Basal and Stress-Induced Network Activity in the Adrenal Medulla In Vivo.

The adrenal medulla plays a critical role in mammalian homeostasis and the stress response. It is populated by clustered chromaffin cells that secrete epinephrine or norepinephrine along with peptides into the bloodstream affecting distant target organs. Despite been heavily studied, the central control of adrenal medulla and in-situ spatiotemporal responsiveness remains poorly understood. For this work, we continuously monitored the electrical activity of individual adrenomedullary chromaffin cells in the living anesthetized rat using multielectrode arrays. We measured the chromaffin cell activity under basal and physiological stress conditions and characterized the functional micro-architecture of the adrenal medulla. Under basal conditions, chromaffin cells fired action potentials with frequencies between ~0.2 and 4 Hz. Activity was almost completely driven by sympathetic inputs coming through the splanchnic nerve. Chromaffin cells were organized into independent local networks in which cells fired in a specific order, with latencies from hundreds of microseconds to a few milliseconds. Electrical stimulation of the splanchnic nerve evoked almost exactly the same spatiotemporal firing patterns that occurred spontaneously. Hypoglycemic stress, induced by insulin administration resulted in increased activity of a subset of the chromaffin cells. In contrast, respiratory arrest induced by lethal anesthesia resulted in an increase in the activity of virtually all chromaffin cells before cessation of all activity. These results suggest a stressor-specific activation of adrenomedullary chromaffin cell networks and revealed a surprisingly complex electrical organization that likely reflects the dynamic nature of the adrenal medulla's neuroendocrine output during basal conditions and during different types of physiological stress.

Validation of new shoulder periprosthetic joint infection criteria.

BACKGROUND: A periprosthetic joint infection (PJI) in the shoulder can be difficult to diagnose. Many variables have been used to determine a PJI. Recently, the 2018 International Consensus Meeting (ICM) on orthopedic infections gave new criteria to help identify PJI in the shoulder. With the new criteria (major and minor), the PJI definition can be categorized into definite, probable, possible, and unlikely. This study was conducted to assess the new criteria for a series of consecutive first stage revision shoulder arthroplasty cases. METHODS: All patients undergoing a first stage revision shoulder arthroplasty using a prosthesis made of antibiotic-loaded acrylic cement (PROSTALAC) spacer from 2016 through 2019 were evaluated retrospectively. All cases were performed by a single surgeon. Each case was reviewed using the 2018 shoulder ICM diagnostic criteria. Secondary factors evaluated were type of organism identified, accuracy of minor criteria, and frozen vs. permanent section accuracy. RESULTS: A total of 87 first-stage revision arthroplasty cases were reviewed. Based on the 2018 ICM criteria, there were 20 definite (30.0%), 19 probable (21.8%), 6 possible (6.9%), and 42 unlikely (48.3%) infections. Cutibacterium acnes was the most common infectious organism overall (77.3% of culture positive cases) and was present in 39.1% of cases overall. Ten patients (25.6%) grew multiple organisms. Thirty-one patients (35.6%) had a loose humeral stem, with 23 of those patients (74.2%) having a definite or probable infection (odds ratio [OR] 7.2, 95% confidence interval [CI] 2.67-19.37, P = .0001). Eleven patients (91.7%) with an elevated intraoperative synovial neutrophil cell count had a definite or probable infection. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) was elevated in patients with a definite or probable infection (OR 9.4, 95% CI 2.47-35.62, P = .0010, and OR 7.7, 95% CI 2.29-25.56, P = .0009), respectively. Discordant results between frozen and permanent sections were found in 4 patients (4.6%). CONCLUSION: The 2018 ICM shoulder infection criteria gave a new scoring system to diagnose PJI. C acnes was the most common infectious organism identified. Patients who had a loose humeral stem, elevated ESR, or elevated CRP were more likely to have either a definite or probable PJI. Frozen sections were able to accurately identify definite infections. Unexpected wound drainage and positive preoperative cultures were low-yield criteria in this series. More research into determining periprosthetic shoulder infection is needed to help identify which patients are more likely to have an infection.

Actions of Rab27B-GTPase on mammalian central excitatory synaptic transmission.

