moreover, highly contrasting features of the cellular architecture could be visualized with the double stained, as well as routinely prepared sections.     

The Vibrio parahaemolyticus effector VopC mediates Cdc42‐dependent invasion of cultured cells but is not required for pathogenicity in an animal model of infection

Vibrio parahaemolyticus is a Gram‐negative marine bacterium that causes acute gastroenteritis in humans. The virulence of V. parahaemolyticus is dependent upon a type III secretion system (T3SS2). One effector for T3SS2, VopC, is a homologue of the catalytic domain of cytotoxic necrotizing factor (CNF), and was recently reported to be a Rho family GTPase activator and to be linked to internalization of V. parahaemolyticus by non‐phagocytic cultured cells. Here, we provide direct evidence that VopC deamidates Rac1 and CDC42, but not RhoA, in vivo. Our results alsosuggest that VopC, through its activation of Rac1, contributes to formation of actin stress fibres in infected cells. Invasion of host cells, which occurs at a low frequency, does not seem linked to Rac1 activation, but instead appears to require CDC42. Finally, using an infant rabbit model of V. parahaemolyticus infection, we show that the virulence of V. parahaemolyticus is not dependent upon VopC‐mediated invasion. Genetic inactivation of VopC did not impair intestinal colonization nor reduce signs of disease, including fluid accumulation, diarrhoea and tissue destruction. Thus, although VopC can promote host cell invasion, such internalization is not a critical step of the disease process, consistent with the traditional view of V. parahaemolyticus as an extracellular pathogen.     

The bactericidal activity of tea and coffee

Extracts of black tea, green tea, pu-erh tea or coffee inhibited the growth of various bacteria known to cause diarrhoeal diseases. Tea or coffee also showed bactericidal activity against Staphylococcus aureus and Vibrio parahaemolyticus.     

Interleukin-2 induces motility of human lymphocytes, but not their mobility

Interleukin-2 induces cytotoxic and antitumor activities of human lymphocytes. For the expression of these activities, the cytoskeletal system is probably activated. This study was do;ne to find if interleukin-2 causes cell movement. Lymphocytes were incubated with interleukin-2, and their morphology and motile activities were studied. After 72 hr of incubation, some 29% of lymphocytes were larger than before; nucleoli had formed and the microvilli were well-developed. The membrane potential of the lymphocytes increased during incubation. Motility under agar after 3 days of incubation with interleukin-2 was examined. Cells aggregated in clumps in the incubation well, and migration was not observed. When mobility was examined with Boyden's method, fewer cells incubated with interleukin-2 migrated than in control preparations. Cells incubated with interleukin-2 were extracted with Triton X-100. The extract obtained had three more bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the controls. The band were at the positions for the molecular weights of 270,000-300,000. We concluded that interleukin-2 activated the motility of lymphocytes, but not their mobility.