Secretion Of Granule Contents Research Articles

Severely impaired CTL killing is a feature of the neurological disorder Niemann-Pick disease type C1

Niemann-Pick disease type C1 (NP-C1) is a rare lysosomal storage disorder resulting from mutations in an endolysosomal cholesterol transporter, NPC1. Despite typically presenting with pronounced neurological manifestations, NP-C1 also resembles long-term congenital immunodeficiencies that arise from impairment of cytotoxic T lymphocyte (CTL) effector function. CTLs kill their targets through exocytosis of the contents of lysosome-like secretory cytotoxic granules (CGs) that store and ultimately release the essential pore-forming protein perforin and proapoptotic serine proteases, granzymes, into the synapse formed between the CTL and target cell. We discovered that NPC1 deficiency increases CG lipid burden, impairs autophagic flux through stalled trafficking of the transcription factor EB (TFEB), and dramatically reduces CTL cytotoxicity. Using a variety of immunological and cell biological techniques, we found that the cytotoxic defect arises specifically from impaired perforin pore formation. We demonstrated defects of CTL function of varying severity in patients with NP-C1, with the greatest losses of function associated with the most florid and/or earliest disease presentations. Remarkably, perforin function and CTL cytotoxicity were restored in vitro by promoting lipid clearance with therapeutic 2-hydroxypropyl-β-cyclodextrin; however, restoration of autophagy through TFEB overexpression was ineffective. Overall, our study revealed that NPC1 deficiency has a deleterious impact on CTL (but not natural killer cell) cytotoxicity that, in the long term, may predispose patients with NP-C1 to atypical infections and impaired immune surveillance more generally.

Effects of Nanoceria on Human Platelet Functions and Blood Coagulation.

PurposeCerium oxide nanoparticles (nanoceria) exist in either 3+ or 4+ oxidation state with interesting redox properties and exhibit both oxidant and antioxidant attributes based on concentration, environment and pH. Thanks to their wide spread use in fuel, cosmetics and other industries as well as in biomedical field, nanoceria particles get released into environment with risk of significant human exposure, and thus necessitate careful examination and optimization of their biomedical applications.MethodsThis research was planned to explore effects of nanoceria on human platelet functions and whole blood coagulation. Platelets and whole blood from volunteer healthy donors were treated with different doses of nanoceria and various platelet functions, fibrin polymerization and blood coagulation were tested.ResultsNanoceria particles were found to reduce platelet aggregation and secretion of granule contents in a dose-dependent manner, impaired fibrin polymerization and retraction of fibrin-rich thrombi, associated with reduced cytosolic filamentous actin. Remarkably, nanoceria instigated early clot formation in whole blood attributable to putative activation of coagulation cascade.ConclusionAbove observations provide cautionary framework to critically re-evaluate and accordingly plan biomedical applications of nanoceria.

A mechanochemical mechanism couples exocrine secretion to endocytic membrane retrieval.

In this issue of Developmental Cell, Kamalesh etal. (2021) reveal a mechanochemical mechanism for the coupling of exocytic release of secretory granule content with endocytic membrane retrieval via actomyosin-driven membrane folding.

Ultrastructural characteristics of the anterior pituitary gland of cattle in the definitive period of postnatal ontogenesis

Today much attention is paid to the study of the pituitary ultrastructure of laboratory animals and humans. But there is not enough available literature to study the most important organ of internal secretion of productive animal. The aim of our work is to study the structure of the ultrastructure of cells of the anterior pituitary gland of cattle in the definitive period of postnatal ontogenesis. Histological, morphometric, and electron microscopic techniques were used to study the cellular composition of the anterior pituitary gland of cattle. It was revealed that the anterior lobe of the pituitary gland occupies 64 % of the entire pituitary parenchyma, while the posterior and middle lobes occupy 27% and 9%, respectively. After using general histological methods there are detected the functionally inactive chromophobes (41%) and chromophilic cells, which include acidophils (38%) and basophils (21%) in the anterior lobe of the pituitary gland. The electron microscopic studies in the parenchyma of the anterior pituitary gland let find Somatotropes that differ in the presence of a large number of secretory granules in the cytoplasm. Lactotropes are less common than somatotropes and differ from them in larger secretory granules. Corticotropes, gonadotropes and thyrotropes with a minimum content of secretory granules are the least detected. All identified endocrine cells are at different stages of functional activity.