Members of the Rab3 gene family are considered central to membrane trafficking of synaptic vesicles at mammalian central excitatory synapses. Recent evidence, however, indicates that the Rab27B-GTPase, which is highly homologous to the Rab3 family, is also enriched on SV membranes and co-localize with Rab3A and Synaptotagmin at presynaptic terminals. While functional roles of Rab3A have been well-established, little functional information exists on the role of Rab27B in synaptic transmission. Here we report on functional effects of Rab27B at SC-CA1 and DG-MF hippocampal synapses. The data establish distinct functional actions of Rab27B and demonstrate functions of Rab27B that differ between SC-CA1 and DG-MF synapses. Rab27B knockout reduced frequency facilitation compared to wild-type (WT) controls at the DG/MF-CA3 synaptic region, while increasing facilitation at the SC-CA1 synaptic region. Remarkably, Rab27B KO resulted in a complete elimination of LTP at the MF-CA3 synapse with no effect at the SC-CA1 synapse. These actions are similar to those previously reported for Rab3A KO. Specificity of action on LTP to Rab27B was confirmed as LTP was rescued in response to lentiviral infection and expression of human Rab27B, but not to GFP, in the DG in the Rab27B KO mice. Notably, the effect of Rab27B KO on MF-CA3 LTP occurred in spite of continued expression of Rab3A in the Rab27B KO. Overall, the results provide a novel perspective in suggesting that Rab27B and Rab3A act synergistically, perhaps via sequential effector recruitment or signaling for presynaptic LTP expression in this hippocampal synaptic region.

Screw Fixation Alone for Scaphoid Fracture Nonunion.

Scaphoid fracture nonunion can often lead to pain, arthrosis, and disability. While typically the result of delayed diagnosis or inadequate treatment, it can sometimes occur even if the initial care was timely and appropriate. Whereas early recognition of acute fractures allows for nonoperative management, nonunions frequently require surgical treatment. Traditionally, this has involved open debridement and bone grafting. However, some publications suggest that certain stable nonunions may be amenable to percutaneous debridement and fixation without formal bone grafting. Although certain characteristics appear to be appropriate indications for such management, well-designed clinical studies are needed to better define them.

SEC23B is required for pancreatic acinar cell function in adult mice.

Mice with germline absence of SEC23B die perinatally, exhibiting massive pancreatic degeneration. We generated mice with tamoxifen-inducible, pancreatic acinar cell-specific Sec23b deletion. Inactivation of Sec23b exclusively in the pancreatic acinar cells of adult mice results in decreased overall pancreatic weights from pancreatic cell loss (decreased pancreatic DNA, RNA, and total protein content), as well as degeneration of exocrine cells, decreased zymogen granules, and alterations in the endoplasmic reticulum (ER), ranging from vesicular ER to markedly expanded cisternae with accumulation of moderate-density content or intracisternal granules. Acinar Sec23b deletion results in induction of ER stress and increased apoptosis in the pancreas, potentially explaining the loss of pancreatic cells and decreased pancreatic weight. These findings demonstrate that SEC23B is required for normal function of pancreatic acinar cells in adult mice.

Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D.

Glucagon-like peptide-1 receptor is present in pancreatic acinar cells and regulates amylase secretion through cAMP.

Glucagon-like peptide-1 (GLP-1) is a glucoincretin hormone that can act through its receptor (GLP-1R) on pancreatic ß-cells and increase insulin secretion and production. GLP-1R agonists are used clinically to treat type 2 diabetes. GLP-1 may also regulate the exocrine pancreas at multiple levels, including inhibition through the central nervous system, stimulation indirectly through insulin, and stimulation directly on acinar cells. However, it has been unclear whether GLP-1R is present in pancreatic acini and what physiological functions these receptors regulate. In the current study we utilized GLP-1R knockout (KO) mice to study the role of GLP-1R in acinar cells. RNA expression of GLP-1R was detected in acutely isolated pancreatic acini. Acinar cell morphology and expression of digestive enzymes were not affected by loss of GLP-1R. GLP-1 induced amylase secretion in wild-type (WT) acini. In GLP-1R KO mice, this effect was abolished, whereas vasoactive intestinal peptide-induced amylase release in KO acini showed a pattern similar to that in WT acini. GLP-1 stimulated cAMP production and increased protein kinase A-mediated protein phosphorylation in WT acini, and these effects were absent in KO acini. These data show that GLP-1R is present in pancreatic acinar cells and that GLP-1 can regulate secretion through its receptor and cAMP signaling pathway.

Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion.

The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1). Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.

ERK activation is required for CCK-mediated pancreatic adaptive growth in mice.

