The objective of this study was to evaluate the effects of dietary lysine on plasma concentrations of amino acid (AAs) and other metabolites of finishing pigs. Eighteen crossbred late-stage finishing pigs (9 barrows and 9 gilts) were assigned to three dietary treatments, Diets I (lysine-deficient), II (lysineequate), and III (lysine-excess), according to a randomized complete block design with gender as block. After 4 weeks on trial, jugular vein blood were collected and centrifuged for plasma samples, which were analyzed for concentrations of 24 AAs using HPLC method and 6 selected metabolites related to major nutrient metabolism using ACE Alera Clinical Chemistry System. Dietary lysine had a significant effect on plasma concentrations of 13 AAs and 3 metabolites (albumin, cholesterol, and urea nitrogen). The patterns of AA and metabolite concentrations alteration indicated complex metabolic interactions between lysine and other AAs, which subsequently had effects on other metabolites and animal growth performance.

Type II diabetes mellitus (TIIDM) causes multiple complications under chronic hyperglycemia. Long term persistent exposure to elevated glucose conditions is considered one of the major factors for diabetic complications. Pathologically, mechanical and biochemical stimuli will induce a signaling cascade in cardiac fibroblasts, which causes myocardial fibrosis and leading to ventricular stiffness. Non-enzymatically, high levels of glucose can react with long-lived proteins, such as collagen to form advanced glycation end-products (AGEs). AGEs have been shown to be associated with many of the diabetic cardiovascular complications due to their interaction with the receptor for AGE (RAGE). AGE/RAGE activation stimulates the secretion of growth factors, promotes increased collagen production that leads to tissue fibrosis, and increased RAGE expression. The purpose of this study is to identify the role for Rap1a in regulating fibrosis under TIIDM conditions, as well as to offer insight into the AGE-RAGE signaling cascade definition for cardiovascular extracellular matrix remodeling under TIIDM condition. To test our hypothesis, both loss-ofunction and gain-ofunction based experiments were performed to manipulate Rap1a protein expression in AGE-RAGE mediated fibrosis. Also, we down-regulated the activity of downstream molecules in the AGE-RAGE signaling cascade, such as protein kinase C-ζ (PKC-ζ) and ERK1/2 by specific inhibitor treatments, to test their positions in AGE-RAGE mediated fibrosis pathway. To perform our experiment in vivo, we used high fat diet to feed Rap1a heterozygous mice in order to build a Rap1a heterozygous diabetic animal model. Our results showed that Rap1a protein plays a key role in AGE-RAGE signaling pathway under TIIDM, and changes in Rap1a activity altered the signaling pathway. Also, we found that PKC-ζ is the upstream player relatively to ERK1/2, and Rap1a is the upstream player for both PKC-ζ and ERK1/2. By understanding the role Rap1a played in AGE-RAGE signaling cascade, a new molecular mechanism is found possibly to reduce the cardiac fibrosis in TIIDM patients.

The cultural significance of effigy pipes among southeastern groups during the Mississippian period (A.D. 1000-1600) has yet to be fully understood. Recent studies, however, have provided new archaeological contexts for framing explanations of their possible use and distribution among such groups. Apart from conjectures about their use as ceremonial objects, selection for effigy pipes in the Mississippian Southeast was directly related to fluctuating environmental and demographic conditions under which such objects were manufactured and distributed. These conditions provided the appropriate context for their emergence as costly signaling devices through which elite or special interest groups advertised fitness levels, typically expressed in displays of power and prestige. As signaling devices, effigy pipes attained their widest distribution in the Southeast during a time of environmental and demographic stability. Their decline was primarily the result of increasing climatic instability and widespread demographic upheaval--events that precipitated major disruptions in commercial and economic relations.