Pancreatic β-Cell-Specific Deletion of VPS41 Causes Diabetes Due to Defects in Insulin Secretion.

Insulin secretory granules (SGs) mediate the regulated secretion of insulin, which is essential for glucose homeostasis. The basic machinery responsible for this regulated exocytosis consists of specific proteins present both at the plasma membrane and on insulin SGs. The protein composition of insulin SGs thus dictates their release properties, yet the mechanisms controlling insulin SG formation, which determine this molecular composition, remain poorly understood. VPS41, a component of the endolysosomal tethering homotypic fusion and vacuole protein sorting (HOPS) complex, was recently identified as a cytosolic factor involved in the formation of neuroendocrine and neuronal granules. We now find that VPS41 is required for insulin SG biogenesis and regulated insulin secretion. Loss of VPS41 in pancreatic β-cells leads to a reduction in insulin SG number, changes in their transmembrane protein composition, and defects in granule-regulated exocytosis. Exploring a human point mutation, identified in patients with neurological but no endocrine defects, we show that the effect on SG formation is independent of HOPS complex formation. Finally, we report that mice with a deletion of VPS41 specifically in β-cells develop diabetes due to severe depletion of insulin SG content and a defect in insulin secretion. In sum, our data demonstrate that VPS41 contributes to glucose homeostasis and metabolism.

The proline-rich tyrosine kinase Pyk2 modulates integrin-mediated neutrophil adhesion and reactive oxygen species generation

Neutrophils are first responders in infection and inflammation. They are able to roll, adhere and transmigrate through the endothelium to reach the site of infection, where they fight pathogens through secretion of granule contents, production of reactive oxygen species, extrusion of neutrophil extracellular traps, and phagocytosis. In this study we explored the role of the non-receptor focal adhesion kinase Pyk2 in neutrophil adhesion and activation. Using a specific Pyk2 pharmacological inhibitor, PF-4594755, as well as Pyk2-deficient murine neutrophils, we found that Pyk2 is activated upon integrin αMβ2-mediated neutrophil adhesion to fibrinogen. This process is triggered by Src family kinases-mediated phosphorylation and supported by Pyk2 autophosphorylation on Y402. In neutrophil adherent to fibrinogen, Pyk2 activates PI3K-dependent pathways promoting the phosphorylation of Akt and of its downstream effector GSK3. Pyk2 also dynamically regulates MAP kinases in fibrinogen-adherent neutrophils, as it stimulates p38MAPK but negatively regulates ERK1/2. Pharmacological inhibition of Pyk2 significantly prevented adhesion of human neutrophils to fibrinogen, and neutrophils from Pyk2-knockout mice showed a reduced ability to adhere compared to wildtype cells. Accordingly, neutrophil adhesion to fibrinogen was reduced upon inhibition of p38MAPK but potentiated by ERK1/2 inhibition. Neutrophil adherent to fibrinogen, but not to polylysine, were able to produce ROS upon lipopolysaccharide challenge and ROS production was completely suppressed upon inhibition of Pyk2. By contrast PMA-induced ROS production by neutrophil adherent to either fibrinogen or polylysine was independent from Pyk2. Altogether these results demonstrate that Pyk2 is an important effector in the coordinated puzzle regulating neutrophil adhesion and activation.

