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Molecules (Basel, Switzerland) Jan 2023The glycosylation of proteins is one of the most common post-translational modifications (PTMs) and plays important regulatory functions in diverse biological processes...
The glycosylation of proteins is one of the most common post-translational modifications (PTMs) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome, maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of tear film might promote the development of chronic eye diseases, indicating glycoproteins as a valuable source for biomarker discovery or drug target identification. Therefore, the present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and to characterize their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we first accumulated native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA, and UEA I, termed 4L) was applied to glycopeptide enrichment from human tear sample digests, followed by MS-based detection and localization of their specific N-glycosylation sites. As the main result, our study identified a total of 26 N glycosylation sites of 11 N-glycoproteins in the tear sample pools of healthy individuals ( = 3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP), and extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, already associated with the pathogenesis of various chronic eye diseases such as dry eye syndrome (DES). In conclusion, the results of the present study will serve as an important tear film N-glycoprotein catalog for future studies focusing on human tear film and ocular surface-related inflammatory diseases.
Topics: Humans; Glycopeptides; Glycoproteins; Glycosylation; Lectins; Mass Spectrometry; Protein Processing, Post-Translational; Tears
PubMed: 36677706
DOI: 10.3390/molecules28020648 -
Journal of Dairy Science Mar 2006Improving the prediction of milk protein yield relies on knowledge of both protein supply and requirement. Definition of protein/amino acid supply in ruminants is a... (Review)
Review
Improving the prediction of milk protein yield relies on knowledge of both protein supply and requirement. Definition of protein/amino acid supply in ruminants is a challenging task, due to feedstuff variety and variability and to the remodeling of nutrient intake by the rumen microflora. The questions arise, therefore, how and where should we measure the real supply of AA in the dairy cow? This review will follow the downstream flow of AA from duodenum to peripheral tissue delivery, with a glance at the efficiency of transfer into milk protein. Duodenal AA flow comprises rumen undegradable feed, microbial protein, and endogenous secretions. Most attention has been directed toward definition of the first two contributions but the latter fraction can represent as much as 20% of duodenal flow. More information is needed on what factors affect its magnitude and overall impact. Once digested, AA are absorbed into the portal vein. The ratio of portal absorption to small intestinal apparent digestion varies among essential AA, from 0.43 (threonine) to 0.76 (phenylalanine), due to the contributions of preduodenal endogenous secretions to the digestive flow, non-reabsorption of endogenous secretions and gut oxidation of AA. Few data are available on these phenomena in dairy cows but the evidence indicates that they alter the profile of AA available for anabolic purposes. Recent comparisons of estimated duodenal flux and measured portal flux have prompted a revisit of the NRC (2001) approach to estimate AA flows at the duodenum. Changes to the model are proposed that yield predictions that better fit the current knowledge of AA metabolism across the gut. After absorption, AA flow first to the liver where substantial and differential net removal occurs, varying from zero for the branched-chain AA to 50% of portal absorption for phenylalanine. This process alters the pattern of net supply to the mammary gland. Overall, intermediary metabolism of AA between the duodenum and the mammary gland biologically explains the decreased efficiency of the transfer of absorbed AA into milk protein as maximal yield is approached. Therefore, variable, rather than fixed, factors for transfer efficiencies must be incorporated into future predictive models.
Topics: Absorption; Amino Acids, Essential; Animal Nutritional Physiological Phenomena; Animals; Biological Availability; Cattle; Dairying; Dietary Proteins; Duodenum; Female; Intestinal Secretions; Intestine, Small; Models, Biological; Oxidation-Reduction; Portal Vein
PubMed: 16527873
DOI: 10.3168/jds.S0022-0302(06)72359-1 -
Pflugers Archiv : European Journal of... Mar 2020Mucin secretion by salivary mucous glands is mediated predominantly by parasympathetic acetylcholine activation of cholinergic muscarinic receptors via increased...
