Carol Norris

Protein quaternary structure and expression levels contribute to peroxisomal-targeting-sequence-1-mediated peroxisomal import of human soluble epoxide hydrolase.

The peroxisomal targeting sequence 1 (PTS1) is a consensus tripeptide 1 (S/C/A)(K/R/H)(L/M) that is found at the C-terminus of most peroxisomal proteins. However, the only known mammalian protein containing a terminal methionine PTS1 (SKM), human soluble epoxide hydrolase (hsEH), shows both peroxisomal and cytosolic localizations in vivo. Mechanisms regulating the subcellular localization of hsEH thus remain unclear. Here we utilized green fluorescent protein-hsEH fusion constructs to study the peroxisomal targeting of hsEH in transiently and stably transfected Chinese hamster ovary cells. Our results suggest that the peroxisomal import of hsEH is regulated by three factors. First, we show that SKM is required, but not sufficient, for peroxisomal import. Second, by manipulating protein expression levels, we show that SKM mediates peroxisomal import of wild-type hsEH only when expression levels are high. Third, we show that amino acid modifications that decrease subunit oligomerization and presumably enhance accessibility of the SKM motif confer peroxisomal targeting even at low protein expression levels. We conclude that, in hsEH, SKM is a necessary but inefficient and context-dependent PTS1. Peroxisomal import occurs when expression levels are high or when the SKM motif is accessible. These results provide a mechanistic basis for understanding the cell-specific and tissue-specific localization of hsEH in vivo.

Effect of Octenidine Hydrochloride on Planktonic Cells and Biofilms of Listeria monocytogenes

ABSTRACT Listeria monocytogenes is a food-borne pathogen capable of forming biofilms and persisting in food processing environments for extended periods of time, thereby potentially contaminating foods. The efficacy of octenidine hydrochloride (OH) for inactivating planktonic cells and preformed biofilms of L. monocytogenes was investigated at 37, 21, 8, and 4°C in the presence and absence of organic matter (rehydrated nonfat dry milk). OH rapidly killed planktonic cells and biofilms of L. monocytogenes at all four temperatures. Moreover, OH was equally effective in killing L. monocytogenes biofilms on polystyrene and stainless steel matrices in the presence and absence of organic matter. The results underscore OHs ability to prevent establishment of L. monocytogenes biofilms by rapidly killing planktonic cells and to eliminate preformed biofilms, thus suggesting that it could be used as a disinfectant to prevent L. monocytogenes from persisting in food processing environments.

Neurochemical Heterogeneity Among Lateral Hypothalamic Hypocretin/Orexin and Melanin-Concentrating Hormone Neurons Identified Through Single-Cell Gene Expression Analysis

Abstract The lateral hypothalamic area (LHA) lies at the intersection of multiple neural and humoral systems and orchestrates fundamental aspects of behavior. Two neuronal cell types found in the LHA are defined by their expression of hypocretin/orexin (Hcrt/Ox) and melanin-concentrating hormone (MCH) and are both important regulators of arousal, feeding, and metabolism. Conflicting evidence suggests that these cell populations have a more complex signaling repertoire than previously appreciated, particularly in regard to their coexpression of other neuropeptides and the machinery for the synthesis and release of GABA and glutamate. Here, we undertook a single-cell expression profiling approach to decipher the neurochemical phenotype, and heterogeneity therein, of Hcrt/Ox and MCH neurons. In transgenic mouse lines, we used single-cell quantitative polymerase chain reaction (qPCR) to quantify the expression of 48 key genes, which include neuropeptides, fast neurotransmitter components, and other key markers, which revealed unexpected neurochemical diversity. We found that single MCH and Hcrt/Ox neurons express transcripts for multiple neuropeptides and markers of both excitatory and inhibitory fast neurotransmission. Virtually all MCH and approximately half of the Hcrt/Ox neurons sampled express both the machinery for glutamate release and GABA synthesis in the absence of a vesicular GABA release pathway. Furthermore, we found that this profile is characteristic of a subpopulation of LHA glutamatergic neurons but contrasts with a broad population of LHA GABAergic neurons. Identifying the neurochemical diversity of Hcrt/Ox and MCH neurons will further our understanding of how these populations modulate postsynaptic excitability through multiple signaling mechanisms and coordinate diverse behavioral outputs.

Differential effects of Akt isoforms on somatic cell reprogramming

ABSTRACT Akt plays an important role in cell growth, proliferation and survival. The specific roles of the three Akt isoforms in somatic cell reprogramming have not been investigated. Here we report that, during iPSC generation, enhanced Akt1 activity promotes complete reprogramming mainly through increased activation of Stat3 in concert with leukemia inhibitory factor (LIF) and, to a lesser extent, through promotion of colony formation. Akt1 augments Stat3 activity through activation of mTOR and upregulation of LIF receptor expression. Similarly, enhanced Akt2 or Akt3 activation also promotes reprogramming and coordinates with LIF to activate Stat3. Blocking Akt1 or Akt3 but not Akt2 expression prohibits cell proliferation and reprogramming. Furthermore, the halt in cell proliferation and reprogramming caused by mTOR and Akt inhibitors can be reversed by inhibition of GSK3. Finally, we found that expressing the GSK3&bgr; target Esrrb overrides inhibition of Akt and restores reprogramming. Our data demonstrated that during reprogramming, Akt promotes establishment of pluripotency through co-stimulation of Stat3 activity with LIF. Akt1 and Akt3 are essential for the proliferation of reprogrammed cells, and Esrrb supports cell proliferation and complete reprogramming during Akt signaling.