УЛЬТРАМІКРОСКОПІЧНІ ПРОЯВИ У СТРУКТУРІ ПІДНИЖНЬОЩЕЛЕПНОЇ СЛИННОЇ ЗАЛОЗИ НАПРИКІНЦІ 4-ГО ТИЖНЯ ЕКСПЕРИМЕНТАЛЬНОГО ОПІОЇДНОГО ВПЛИВУ

This publication demonstrates the ultrastructural changes of the submandibular salivary gland under the opioid effect at the 4th week of the experimental research. After 28 days of experimental opioid effect, we found that in exocrinocytes of the end secretory departments of acinar cells revealed the destruction of organelles, especially granular and smooth endoplasmic reticulum, mitochondria, as well as reducing the content of secretory granules in the apical area of glandulocytes. In some exocrinocytes, as a result of the increase in destructive changes, a decrease in the volume of the nucleus was noted, which moved to the peripheral parts of the cell. Necrotic changes developed in individual exocrinocytes of the final secretory protein divisions. In such cells, the nucleus was reduced in volume, filled with heterochromatin.

Stearoyl‐CoA desaturase 1 determines pancreatic β‐cell fate through regulation of DNA methylation pattern

Study objectiveStearoyl CoA desaturase 1 (SCD1) is a pivotal enzyme involved in saturated fatty acids metabolism that exhibits a protective role against lipotoxicity in pancreatic β‐cells. It has been shown that SCD1 deficiency contributes to impaired insulin secretion, β‐cell failure and development of type 2 diabetes (T2D). In the present study we investigated the involvement of SCD1 in regulation of DNA methylation and expression of transcription factors (TFs) responsible for maintenance of mature β‐cells function and identity.MethodsThe experiments were carried out ex vivo on pancreatic islets isolated from wild type (WT) and SCD1 knock‐out (SCD1 KO) mice, and in vitro on INS‐1E pancreatic β‐cell line, where lipotoxicity was induced by palmitic acid treatment. Morphological and functional characteristics of islets was evaluated by ELISA assays, immunofluorescence labeling and transmission electron microscopy techniques. DNA methylation and gene expression levels were assessed by bisulfite sequencing and real‐time quantitative PCR analyses, respectively.ResultsOur data demonstrated that pancreatic islets of SCD1 KO mice are characterized by different microarchitecture, secretory granules content and functional abnormalities manifested by diminished insulin secretion and greater proinsulin secretion in comparison to WT islets. On both ex vivo and in vitro models we observed significantly reduced expression of TFs crucial to maintenance of β‐cell identity such as Pdx1, MafA, Nkx6.1 and Neurod1. Interestingly, transcriptional repression of Pdx1 and MafA was related to excessive methylation of promoter regions of those TFs. In addition, we showed that downregulation of SCD1 activity/gene expression affects global DNA methylation pattern and changes methyl groups distribution within chromosomes in pancreatic β‐cells. DNA hypomethylation which occurred in SCD1‐deficient β‐cells was followed by decreased expression of DNA methyltransferase 1 (Dnmt1). We indicated that activation of AMP‐activated protein kinase (AMPK) axis influences post‐translational regulation of Dnmt1, thereby mediating the effect of SCD1 inhibition on global DNA methylation profile in β‐cells.ConclusionsTogether, our findings suggest that SCD1 is a gatekeeper that preserves epigenetic inheritance of DNA methylation pattern in β‐cells. Thus, SCD1 protects β‐cells from lipid‐derived loss of identity in methylation‐dependent manner through regulation of expression of TFs responsible for maintenance of β‐cell identity. Our results provide additional mechanistic insights toward understanding the role of SCD1 in T2D pathogenesis.Support or Funding InformationThis work was supported by grants from the National Science Center, Poland, grants no UMO‐2017/27/N/NZ3/01987 (AMD) and UMO‐2015/19/D/NZ4/03705 (JJ), and by The National Centre for Research and Development, grant no STRATEGMED 3/305813/2/NCBR/2017 (AD).