Mucin secretion by salivary mucous glands is mediated predominantly by parasympathetic acetylcholine activation of cholinergic muscarinic receptors via increased intracellular free calcium ([Ca]) and activation of conventional protein kinase C isozymes (cPKC). However, the parasympathetic co-neurotransmitter, vasoactive intestinal peptide (VIP), also initiates secretion, but to a lesser extent. In the present study, cross talk between VIP- and muscarinic-induced mucin secretion was investigated using isolated rat sublingual tubuloacini. VIP-induced secretion is mediated by cAMP-activated protein kinase A (PKA), independently of increased [Ca]. Synergistic secretion between VIP and the muscarinic agonist, carbachol, was demonstrated but only with submaximal carbachol. Carbachol has no effect on cAMP ± VIP. Instead, PKA activated by VIP releases Ca from an intracellular pool maintained by the sarco/endoplasmic reticulum Ca-ATPase pump. Calcium release was independent of phospholipase C activity. The resultant sustained [Ca] increase is additive to submaximal, but not maximal carbachol-induced [Ca]. Synergistic mucin secretion was mimicked by VIP plus either phorbol 12-myristate 13-acetate or 0.01 μM thapsigargin, and blocked by the PKC inhibitor, Gö6976. VIP-induced Ca release also promoted store-operated Ca entry. Synergism is therefore driven by VIP-mediated [Ca] augmenting cPKC activity to enhance muscarinic mucin secretion. Additional data suggest ryanodine receptors control VIP/PKA-mediated Ca release from a Ca pool also responsive to maximal carbachol. A working model of muscarinic and VIP control of mucous cell exocrine secretion is presented. Results are discussed in relation to synergistic mechanisms in other secretory cells, and the physiological and therapeutic significance of VIP/muscarinic synergism controlling salivary mucous cell exocrine secretion.
Topics: Adenosine Triphosphatases; Animals; Bodily Secretions; Calcium; Cholinergic Agents; Isoenzymes; Male; Mucins; Muscarinic Agonists; Phorbol Esters; Protein Kinase C; Rats; Rats, Wistar; Receptors, Muscarinic; Salivary Glands; Thapsigargin; Vasoactive Intestinal Peptide
PubMed: 31932898
DOI: 10.1007/s00424-020-02348-7 -
Life Science Alliance Dec 2020This study reports that parathymosin (PTMS) is secreted by hypothalamic stem/progenitor cells (htNSC) to inhibit senescence of recipient cells such as fibroblasts. Upon...
This study reports that parathymosin (PTMS) is secreted by hypothalamic stem/progenitor cells (htNSC) to inhibit senescence of recipient cells such as fibroblasts. Upon release, PTMS is rapidly transferred into the nuclei of various cell types, including neuronal GT1-7 cells and different peripheral cells, and it is effectively transferred into neuronal nuclei in various brain regions in vivo. Notably, brain neurons also produce and release PTMS, and because neuronal populations are large, they are important for maintaining PTMS in the cerebrospinal fluid which is further transferable into the blood. Compared with several other brain regions, the hypothalamus is stronger for long-distance PTMS transfer, supporting a key hypothalamic role in this function. In physiology, aging is associated with declines in PTMS production and transfer in the brain, and knockdown in the hypothalamus versus hippocampus were studied showing different contributions to neurobehavioral physiology. In conclusion, the brain is an endocrine organ through secretion and nuclear transfer of PTMS, and the hypothalamus-brain orchestration of this function is protective in physiology and counteractive against aging-related disorders.
Topics: Animals; Bodily Secretions; Brain; Endocrine Glands; Fibroblasts; Hippocampus; Hypothalamus; Mice; Mice, Inbred C57BL; Neurons; Stem Cells; Thymosin
PubMed: 33087487
DOI: 10.26508/lsa.202000917 -
Molecular and Cellular Biology May 2014Sebaceous glands are skin appendages that secrete sebum onto hair follicles to lubricate the hair and maintain skin homeostasis. In this study, we demonstrated that...
Sebaceous glands are skin appendages that secrete sebum onto hair follicles to lubricate the hair and maintain skin homeostasis. In this study, we demonstrated that Cidea is expressed at high levels in lipid-laden mature sebocytes and that Cidea deficiency led to dry hair and hair loss in aged mice. In addition, Cidea-deficient mice had markedly reduced levels of skin surface lipids, including triacylglycerides (TAGs) and wax diesters (WDEs), and these mice were defective in water repulsion and thermoregulation. Furthermore, we observed that Cidea-deficient sebocytes accumulated a large number of smaller-sized lipid droplets (LDs), whereas overexpression of Cidea in human SZ95 sebocytes resulted in increased lipid storage and the accumulation of large LDs. Importantly, Cidea was highly expressed in human sebaceous glands, and its expression levels were positively correlated with human sebum secretion. Our data revealed that Cidea is a crucial regulator of sebaceous gland lipid storage and sebum lipid secretion in mammals and humans.