High levels of cholecystokinin (CCK) can stimulate pancreatic adaptive growth in which mature acinar cells divide, leading to enhanced pancreatic mass with parallel increases in protein, DNA, RNA, and digestive enzyme content. Prolonged release of CCK can be induced by feeding trypsin inhibitor (TI) to disrupt normal feedback control. This leads to exocrine growth in a CCK-dependent manner. The extracellular signal-related kinase (ERK) pathway regulates many proliferative processes in various tissues and disease models. The aim of this study was to evaluate the role of ERK signaling in pancreatic adaptive growth using the MEK inhibitors PD-0325901 and trametinib (GSK-1120212). It was determined that PD-0325901 given two times daily by gavage or mixed into powdered chow was an effective and specific inhibitor of ERK signaling in vivo. TI-containing chow led to a robust increase in pancreatic mass, protein, DNA, and RNA content. This pancreatic adaptive growth was blocked in mice fed chow containing the MEK inhibitors. PD-0325901 blocked TI-induced ERK-regulated early response genes, cell-cycle proteins, and mitogenesis by acinar cells. It was determined that ERK signaling is necessary for the initiation of pancreatic adaptive growth but not necessary to maintain it. PD-0325901 blocked adaptive growth when given before cell-cycle initiation but not after mitogenesis had been established. Furthermore, GSK-1120212, a chemically distinct inhibitor of the ERK pathway that is now approved for clinical use, inhibited growth similar to PD-0325901. These data demonstrate that the ERK pathway is required for CCK-stimulated pancreatic adaptive growth.

Circulating lethal toxin decreases the ability of neutrophils to respond to Bacillus anthracis.

Polymorphonuclear leucocytes (PMNs) play a protective role during Bacillus anthracis infection. However, B. anthracis is able to subvert the PMN response effectively as evidenced by the high mortality rates of anthrax. One major virulence factor produced by B. anthracis, lethal toxin (LT), is necessary for dissemination in the BSL2 model of mouse infection. While human and mouse PMNs kill vegetative B. anthracis, short in vitro half-lives of PMNs have made it difficult to determine how or if LT alters their bactericidal function. Additionally, the role of LT intoxication on PMN's ability to migrate to inflammatory signals remains controversial. LF concentrations in both serum and major organs were determined from mice infected with B. anthracis Sterne strain at defined stages of infection to guide subsequent administration of purified toxin. Bactericidal activity of PMNs assessed using ex vivo cell culture assays showed significant defects in killing B. anthracis. In vivo PMN recruitment to inflammatory stimuli was significantly impaired at 24 h as assessed by real-time analysis of light-producing PMNs within the mouse. The observations described above suggest that LT serves dual functions; it both attenuates accumulation of PMNs at sites of inflammation and impairs PMNs bactericidal activity against vegetative B. anthracis.

Bacillus anthracis has two independent bottlenecks that are dependent on the portal of entry in an intranasal model of inhalational infection.

Bacillus anthracis can cause inhalational anthrax. Murine inhalational B. anthracis infections have two portals of entry, the nasal mucosa-associated lymphoid tissue (NALT) and the lumen of the lungs. Analysis of the dissemination from these sites is hindered because infections are asynchronous and asymptomatic until the hosts near death. To further understand and compare how B. anthracis disseminates from these two different environments, clonal analysis was employed using a library of equally virulent DNA-tagged clones of a luminescent Sterne strain. Luminescence was used to determine the origin of the infection and monitor the dissemination in vivo. The number of clones and their proportions in the portals of entry, lymph nodes draining the portals, and kidneys were analyzed. Clonal analysis indicated a bottleneck for both portals of entry, yet the extent and location of the reduction in represented clones differed between the routes. In NALT-based infections, all clones were found to germinate in the NALT, but they underwent a bottleneck as the infection spread to the cervical lymph node. However, lung-based infections underwent a bottleneck in a focal region of growth within the lung lumen and did not need to spread through the mediastinal lymph nodes to cause a systemic infection. Further, the average number of clones found in the kidney and the rate at which genetic drift was affecting the disseminated populations were significantly higher in lung-based infections. Collectively, the data suggested that differences in the host environment alter dissemination of B. anthracis depending on the site of initial colonization and growth.

EPI64B acts as a GTPase-activating protein for Rab27B in pancreatic acinar cells.