Mechanism of action of catestatin as an autocrine modulator regulating catecholamine secretion from chromaffin cells at the of splanchnicoadrenomedullary synapse

ObjectiveThe chromogranin A (CgA)‐derived peptide catestatin (CST: human CgA352–372) is thought to act as an autocrine modulator of catecholamine secretion from the adrenal medulla in vivo. Here, we examined how CST affects nicotine‐ and PACAP‐induced secretion from, and protein resynthesis within, dense core (DC) vesicle (DCV) quanta containing catecholamines and chromogranin/secretogranin proteins in PC12 cells.Methods and ResultsDepletion of norepinephrine (NE) from PC12 cells treated acutely (0.5 hours) with either nicotine (60 μM)‐ or PACAP (0.1 μM) was reversed by CST (2 μM), as was 3H‐NE release elicited by both secretagogues. DCV and DC area and diameter, evaluated by Transmission Electron Microscopy, were both decreased after treatment with either nicotine or PACAP, and this morphological effect was likewise reversed by CST. Nicotine or CST alone increased expression of CgA protein (but not CgB), and, in combination, elicited an additional increase in CgA protein, implying that nicotine and CST utilize separate signaling pathways to activate expression of CgA. In contrast, PACAP increased expression of CgB protein (but not CgA), indicating differential regulation of CgA and CgB proteins by cholinergic and peptidergic stimulation. Although CST augmented expression of tyrosine hydroxylase (TH) protein, it did not induce synthesis of catecholamines, presumably due to its inability to cause post‐translation activation of TH via serine phosphorylation. Increased area and diameter of DCVs, and their DCs after CST, CST+nicotine and CST+PACAP are consistent with increased quantal size. Decreased quantal size is possibly mediated by increased synthesis of granulogenic proteins: CgA after CST treatment; CgA after CST+nicotine treatment; and CgB after CST+PACAP treatment.ConclusionBased on our results obtained in PC12 cells, we suggest that the CgA‐derived peptide CST acts as a paracrine factor during ACh or PACAP stimulation of secretion from chromaffin cells of the adrenal medulla, modulating both the extent of secretory stimulation by splanchnic nerve firing, and the repletion of secretory granule content after bouts of secretory activity.Support or Funding InformationSupported by grants from the VA (I01BX000323 to S.K.M.; I01BX002709 to N.J.G.W), and the NIH (MH002386 to L.E.E.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Extended cleavage specificities of mast cell proteases 1 and 2 from golden hamster: Classical chymase and an elastolytic protease comparable to rat and mouse MCP-5.

Serine proteases constitute the major protein content of mast cell secretory granules. Here we present the extended cleavage specificity of two such proteases from the golden hamster, Mesocricetus auratus. Analysis by phage display technique showed that one of them (HAM1) is a classical chymase with a specificity similar to the human mast cell chymase. However, in contrast to the human chymase, it does not seem to have a particular preference for any of the three aromatic amino acids, Phe, Tyr and Trp, in the P1 position of substrates. HAM1 also efficiently cleaved after Leu similarly to human and many other mast cell chymases. We observed only a 3-fold lower cleavage activity on Leu compared to substrates with P1 aromatic amino acids. Chymotryptic enzymes seem to be characteristic for connective tissue mast cells in mammalian species from opossums to humans, which indicates a very central role of these enzymes in mast cell biology. HAM1 also seems to have the strongest preference for negatively charged amino acids in the P2´position of all mast cell chymases so far characterized. The second hamster chymase, HAM2, is an elastolytic in its activity, similarly to the α-chymases in rats and mice (rMCP-5 and mMCP-5, respectively). The presence of an α-chymase that developed elastase activity thereby seems to be a relatively early modification of the α-chymase within the rodent branch of the mammalian evolutionary tree.

Mitogen-Activated Protein Kinase Signaling Regulates Proteoglycan Composition of Mast Cell Secretory Granules.

