Forced involution of the functionally differentiated mammary gland by overexpression of the pro-apoptotic protein bax

The mammary gland is a developmentally dynamic, hormone-responsive organ that undergoes proliferation and differentiation within the secretory epithelial compartment during pregnancy. The epithelia are maintained by pro-survival signals (e.g., Stat5, Akt1) during lactation, but undergo apoptosis during involution through inactivation of cell survival pathways and upregulation of pro-apoptotic proteins. To assess if the survival signals in the functionally differentiated mammary epithelial cells can override a pro-apoptotic signal, we generated transgenic mice that express Bax under the whey acidic protein (WAP) promoter. WAP-Bax females exhibited a lactation defect and were unable to nourish their offspring. Mammary glands demonstrated: (1) a reduction in epithelial content, (2) hallmark signs of mitochondria-mediated cell death, (3) an increase in apoptotic cells by TUNEL assay, and (4) precocious Stat3 activation. This suggests that upregulation of a single pro-apoptotic factor of the Bcl-2 family is sufficient to initiate apoptosis of functionally differentiated mammary epithelial cells in vivo.     

Altering pyrroloquinoline quinone nutritional status modulates mitochondrial, lipid, and energy metabolism in rats

We have reported that pyrroloquinoline quinone (PQQ) improves reproduction, neonatal development, and mitochondrial function in animals by mechanisms that involve mitochondrial related cell signaling pathways. To extend these observations, the influence of PQQ on energy and lipid relationships and apparent protection against ischemia reperfusion injury are described herein. Sprague-Dawley rats were fed a nutritionally complete diet with PQQ added at either 0 (PQQ-) or 2 mg PQQ/Kg diet (PQQ+). Measurements included: 1) serum glucose and insulin, 2) total energy expenditure per metabolic body size (Wt(3/4)), 3) respiratory quotients (in the fed and fasted states), 4) changes in plasma lipids, 5) the relative mitochondrial amount in liver and heart, and 6) indices related to cardiac ischemia. For the latter, rats (PQQ- or PQQ+) were subjected to left anterior descending occlusions followed by 2 h of reperfusion to determine PQQ's influence on infarct size and myocardial tissue levels of malondialdehyde, an indicator of lipid peroxidation. Although no striking differences in serum glucose, insulin, and free fatty acid levels were observed, energy expenditure was lower in PQQ- vs. PQQ+ rats and energy expenditure (fed state) was correlated with the hepatic mitochondrial content. Elevations in plasma di- and triacylglyceride and β-hydroxybutryic acid concentrations were also observed in PQQ- rats vs. PQQ+ rats. Moreover, PQQ administration (i.p. at 4.5 mg/kg BW for 3 days) resulted in a greater than 2-fold decrease in plasma triglycerides during a 6-hour fast than saline administration in a rat model of type 2 diabetes. Cardiac injury resulting from ischemia/reperfusion was more pronounced in PQQ- rats than in PQQ+ rats. Collectively, these data demonstrate that PQQ deficiency impacts a number of parameters related to normal mitochondrial function.     

Probenecid inhibits the human bitter taste receptor TAS2R16 and suppresses bitter perception of salicin

Bitter taste stimuli are detected by a diverse family of G protein-coupled receptors (GPCRs) expressed in gustatory cells. Each bitter taste receptor (TAS2R) responds to an array of compounds, many of which are toxic and can be found in nature. For example, human TAS2R16 (hTAS2R16) responds to β-glucosides such as salicin, and hTAS2R38 responds to thiourea-containing molecules such as glucosinolates and phenylthiocarbamide (PTC). While many substances are known to activate TAS2Rs, only one inhibitor that specifically blocks bitter receptor activation has been described. Here, we describe a new inhibitor of bitter taste receptors, p-(dipropylsulfamoyl)benzoic acid (probenecid), that acts on a subset of TAS2Rs and inhibits through a novel, allosteric mechanism of action. Probenecid is an FDA-approved inhibitor of the Multidrug Resistance Protein 1 (MRP1) transporter and is clinically used to treat gout in humans. Probenecid is also commonly used to enhance cellular signals in GPCR calcium mobilization assays. We show that probenecid specifically inhibits the cellular response mediated by the bitter taste receptor hTAS2R16 and provide molecular and pharmacological evidence for direct interaction with this GPCR using a non-competitive (allosteric) mechanism. Through a comprehensive analysis of hTAS2R16 point mutants, we define amino acid residues involved in the probenecid interaction that result in decreased sensitivity to probenecid while maintaining normal responses to salicin. Probenecid inhibits hTAS2R16, hTAS2R38, and hTAS2R43, but does not inhibit the bitter receptor hTAS2R31 or non-TAS2R GPCRs. Additionally, structurally unrelated MRP1 inhibitors, such as indomethacin, fail to inhibit hTAS2R16 function. Finally, we demonstrate that the inhibitory activity of probenecid in cellular experiments translates to inhibition of bitter taste perception of salicin in humans. This work identifies probenecid as a pharmacological tool for understanding the cell biology of bitter taste and as a lead for the development of broad specificity bitter blockers to improve nutrition and medical compliance.     