The small GTPase Rab27B localizes to the zymogen granule membranes and plays an important role in regulating protein secretion by pancreatic acinar cells, as does Rab3D. A common guanine nucleotide exchange factor (GEF) for Rab3 and Rab27 has been reported; however, the GTPase-activating protein (GAP) specific for Rab27B has not been identified. In this study, the expression in mouse pancreatic acini of two candidate Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, EPI64 (TBC1D10A) and EPI64B (TBC1D10B), was first demonstrated. Their GAP activity on digestive enzyme secretion was examined by adenovirus-mediated overexpression of EPI64 and EPI64B in isolated pancreatic acini. EPI64B almost completely abolished the GTP-bound form of Rab27B, without affecting GTP-Rab3D. Overexpression of EPI64B also enhanced amylase release. This enhanced release was independent of Rab27A, but dependent on Rab27B, as shown using acini from genetically modified mice. EPI64 had a mild effect on both GTP-Rab27B and amylase release. Co-overexpression of EPI64B with Rab27B can reverse the inhibitory effect of Rab27B on amylase release. Mutations that block the GAP activity decreased the inhibitory effect of EPI64B on the GTP-bound state of Rab27B and abolished the enhancing effect of EPI64B on the amylase release. These data suggest that EPI64B can serve as a potential physiological GAP for Rab27B and thereby participate in the regulation of exocytosis in pancreatic acinar cells.

Lamin aggregation is an early sensor of porphyria-induced liver injury.

Oxidative liver injury during steatohepatitis results in aggregation and transglutaminase-2 (TG2)-mediated crosslinking of the keratin cytoplasmic intermediate filament proteins (IFs) to form Mallory-Denk body (MDB) inclusions. The effect of liver injury on lamin nuclear IFs is unknown, though lamin mutations in several human diseases result in lamin disorganization and nuclear shape changes. We tested the hypothesis that lamins undergo aggregation during oxidative liver injury using two MDB mouse models: (i) mice fed the porphyrinogenic drug 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and (ii) mice that harbor a mutation in ferrochelatase (fch), which converts protoporphyrin IX to heme. Dramatic aggregation of lamin A/C and B1 was noted in the livers of both models in association with changes in lamin organization and nuclear shape, as determined by immunostaining and electron microscopy. The lamin aggregates sequester other nuclear proteins including transcription factors and ribosomal and nuclear pore components into high molecular weight complexes, as determined by mass-spectrometry and confirmed biochemically. Lamin aggregate formation is rapid and precedes keratin aggregation in fch livers, and is seen in liver explants of patients with alcoholic cirrhosis. Exposure of cultured cells to DDC, protoporphyrin IX or N-methyl-protoporphyrin, or incubation of purified lamins with protoporphyrin IX, also results in lamin aggregation. In contrast, lamin aggregation is ameliorated by TG2 inhibition. Therefore, lamin aggregation is an early sensor of porphyria-associated liver injury and might serve to buffer oxidative stress. The nuclear shape and lamin defects associated with porphyria phenocopy the changes seen in laminopathies and could result in transcriptional alterations due to sequestration of nuclear proteins.

c-Jun/AP-1 is required for CCK-induced pancreatic acinar cell dedifferentiation and DNA synthesis in vitro.

Endogenous CCK plays an important role in pancreatic regeneration after pancreatitis. We used primary culture of mouse pancreatic acinar cells to evaluate the effect of CCK on acinar cell morphology and gene expression and to determine signaling pathways required for proliferation of acinar cells in vitro. Over 4 days in culture, cells grew out from acini and formed patches of monolayer, which displayed a reduced expression of acinar cell markers including digestive enzymes and Mist1 and an increased expression of ductal and embryonic markers, including cytokeratin 7, ß-catenin, E-cadherin, pdx-1, and nestin. There was no appearance of stellate cell markers. CCK enhanced cellular spreading, DNA synthesis, and cyclin D1 expression. When signaling pathways were evaluated, CCK stimulation increased c-Jun expression, JNK and ERK activity, and AP-1 activation. Chemical inhibitors of JNK and ERK pathways, dominant-negative JNK and c-Jun, and c-Jun shRNA significantly inhibited CCK-induced DNA synthesis, CCK-induced AP-1 activation, and cyclin D1 expression. Furthermore, dominant-negative c-Jun reduced the increased expression of ß-catenin and the decreased expression of amylase during culture. These results show that MAPK/c-Jun/AP-1 pathway plays an important role in pancreatic acinar cell dedifferentiation and proliferation in culture. Monolayer culture can serve as a model to study acinar cell proliferation similar to regeneration after pancreatitis in vivo.

Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells.