Mast cells (MCs) are characterized by an abundance of lysosome-like secretory granules filled with immunomodulatory compounds including histamine, cytokines, lysosomal hydrolases, MC-restricted proteases, and serglycin proteoglycans. The latter are essential for promoting the storage of other granule compounds and are built up of the serglycin core protein to which highly sulfated and thereby negatively charged glycosaminoglycan (GAG) side chains of heparin or chondroitin sulfate type are attached. In the search for mechanisms operating in regulating MC granule homeostasis, we here investigated the role of mitogen-activated protein kinase (MAPK) signaling. We show that inhibition of MEK1/2 (a MAPK kinase) leads to increased metachromatic staining of MC granules, indicative of increased proteoglycan content. Indeed, MEK1/2 inhibition caused a profound increase in the expression of the gene coding for the serglycin core protein and of genes coding for various enzymes involved in the biosynthesis/sulfation of the GAGs attached to the serglycin core protein. This was accompanied by corresponding increases in the levels of the respective GAGs. Deletion of the serglycin core protein abrogated the induction of enzymes operative in proteoglycan synthesis, indicating that availability of the serglycin proteoglycan core protein has a regulatory function impacting on the expression of the various serglycin-modifying enzymes. MEK1/2 inhibition also caused a substantial increase in the expression of granule-localized, proteoglycan-binding proteases. Altogether, this study identifies a novel role for MAPK signaling in regulating the content of secretory granules in MCs.

Infradian Rhythm of the Content of Secretory Granules in Pinealocyte Cytoplasm in Mice and Rats.

The numerical density of secretory granules dense-core vesicles (DCV) in the cytoplasm of pinealocytes of the pineal gland was estimated by transmission electron microscopy in male white mice and Wistar rats. The 3-day biorhythm and lunaphase changes in the DCV content in the perikaryon and the processes of pinealocytes, which are manifested significantly in different seasons of the year, are established. The three-day biorhythm in adult male mice in comparison with younger male rats is not expressed uniformly in different phases of the moon. The in-phase manifestation of infradian biorhythms in different species of animals during the year with an unchanged daily photophase indicates the existence of common external synchronizers for mammals of these biorhythms that are not associated with the light/dark cycle.

ULTRASTRUCTURAL FEATURES OF THE DIFFUSE NEUROENDOCRINE SYSTEM OF THE DUODENUM IN ACUTE INTESTINAL OBSTRUCTION

Study of changes in the structure of the duodenal endocrinocytes with intestinal obstruction is a step towards finding out the pathogenesis of the disease. The aim is to establish the ultrastructural features of the diffuse neuroendocrine system of the duodenum in the modeling of high acute obstructive intestinal obstruction. Material and methods. Experimental studies were performed on 20 white mature male rats weighing 300350 g. Animals were divided into 2 groups: control (5) and experimental (15). Animals of the experimental group were created a model of high acute obstructive intestinal obstruction. Euthanasia was performed by overdose of thiopental sodium at 24, 48 and 72 hours.. Pieces of the duodenum was used for microscopic examination, they were fixed in 10% neutral formalin solution. The preparations were stained by van Gison method, hematoxylin-eosin, for studying endocrinocytes, stained using the Grimmel and Mason-Hamperl method. For electron microscopic examination, the pieces of the duodenum were fixed in a 2.5% solution of glutaraldehyde in 0.1 g phosphate buffer. Results and discussion. Endocrinocytes of the mucous membranes of the small intestine under conditions of high intestinal obstruction above the blockade site differ sharply from similar cells in the villi and crypts of the gut of the animals of the control group. Characteristics of endocrine cells in the content of secretory granules sharply changes. In this group, we observed endocrinocytes whose cytoplasm was slightly saturated with granules up to complete degranulation. The integrity of the membranes of individual granules has been broken, which led to the possibility of secretion in the cytoplasm. Conclusions. In the dynamics of the development of high obstructive acute narrow-hearth obstruction in the cells of the diffuse neuroendocrine system, destructive changes have been identified, which are manifested by a decrease in the total number of endocrine cells and, at the same time, an increase in the number of destroyed endocrinocytes. Ultrastructural changes are characterized by a violation of the integrity of the membranes of the individual secretory granules with the possibility of secretion in the cytoplasm, violation of the structure of mitochondria, illumination of their matrix, and cristae disintegration.