A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C

CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.     

Mosaic gene expression in nuclear transfer-derived embryos and the production of cloned transgenic pigs from ear-derived fibroblasts

Genetically modified domestic animals have many potential applications ranging from basic research to production agriculture. One of the goals in transgenic animal production schemes is to reliably predict the expression pattern of the foreign gene. Establishing a method to screen genetically modified embryos for transgene expression before transfer to surrogates may improve the likelihood of producing offspring with the desired expression pattern. In order to determine how transgene expression may be regulated in the early embryo, we generated porcine embryos from two distinct genetically modified cell lines by using the nuclear transfer (NT) technique. Both cell lines expressed the enhanced green fluorescent protein (eGFP); the first was a fibroblast cell line derived from the skin of a newborn pig that expressed eGFP, whereas the second was a fetal derived fibroblast cell line into which the eGFP gene was introduced by a retroviral vector. The reconstructed embryos were activated by electrical pulses and cultured in NCSU23. Although the in vitro developmental ability of each group of NT embryos was not different, the eGFP expression pattern was different. All embryos produced from the transduced fetal cell line fluoresced, but only 26% of the embryos generated from the newborn cell line fluoresced, and among those that did express eGFP, more than half had a mosaic expression pattern. This was unexpected because the fetal cell line was not clonally selected, and each cell had potentially different sites of integration. Embryos generated from the newborn cell line were surgically transferred to five surrogate gilts. One gilt delivered four female piglets, all of which expressed eGFP, and all had microsatellites identical to the donor. Here we demonstrate that transgene expression in all the blastomeres of an NT embryo is not uniform. In addition, transgene expression in a genetically manipulated embryo may not be an accurate indicator of expression in the resulting offspring.     

Electrolyte,Blood Urea Nitrogen and Glucose Level Screening in Medical Admissions: Impact on Patient Management

Investigators have failed to show the usefulness of screening electrolyte—sodium, potassium, chloride and bicarbonate—blood urea nitrogen and glucose levels. In spite of this, we observed that that practice continues to be widely used at our university medical center. Using a form of consensus analysis, we examined the records of 301 admissions to the medicine service to determine whether laboratory tests were done for diagnostic or screening purposes and whether screening test results led to changes in patient management. Of the 1,764 tests done, 716 (40.6%) were for screening purposes. Only 2 (0.3%) screening test abnormalities led to any therapeutic changes, and many false-positive tests led to unnecessary diagnostic retesting.     

A Carboxyl Terminal Leucine Zipper Is Required for Tyrosine Hydroxylase Tetramer Formation

Abstract: Tyrosine hydroxylase catalyzes the rate-limiting reaction in the biosynthesis of the catecholamine neurotransmitters and hormones (dopamine, norepinephrine, and epinephrine). Rat tyrosine hydroxylase exists, in its native form, as a tetramer composed of identical 498 amino acid subunits. There is currently no information describing the molecular interactions by which the four monomeric tyrosine hydroxylase subunits assemble into an active tetramer. Mutational analysis was performed on bacterially expressed enzyme to assess the role of a putative C-terminal leucine zipper in the assembly of subunits into the tetrameric holoenzyme. Deletion of the C-terminal 19 amino acids, or mutation of a leucine residue (to an alanine), converts the enzyme from a tetrameric to a dimeric form that exhibits greater structural heterogeneity. This change in macromolecular form is accompanied by a 75% (deletion mutation) to 20% (Leu → Ala mutation) reduction in specific activity of the enzyme. This represents the first report of the functional involvement of a region containing a leucine zipper motif in the assembly and activity of a neuronal enzyme.     

Role of internal thermodynamics in determining hydrogen tunneling in enzyme-catalyzed hydrogen transfer reactions.

Previous investigations have indicated a role for hydrogen tunneling in the yeast alcohol dehydrogenase catalyzed oxidation of benzyl alcohol [Cha, Y., Murray, C. J., & Klinman, J. P. (1989) Science 243, 1325] and the bovine plasma amine oxidase catalyzed oxidation of benzylamine [Grant, K.L., & Klinman, J. P. (1989) Biochemistry 28,6597]. In the present studies, values of protium to tritium and deuterium to tritium isotope effects and their temperature dependencies have been measured using ring-substituted substrates for yeast alcohol dehydrogenase and bovine plasma amine oxidase, revealing tunneling in each case. The results of these studies indicate that hydrogen tunneling is a general phenomenon and is not limited to enzyme reactions with degenerate energy levels for bound substrates and products. An analysis of internal thermodynamics in the yeast alcohol dehydrogenase reaction shows that tunneling occurs when delta H degrees is endothermic and that the degree of tunneling appears to increase as delta H degrees decreases toward zero.