Notch signaling is known to regulate the proliferation and differentiation of intestinal stem and progenitor cells; however, direct cellular targets and specific functions of Notch signals had not been identified. We show here in mice that Notch directly targets the crypt base columnar (CBC) cell to maintain stem cell activity. Notch inhibition induced rapid CBC cell loss, with reduced proliferation, apoptotic cell death and reduced efficiency of organoid initiation. Furthermore, expression of the CBC stem cell-specific marker Olfm4 was directly dependent on Notch signaling, with transcription activated through RBP-Jκ binding sites in the promoter. Notch inhibition also led to precocious differentiation of epithelial progenitors into secretory cell types, including large numbers of cells that expressed both Paneth and goblet cell markers. Analysis of Notch function in Atoh1-deficient intestine demonstrated that the cellular changes were dependent on Atoh1, whereas Notch regulation of Olfm4 gene expression was Atoh1 independent. Our findings suggest that Notch targets distinct progenitor cell populations to maintain adult intestinal stem cells and to regulate cell fate choice to control epithelial cell homeostasis.

Profiling CCK-mediated pancreatic growth: the dynamic genetic program and the role of STATs as potential regulators.

Feeding mice with protease inhibitor (PI) leads to increased endogenous cholecystokinin (CCK) release and results in pancreatic growth. This adaptive response requires calcineurin (CN)-NFAT and AKT-mTOR pathways, but the genes involved, the dynamics of their expression, and other regulatory pathways remain unknown. Here, we examined the early (1-8 h) transcriptional program that underlies pancreatic growth. We found 314 upregulated and 219 downregulated genes with diverse temporal and functional profiles. Several new identifications include the following: stress response genes Gdf15 and Txnip, metabolic mediators Pitpnc1 and Hmges2, as well as components of growth factor response Fgf21, Atf3, and Egr1. The genes fell into seven self-organizing clusters, each with a distinct pattern of expression; a representative gene within each of the upregulated clusters (Egr1, Gadd45b, Rgs2, and Serpinb1a) was validated by qRT-PCR. Genes up at any point throughout the time course and CN-dependent genes were subjected to further bioinformatics-based networking and promoter analysis, yielding STATs as potential transcriptional regulators. As shown by PCR, qPCR, and Western blots, the active phospho-form of STAT3 and the Jak-STAT feedback inhibitor Socs2 were both increased throughout early pancreatic growth. Moreover, immunohistochemistry showed a CCK-dependent and acinar cell-specific increase in nuclear localization of p-STAT3, with >75% nuclear occupancy in PI-fed mice vs. <0.1% in controls. Thus, the study identified novel genes likely to be important for CCK-driven pancreatic growth, characterized and biologically validated the dynamic pattern of their expression and investigated STAT-Socs signaling as a new player in this trophic response.

Secretin is not necessary for exocrine pancreatic development and growth in mice.

Adaptive exocrine pancreatic growth is mediated primarily by dietary protein and the gastrointestinal hormone cholecystokinin (CCK). Feeding trypsin inhibitors such as camostat (FOY-305) is known to induce CCK release and stimulate pancreatic growth. However, camostat has also been reported to stimulate secretin release and, because secretin often potentiates the action of CCK, it could participate in the growth response. Our aim was to test the role of secretin in pancreatic development and adaptive growth through the use of C57BL/6 mice with genetic deletion of secretin or secretin receptor. The lack of secretin in the intestine or the secretin receptor in the pancreas was confirmed by RT-PCR. Other related components, such as vasoactive intestinal polypeptide (VIP) receptors (VPAC(1) and VPAC(2)), were not affected. Secretin increased cAMP levels in acini from wild-type (WT) mice but had no effect on acini from secretin receptor-deleted mice, whereas VIP and forskolin still induced a normal response. Secretin in vivo failed to induce fluid secretion in receptor-deficient mice. The pancreas of secretin or secretin receptor-deficient mice was of normal size and histology, indicating that secretin is not necessary for normal pancreatic differentiation or maintenance. When WT mice were fed 0.1% camostat in powdered chow, the pancreas doubled in size in 1 wk, accompanied by parallel increases in protein and DNA. Camostat-fed littermate secretin and secretin receptor-deficient mice had similar pancreatic mass to WT mice. These results indicate that secretin is not required for normal pancreatic development or adaptive growth mediated by CCK.

Structural and functional analysis of tomosyn identifies domains important in exocytotic regulation.