Intracellular Trafficking, Localization, and Mobilization of Platelet-Borne Thiol Isomerases.

Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation. Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13d(Jinx) mice). Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.

Beta cells transfer vesicles containing insulin to phagocytes for presentation to T cells

Beta cells from nondiabetic mice transfer secretory vesicles to phagocytic cells. The passage was shown in culture studies where the transfer was probed with CD4 T cells reactive to insulin peptides. Two sets of vesicles were transferred, one containing insulin and another containing catabolites of insulin. The passage required live beta cells in a close cell contact interaction with the phagocytes. It was increased by high glucose concentration and required mobilization of intracellular Ca2+. Live images of beta cell-phagocyte interactions documented the intimacy of the membrane contact and the passage of the granules. The passage was found in beta cells isolated from islets of young nonobese diabetic (NOD) mice and nondiabetic mice as well as from nondiabetic humans. Ultrastructural analysis showed intraislet phagocytes containing vesicles having the distinct morphology of dense-core granules. These findings document a process whereby the contents of secretory granules become available to the immune system.

SORCS1 is necessary for normal insulin secretory granule biogenesis in metabolically stressed β cells.

We previously positionally cloned Sorcs1 as a diabetes quantitative trait locus. Sorcs1 belongs to the Vacuolar protein sorting-10 (Vps10) gene family. In yeast, Vps10 transports enzymes from the trans-Golgi network (TGN) to the vacuole. Whole-body Sorcs1 KO mice, when made obese with the leptin(ob) mutation (ob/ob), developed diabetes. β Cells from these mice had a severe deficiency of secretory granules (SGs) and insulin. Interestingly, a single secretagogue challenge failed to consistently elicit an insulin secretory dysfunction. However, multiple challenges of the Sorcs1 KO ob/ob islets consistently revealed an insulin secretion defect. The luminal domain of SORCS1 (Lum-Sorcs1), when expressed in a β cell line, acted as a dominant-negative, leading to SG and insulin deficiency. Using syncollin-dsRed5TIMER adenovirus, we found that the loss of Sorcs1 function greatly impairs the rapid replenishment of SGs following secretagogue challenge. Chronic exposure of islets from lean Sorcs1 KO mice to high glucose and palmitate depleted insulin content and evoked an insulin secretion defect. Thus, in metabolically stressed mice, Sorcs1 is important for SG replenishment, and under chronic challenge by insulin secretagogues, loss of Sorcs1 leads to diabetes. Overexpression of full-length SORCS1 led to a 2-fold increase in SG content, suggesting that SORCS1 is sufficient to promote SG biogenesis.

Mast cell secretory granules: armed for battle.

Mast cells are important effector cells of the immune system and recent studies show that they have immunomodulatory roles in diverse processes in both health and disease. Mast cells are distinguished by their high content of electron-dense secretory granules, which are filled with large amounts of preformed and pre-activated immunomodulatory compounds. When appropriately activated, mast cells undergo degranulation, a process by which these preformed granule compounds are rapidly released into the surroundings. In many cases, the effects that mast cells have on an immune response are closely associated with the biological actions of the granule compounds that they release, as exemplified by the recent studies showing that mast cell granule proteases account for many of the protective and detrimental effects of mast cells in various inflammatory settings. In this Review, we discuss the current knowledge of mast cell secretory granules.