Changes in gene expression in response to mitochondrial dysfunction are mediated by components of the retrograde signaling pathway. The mitochondrial signal is recognized and transferred to the nucleus by dynamic interactions between regulatory proteins Rtg2p, Mks1p and Bmh1p. Retrograde signaling genes have been well characterized in the budding yeast Saccharomyces cerevisiae but very little is known about the retrograde response of other fungi. To identify retrograde signaling proteins in other fungi, the protein sequence encoded by the S. cerevisiae RTG2 gene was used to search for fungal homologs using NCBI BlastP and the T-Coffee Multiple Sequence Alignment program. We selected four species having uncharacterized ORFs with more than 66% amino acid identity to Rtg2p for further analysis: Ashbya gossypii, Candida glabrata, Vanderwaltozyma polyspora and Kluyveromyces lactis. In S. cerevisiae, cells deleted for RTG2 are glutamate auxotrophs, and have reduced expression of Aco1p and Cit2p proteins. To determine whether the putative RTG2 genes we identified encode bonefide regulators of the retrograde response pathway, we used standard yeast genetic approaches and molecular biology tools to investigate their ability to complement the defects associated with the rtg2Ä mutant using our S. cerevisiae RTG2 shuffle strain. We investigated functional roles of Rtg2p homologs by comparing Cit2p and Aco1p protein levels, glutamate auxotrophy, as well as analyzing the interaction between Rtg2p homologs and Mks1p. We also analyzed sensitivity of mutant strains under various stress conditions to address possible signaling cross talk between the retrograde signaling pathway and the TOR pathway. Our data show that the fungal Rtg2p homologs from C. glabrata, V. polyspora and K. lactis are functional in mediating the mitochondrial signal through known components of the retrograde signaling cascade. Our immunoprecipitation data suggest that TOR and retrograde signaling may exhibit cross pathway activation under rapamycin treatment. We show that Mks1p, the negative regulator of retrograde signaling pathway is required for Cit2p expression under rapamycin treatment. Given that all Rtg2p homologs showed low affinity for Mks1p which was in turn paralleled by a higher affinity of Mks1p for Bmh1p suggests that Rtg2p may have an additional functional role in influencing the association of Mks1p with Bmh1p.

No description available.

Acute myeloid leukemia (AML) is characterized by a blockage in the differentiation of myeloid cells at different stages. CD44-ligation using anti-CD44 monoclonal antibodies (mAbs) has been shown to reverse the blockage of differentiation and to inhibit the proliferation of blasts in most AML-subtypes. However, the molecular mechanisms underlying this property have not been fully elucidated. Here, we sought to I) analyze the effects of anti-CD44 mAbs on downstream signaling pathways, including the ERK1/2 (extracellular signal-regulated kinase 1 and 2) and mTOR (mammalian target of rapamycin) pathways and II) use state-of-the-art Nuclear Magnetic Resonance (NMR) technology to determine the global metabolic changes during differentiation induction of AML cells using anti-CD44 mAbs and other two previously reported differentiation agents. In the first objective (Chapter 4), our studies provide evidence that CD44-ligation with specific mAbs in AML cells induced an increase in ERK1/2 phosphorylation. The use of the MEK inhibitor (U0126) significantly inhibited the CD44-induced differentiation of HL60 cells, suggesting that ERK1/2 is critical for the CD44-triggered differentiation in AML. In addition, this was accompanied by a marked decrease in the phosphorylation of the mTORC1 and mTORC2 complexes, which are strongly correlated with the inhibition of the PI3K/Akt pathway. In the second objective (Chapter 5), 1H NMR experiments demonstrated that considerable changes in the metabolic profiles of HL60 cells were induced in response to each differentiation agent. These most notable metabolites that significantly changed upon CD44 ligation were involved in the tricarboxylic acid (TCA) cycle and glycolysis such as, succinate, fumarate and lactate. Therefore, we sought to analyze the mechanisms underlying their alterations. Our results revealed that anti-CD44 mAbs treatment induced upregulation in fumarate hydratase (FH) expression and its activity which was accompanied by a decrease in succinate dehydrogenase (SDH) activity. Interestingly, our results indicated that FH induced by anti-CD44 mAb is regulated through the activation of the ERK1/2 pathway. Therefore, our findings highlight new elements in support for the use of anti-CD44 mAbs in AML therapies and open new perspectives to use metabolic profiling as a tool to support the potential possibilities for the development of CD44-targeted therapy of AML.