Tomosyn is a 130-kDa cytosolic R-SNARE protein that associates with Q-SNAREs and reduces exocytotic activity. Two paralogous genes, tomosyn-1 and -2, occur in mammals and produce seven different isoforms via alternative splicing. Here, we map the structural differences between the yeast homologue of m-tomosyn-1, Sro7, and tomosyn genes/isoforms to identify domains critical to the regulation of exocytotic activity to tomosyn that are outside the soluble N-ethylmaleimide-sensitive attachment receptor motif. Homology modeling of m-tomosyn-1 based on the known structure of yeast Sro7 revealed a highly conserved functional conformation but with tomosyn containing three additional loop domains that emanate from a ß-propeller core. Notably, deletion of loops 1 and 3 eliminates tomosyn inhibitory activity on secretion without altering its soluble N-ethylmaleimide-sensitive attachment receptor pairing with syntaxin1A. By comparison, deletion of loop 2, which contains the hypervariable splice region, did not reduce the ability of tomosyn to inhibit regulated secretion. However, exon variation within the hypervariable splice region resulted in significant differences in protein accumulation of tomosyn-2 isoforms. Functional analysis of s-tomosyn-1, m-tomosyn-1, m-tomosyn-2, and xb-tomosyn-2 demonstrated that they exert similar inhibitory effects on elevated K(+)-induced secretion in PC12 cells, although m-tomosyn-2 was novel in strongly augmenting basal secretion. Finally, we report that m-tomosyn-1 is a target substrate for SUMO 2/3 conjugation and that mutation of this small ubiquitin-related modifier target site (Lys-730) enhances m-tomosyn-1 inhibition of secretion without altering interaction with syntaxin1A. Together these results suggest that multiple domains outside the R-SNARE of tomosyn are critical to the efficacy of inhibition by tomosyn on exocytotic secretion.

Crystal structures of native and inactivated cis-3-chloroacrylic acid dehalogenase: Implications for the catalytic and inactivation mechanisms.

The isomeric mixture of cis- and trans-1,3-dichloropropene constitutes the active component of a widely used nematocide known as Telone II®. The mixture is processed by various soil bacteria to acetaldehyde through the 1,3-dichloropropene catabolic pathway. The pathway relies on an isomer-specific hydrolytic dehalogenation reaction catalyzed by cis- or trans-3-chloroacrylic acid dehalogenase, known respectively as cis-CaaD and CaaD. Previous sequence analysis and crystallographic studies of the native and covalently modified enzymes identified Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114 as key binding and catalytic residues in cis-CaaD. Mutagenesis of these residues confirmed their importance to the dehalogenation reaction. Crystal structures of the native enzyme (2.01Å resolution) and the enzyme covalently modified at the Pro-1 nitrogen by 2-hydroxypropanoate (1.65Å resolution) are reported here. Both structures are at a resolution higher than previously reported (2.75Å and 2.1Å resolution, respectively). The conformation of the covalent adduct is strikingly different from that previously reported due to its interaction with a 7-residue loop (Thr-32 to Leu-38). The participation of another active site residue, Arg-117, in catalysis and inactivation was also examined. The implications of the combined findings for the mechanisms of catalysis and inactivation are discussed.

CCK-independent mTORC1 activation during dietary protein-induced exocrine pancreas growth.

Dietary protein can stimulate pancreatic growth in the absence of CCK release, but there is little data on the regulation of CCK-independent growth. To identify mechanisms whereby protein stimulates pancreatic growth in the absence of CCK release, C57BL/6 control and CCK-null male mice were fed normal-protein (14% casein) or high-protein (75% casein) chow for 7 days. The weight of the pancreas increased by 32% in C57BL/6 mice and 26% in CCK-null mice fed high-protein chow. Changes in pancreatic weight in control mice were due to both cell hypertrophy and hyperplasia since there was an increase in protein-to-DNA ratio, total DNA content, and DNA synthesis. In CCK-null mice pancreatic growth was almost entirely due to hypertrophy with both protein-to-DNA ratio and cell size increasing without significant increases in DNA content or DNA synthesis. ERK, calcineurin, and mammalian target of rapamycin complex 1 (mTORC1) are activated in models of CCK-induced growth, but there were no differences in ERK or calcineurin activation between fasted and fed CCK-null mice. In contrast, mTORC1 activation was increased after feeding and the duration of activation was prolonged in mice fed high-protein chow compared with normal-protein chow. Changes in pancreatic weight and RNA content were completely inhibited, and changes in protein content were partially abated, when the mTORC1 inhibitor rapamycin was administered during high-protein chow feeding. Prolonged mTORC1 activation is thus required for dietary protein-induced pancreatic growth in the absence of CCK.