Topics: Animals; Apoptosis Regulatory Proteins; Body Temperature Regulation; Cell Line; Epidermis; Hair; Humans; Lipid Metabolism; Mice; Mice, Knockout; Organ Specificity; Sebaceous Glands; Sebum; Triglycerides
PubMed: 24636991
DOI: 10.1128/MCB.01723-13 -
Cellular Microbiology Nov 2019Acid sphingomyelinase (ASM) is a lysosomal enzyme that cleaves the phosphorylcholine head group of sphingomyelin, generating ceramide. Recessive mutations in SMPD1, the... (Review)
Review
Acid sphingomyelinase (ASM) is a lysosomal enzyme that cleaves the phosphorylcholine head group of sphingomyelin, generating ceramide. Recessive mutations in SMPD1, the gene encoding ASM, cause Niemann-Pick Disease Types A and B. These disorders are attributed not only to lipid accumulation inside lysosomes but also to changes on the outer leaflet of the plasma membrane, highlighting an extracellular role for ASM. Secretion of ASM occurs under physiological conditions, and earlier studies proposed two forms of the enzyme, one resident in lysosomes and another form that would be diverted to the secretory pathway. Such differential intracellular trafficking has been difficult to explain because there is only one SMPD1 transcript that generates an active enzyme, found primarily inside lysosomes. Unexpectedly, studies of cell invasion by the protozoan parasite Trypanosoma cruzi revealed that conventional lysosomes can fuse with the plasma membrane in response to elevations in intracellular Ca , releasing their contents extracellularly. ASM exocytosed from lysosomes remodels the outer leaflet of the plasma membrane, promoting parasite invasion and wound repair. Here, we discuss the possibility that ASM release during lysosomal exocytosis, in response to various forms of stress, may represent a major source of the secretory form of this enzyme.
Topics: Animals; Bodily Secretions; Calcium; Cell Membrane; Ceramides; Exocytosis; Humans; Lysosomes; Niemann-Pick Disease, Type A; Niemann-Pick Disease, Type B; Protein Transport; Sphingomyelin Phosphodiesterase; Sphingomyelins; Trypanosoma cruzi
PubMed: 31155842
DOI: 10.1111/cmi.13065 -
European Journal of Pharmaceutics and... Jun 2021Accurate in vivo predictions of intestinal absorption of low solubility drugs require knowing their solubility in physiologically relevant dissolution media. Aspirated...
Accurate in vivo predictions of intestinal absorption of low solubility drugs require knowing their solubility in physiologically relevant dissolution media. Aspirated human intestinal fluids (HIF) are the gold standard, followed by simulated intestinal HIF in the fasted and fed state (FaSSIF/FeSSIF). However, current HIF characterization data vary, and there is also some controversy regarding the accuracy of FaSSIF and FeSSIF for predicting drug solubility in HIF. This study aimed at characterizing fasted and fed state duodenal HIF from 16 human volunteers with respect to pH, buffer capacity, osmolarity, surface tension, as well as protein, phospholipid, and bile salt content. The fasted and fed state HIF samples were further used to investigate the equilibrium solubility of 17 representative low-solubility small-molecule drugs, six of which were confidential industry compounds and 11 were known and characterized regarding chemical diversity. These solubility values were then compared to reported solubility values in fasted and fed state HIF, FaSSIF and FeSSIF, as well as with their human bioavailability for both states. The HIF compositions corresponded well to previously reported values and current FaSSIF and FeSSIF compositions. The drug solubility values in HIF (both fasted and fed states) were also well in line with reported solubility data for HIF, as well as simulated FaSSIF and FeSSIF. This indicates that the in vivo conditions in the proximal small intestine are well represented by simulated intestinal fluids in both composition and drug equilibrium solubility. However, increased drug solubility in the fed vs. fasted states in HIF did not correlate with the human bioavailability changes of the same drugs following oral administration in either state.
Topics: Administration, Oral; Biological Availability; Eating; Fasting; Humans; Intestinal Absorption; Intestinal Secretions; Intestine, Small; Pharmaceutical Preparations; Solubility
PubMed: 33872761
DOI: 10.1016/j.ejpb.2021.04.005 -
Frontiers in Immunology 2018S100A8 and S100A9 are members of the S100 family of cytoplasmic EF-hand Ca-binding proteins and are abundantly expressed in the cytosol of neutrophils. In addition to...