The Hippo pathway is frequently deregulated in human cancers, but mutations and deletions of core signalome members are rare, suggesting that our understanding of its upstream regulators remains incomplete. A focused RNAi-based kinome screen identified novel candidate regulators of Hippo signaling, including STK25 and MST4. Here, we characterize the kinase STK25 as a novel upstream activator of LATS signaling. Depletion of STK25 was found to significantly reduce YAP phosphorylation in response to Hippo-activating stimuli, with consequent increases in YAP/TAZ activity and increased proliferation and resistance to cell cycle arrest. Mechanistically, STK25 activates LATS independently of the canonical MST/MAP4K axis, wherein STK25 directly promotes the phosphorylation of the LATS kinase activation loop in the absence of a preceding hydrophobic motif phosphorylation event. This differentiates STK25 from all other identified Hippo kinases to date, which may explain why singular loss of this kinase cannot be compensated for by the presence of other Hippo kinases. We also find that loss of STK25 increases YAP/TAZ signaling in vivo and that this promotes organ overgrowth in murine models. Interestingly, STK25 is frequently focally deleted in a spectrum of human cancers, suggesting that its loss might represent a way by which cancer cells functionally inactivate Hippo signaling. We also report that STK25 may be a novel regulator of mTOR signaling, as loss of STK25 hyper-activates mTOR signaling in response to amino acids and growth factors, but not to energy stresses. Interestingly, we find that MST4, a kinase closely related to STK25, appears to have highly context-specific Hippo regulatory functions; loss of MST4 was found to modulate Hippo signaling only in non-polarized cell lines, suggesting that polarity-responsive subcellular localization of MST4 may dictate its ability to interact with Hippo signaling. Lastly, we describe a novel role for Hippo signaling as a surveillance system for abnormal prolongation of mitosis, in which the LATS kinases regulate cell fate following abnormal mitosis via its control over p53-p21 signaling and YAP/TAZ signaling. This work thus identifies new roles and mechanisms by which kinases interact in the context of this major tumor suppressor pathway to control cellular processes critical to physiologic homeostasis. / 2021-10-07T00:00:00Z

Considering that both cardiovascular disease (CVD) and congenital heart diseases (CHD) are still the leading cause of morbidity and mortality worldwide, there is a need for a robust and reliable cardiac model. Cardiac organoids are complex, three-dimensional cellular constructs that recapitulate the processes of the human embryonic heart. However, certain vital morphological features within the fetus are not yet replicable with cardiac organoids. Here we report our investigation to generate cardiac organoids with chamber formations. Our method involves modulating the Wnt pathway at two different instances while also implementing two cell seeding densities, all to determine the most optimized that to produce chamber formations within cardiac organoids.

Molecular analysis of the cross talk between Angiotensin II (Ang II) and insulin signaling systems reveal that they are multifaceted and occur at cellular level and intracellular level. Experiments were carried out to evaluate the crosstalk between the Ang II receptors-AT1 and AT2 and the Insulin Receptor (IR) to understand the changes in the signaling pathway that could lead to the transition from hypertension to insulin resistance. Transient expression of rat AT2 in CHO cells induced co-immunoprecipitation of the AT2R with IRâ and inhibition of IRâ tyrosine phosphorylation. An AT2-peptide carrying the amino acids 226-363 (that spans 3rd intracellular loop (ICL) and C-terminal cytoplasmic domain) was sufficient for AT2- IRâ interaction in a yeast two-hybrid assay. An orthovanadate-insensitive AT2- IRâ association was also observed in human breast cancer cell line MCF-7. Interestingly, while AT2- IRâ complex formation was insensitive to pertussis toxin (PTX), AT2-mediated inhibition of IRâ phosphorylation was partially sensitive to PTX treatment in MCF-7. To address the mechanism behind the transition of an early hypertensive heart to an insulin resistant status, we investigated the changes that occur at post translational level in the IR and its downstream signaling molecules that modulate insulin signaling. Early hypertension was induced in 10-week old SD rats by 2% NaCl diet in combination with Ang II infusion. Enhanced serine phosphorylation of the IRâ suggestive of dysfunctional insulin signaling was observed in cardiac tissues as a result of the treatment. In addition, an enhanced association of both AT1R and AT2R with IRâ was observed in the heart tissue lysates from hypertensive rat heart. To evaluate the tissue effects of Ang II, we compared the transcriptome of hypertensive rat hearts to the controls. Analysis suggests that the Ang II induces multiple responses in heart tissue that result in changes to the gene expression pattern intended to promote insulin sensitivity and insulin resistance. Taken together our results suggest that exogenous Ang II and moderately high salt diet promote metabolic abnormalities in heart tissue that result in sequestration of IR and modulation of IR signaling, and significant changes in gene expression profile in the hypertensive heart.

No description available.