Discovery of Two Distinct Small Heat Shock Protein (HSP) Families in the Desert Fish Poeciliopsis

The availability of a genetically pedigreed fish colony at the University of Connecticut has made possible a detailed investigation of the heat shock response of a Poikilothermic vertebrate. During the past 12 years, two tropical species, six desert species and numerous interspecific hybrids of viviparous fishes in the genus Poeciliopsis have been studied. In the natural environment, these animals occupy thermally distinct niches that range in latitude from southern Arizona, through the Sonora Desert of northwestern Mexico across tropical southern Mexico and Central America into Columbia in South America. The diverse habitats include: (1) relatively stable tropical rivers with narrow annual and daily temperature ranges, (2) unstable streams that cross subtropical desert where temperatures range from 4 to 40°C seasonally and undergo rapid daily changes of as much as 22°C in 3h, and (3) pristine headwaters at higher elevations in the foothills of the Sierra Madre Occidental. When we began these studies, no detailed analysis of diversity in HSP families had been attempted. In fact, many investigators considered heat shock genes to be so highly conserved evolutionarily, that a search for variation would be fruitless. This view turned out to be overly pessimistic. Our approach to exploring HSP diversity employed high resolution two-dimensional Polyacrylamide gels to display protein isoforms at the level of denatured polypeptide chains. This strategy allowed a relatively rapid screening of multiple HSP families compared with the time required to clone and sequence all of the members of even one multigene family. Analyses of protein patterns also had the advantage of screening out nonfunctional genes and allowing a comparison of the levels of expression of different isoforms.

Akt3 is responsible for the survival and proliferation of embryonic stem cells

ABSTRACT The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway plays an important role in regulating cell proliferation, metabolism, and survival. However, the distinct roles of Akt isoforms (Akt1, Akt2, and Akt3) in pluripotent stem cell maintenance are not fully defined. Using mouse embryonic stem cells (ESCs), we show that direct inhibition of Akt activity leads to ESC apoptosis. The Akt3, but not Akt1 or Akt2, activity specifically regulates this effect. Inhibiting Akt3 also leads to a cell cycle arrest at G1 phase. These regulatory roles of Akt3 are dependent on its kinase activity. Blocking the expression of Akt1 plus Akt2 in ESCs does not affect cell survival or proliferation, although blocking Akt1 aggravates the apoptotic effect induced by depletion of Akt3. We further show that blocking Akt3 in ESCs results in significant nuclear accumulation of p53, as well as the activation of its downstream targets, such as Mdm2, p21, and Fas. Inhibiting p53 and its downstream targets partially rescued the effects caused by Akt3-depletion. Our results revealed an Akt3 isoform-specific mechanism for ESC survival and proliferation involving the control of p53 activity. Summary: We identified that Akt isoform 3, but not Akt1 or Akt2, specifically regulates embryonic stem cell survival and proliferation. Mechanistically, this is achieved partially through controlling the p53 pathway activity.

Selenium reduces enterohemorrhagic Escherichia coli O157:H7 verotoxin production and globotriaosylceramide receptor expression on host cells

AIM This study investigated the efficacy of selenium (Se) in reducing Escherichia coli O157:H7 verotoxin production and toxin gene expression. Additionally, the effect of Se on globotriaosylceramide (Gb3) receptor in human lymphoma cells was determined. MATERIALS & METHODS The effect of Se on verotoxin synthesis was determined by standard ELISA, whereas its effect on Gb3 receptor was determined by flow cytometry and real-time quantitative PCR. RESULTS & CONCLUSIONS Se reduced extracellular and intracellular verotoxin concentration by 40-60% and 80-90%, respectively (p < 0.05), and downregulated verotoxin genes (p < 0.05). Se reduced Gb3 receptor synthesis in lymphoma cells, and real-time quantitative PCR data revealed a significant downregulation of LacCer synthase gene (GalT2) involved in Gb3 synthesis. Further studies are warranted to validate these results in an appropriate animal model.

Chapter 16 - The Heat Shock Response of Tropical and Desert Fish (genus Poeciliopsis)

In the unstressed cell, constitutively expressed chaperones such as heat shock cognate 70 protein (HSC70) play a vital role in the folding of newly synthesized proteins, in other processes such as membrane translocation that require proteins to be maintained in an extended conformation, and in modulating the activity of multi protein complexes. During exposure to stress, an accumulation of unfolded protein exceeding the capacity of chaperones leads to the induction of heat shock protein (HSP) synthesis. Inducible HSPs such as HSP70 prevent irreversible protein aggregation during stress, and assist in the refolding process or in targeting nonfunctional proteins for degradation during recovery. Thermotolerance has been defined as the ability of a cell or organism to survive a normally lethal heat stress. The establishment and decay of the thermotolerant state has been correlated in both cultured cells and at the organismal level with changes in the levels of HSPs.