S100A8 and S100A9 are members of the S100 family of cytoplasmic EF-hand Ca-binding proteins and are abundantly expressed in the cytosol of neutrophils. In addition to their intracellular roles, S100A8/A9 can be secreted in the extracellular environment and are considered as alarmins able to amplify the inflammatory response. The intracellular activity of S100A8/A9 was shown to be regulated by S100A9 phosphorylation, but the importance of this phosphorylation on the extracellular activity of S100A8/A9 has not yet been extensively studied. Our work focuses on the impact of the phosphorylation state of secreted S100A9 on the proinflammatory function of neutrophils. In a first step, we characterized the secretion of S100A8/A9 in different stimulatory conditions and investigated the phosphorylation state of secreted S100A9. Our results on neutrophil-like differentiated HL-60 (dHL-60) cells and purified human neutrophils showed a time-dependent secretion of S100A8/A9 when induced by phorbol 12-myristoyl 13-acetate and this secreted S100A9 was found in a phosphorylated form. Second, we evaluated the impact of this phosphorylation on proinflammatory cytokine expression and secretion in dHL-60 cells. Time course experiments with purified unphosphorylated or phosphorylated S100A8/A9 were performed and the expression and secretion levels of interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor alpha, CCL2, CCL3, CCL4, and CXCL8 were measured by real-time PCR and cytometry bead array, respectively. Our results demonstrate that only the phosphorylated form of the complex induces proinflammatory cytokine expression and secretion. For the first time, we provide evidence that S100A8/PhosphoS100A9 is inducing cytokine secretion through toll-like receptor 4 signaling.
Topics: Alarmins; Bodily Secretions; Calgranulin A; Calgranulin B; Cytokines; Extracellular Space; HL-60 Cells; Humans; Inflammation Mediators; Neutrophil Activation; Neutrophils; Phosphorylation; Signal Transduction; Toll-Like Receptor 4
PubMed: 29593718
DOI: 10.3389/fimmu.2018.00447 -
The European Respiratory Journal Jul 1997The airway mucosa is lined by a continuous epithelium comprised of multiple cell phenotypes, several of which are secretory. Secretions produced by these cells mix with... (Review)
Review
The airway mucosa is lined by a continuous epithelium comprised of multiple cell phenotypes, several of which are secretory. Secretions produced by these cells mix with a variety of macromolecules, ions and water to form a respiratory tract fluid that protects the more distal airways and alveoli from injury and infection. The present article highlights the structure of the mucosa, particularly its secretory cells, gives a synopsis of the structure of mucus, and provides new information on the localization of mucin (MUC) genes that determine the peptide sequence of the protein backbone of the glycoproteins, which are a major component of mucus. Airway secretory cells comprise the mucous, serous, Clara and dense-core granulated cells of the surface epithelium, and the mucous and serous acinar cells of the submucosal glands. Several transitional phenotypes may be found, especially during irritation or disease. Respiratory tract mucins constitute a heterogeneous group of high molecular weight, polydisperse richly glycosylated molecules: both secreted and membrane-associated forms of mucin are found. Several mucin (MUC) genes encoding the protein core of mucin have been identified. We demonstrate the localization of MUC gene expression to a number of distinct cell types and their upregulation both in response to experimentally administered lipopolysaccharide and cystic fibrosis.
Topics: Animals; Gene Expression; Humans; Mucins; Mucous Membrane; Mucus; Respiratory System
PubMed: 9230262
DOI: 10.1183/09031936.97.10071655 -
Molecules (Basel, Switzerland) Apr 2021Saliva secretion changes in response to different stimulation. Studies performed in animals and humans suggest that dietary constituents may influence saliva...
Saliva secretion changes in response to different stimulation. Studies performed in animals and humans suggest that dietary constituents may influence saliva composition, although the dynamics of these changes, and how they are specific for each type of food, are little known. The objective of the present study was to access the short-term effects of different foods in salivation and salivary protein composition. Twelve participants were tested for four snacks (yoghurt, bread, apple and walnuts). Non-stimulated saliva was collected before and at 0', 5' and 30' after each snack intake. Flow rate, total protein, alpha-amylase enzymatic activity and salivary protein profile were analyzed. Yoghurt and apple were the snacks resulting in higher salivary changes, with higher increases in flow rate and alpha-amylase activity immediately after intake. The expression levels of immunoglobulin chains decreased after the intake of all snacks, whereas cystatins and one pink band (proline-rich proteins-PRPs) increased only after yoghurt intake. Walnut's snack was the one resulting in lower changes, probably due to lower amounts eaten. Even so, it resulted in the increase in one PRPs band. In conclusion, changes in saliva composition varies with foods, with variable changes in proteins related to oral food processing and perception.
Topics: Biomarkers; Enzyme Activation; Humans; Proteome; Proteomics; Saliva; Salivary Glands; Salivary Proteins and Peptides; Salivation; Snacks; alpha-Amylases
PubMed: 33919042
DOI: 10.3390/molecules26092403