Rab5 Is a Novel Regulator of Mast Cell Secretory Granules: Impact on Size, Cargo, and Exocytosis

Secretion of inflammatory mediators prestored in mast cells secretory granules (SGs) enhances immune responses such as in allergy and host defense. However, the mechanisms underlying the biogenesis of the SGs remain largely unresolved. By combining high-resolution live cell imaging and quantitative morphometric analyses, we show that the small GTPase Rab5 controls the SG size and cargo composition by a VAMP8-dependent fusion mechanism. Knockdown of the endogenous Rab5, or expression of constitutively negative mutants, significantly reduces the size of SGs and increases their number. Conversely, expression of constitutively active Rab5 mutants induces few, but giant, SGs. Both the small and giant SGs maintain their exocytosis competence. Finally, we show that Rab5-mediated fusion between Golgi-derived SGs and early endosomes precedes the maturation of the SGs, as reflected by the recruitment of Rab27B, and allows the incorporation of cargo, such as CD63, that traffics through endosomes. Collectively, our results assign Rab5 a key role in mediating mast cell SG fusion during biogenesis, thereby controlling the amount and composition of the SGs content and maintaining the communication between new and pre-existing SGs.

Proteolytic Histone Modification by Mast Cell Tryptase, a Serglycin Proteoglycan-dependent Secretory Granule Protease

A hallmark feature of mast cells is their high content of cytoplasmic secretory granules filled with various preformed compounds, including proteases of tryptase-, chymase-, and carboxypeptidase A3 type that are electrostatically bound to serglycin proteoglycan. Apart from participating in extracellular processes, serglycin proteoglycan and one of its associated proteases, tryptase, are known to regulate cell death by promoting apoptosis over necrosis. Here we sought to outline the underlying mechanism and identify core histones as primary proteolytic targets for the serglycin-tryptase axis. During the cell death process, tryptase was found to relocalize from granules into the cytosol and nucleus, and it was found that the absence of tryptase was associated with a pronounced accumulation of core histones both in the cytosol and in the nucleus. Intriguingly, tryptase deficiency resulted in defective proteolytic modification of core histones even at baseline conditions, i.e. in the absence of cytotoxic agent, suggesting that tryptase has a homeostatic impact on nuclear events. Indeed, tryptase was found in the nucleus of viable cells and was shown to cleave core histones in their N-terminal tail. Moreover, it was shown that the absence of the serglycin-tryptase axis resulted in altered chromatin composition. Together, these findings implicate histone proteolysis through a secretory granule-derived serglycin-tryptase axis as a novel principle for histone modification, during both cell homeostasis and cell death.

Resistance exercise induces region-specific adaptations in anterior pituitary gland structure and function in rats

The anterior pituitary gland (AP) increases growth hormone (GH) secretion in response to resistance exercise (RE), but the nature of AP adaptations to RE is unknown. To that end, we examined the effects of RE on regional AP somatotroph GH release, structure, and relative quantity. Thirty-six Sprague-Dawley rats were assigned to one of four groups: 1) no training or acute exercise (NT-NEX); 2) no training with acute exercise (NT-EX); 3) resistance training without acute exercise (RT-NEX); 4) resistance training with acute exercise (RT-EX). RE incorporated 10, 1 m-weighted ladder climbs at an 85° angle. RT groups trained 3 days/wk for 7 wk, progressively. After death, trunk blood was collected, and each AP was divided into quadrants (ventral-dorsal and left-right). We measured: 1) trunk plasma GH; 2) somatotroph GH release; 3) somatotroph size; 4) somatotroph secretory content; and 5) percent of AP cells identified as somatotrophs. Trunk GH differed by group (NT-NEX, 8.9 ± 2.4 μg/l; RT-NEX, 9.2 ± 3.5 μg/l; NT-EX, 15.6 ± 3.4 μg/l; RT-EX, 23.4 ± 4.6 μg/l). RT-EX demonstrated greater somatotroph GH release than all other groups, predominantly in ventral regions (P < 0.05-0.10). Ventral somatotrophs were larger in NT-EX and RT-NEX compared with RT-EX (P < 0.05-0.10). RT-NEX exhibited significantly greater secretory granule content than all other groups but in the ventral-right region only (P < 0.05-0.10). Our findings indicate reproducible patterns of spatially distinct, functionally different somatotroph subpopulations in the rat pituitary gland. RE training appears to induce dynamic adaptations in somatotroph structure and function.