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A major component of the extracellular matrix (ECM), laminins, modulates cells via diverse receptors. Their fragments have emerging utility as components of "ECM-mimetics" optimized to promote cell-based therapies. Recently, we reported that a bioactive laminin peptide known as A99 enhanced cell binding and spreading via fusion to an elastin-like polypeptide (ELP). The ELP "handle" serves as a rapid, noncovalent strategy to concentrate bioactive peptide mixtures onto a surface. We now report that this strategy can be further generalized across an expanded panel of additional laminin-derived elastin-like polypeptides (LELPs). A99 (AGTFALRGDNPQG), A2G80 (VQLRNGFPYFSY), AG73 (RKRLQVQLSIRT), and EF1m (LQLQEGRLHFMFD) all promote cell spreading while showing morphologically distinct F-actin formation. Equimolar mixtures of A99:A2G80-LELPs have synergistic effects on adhesion and spreading. Finally, three of these ECM-mimetics promote the neurite outgrowth of PC-12 cells. The evidence presented here demonstrates the potential of ELPs to deposit ECM-mimetics with applications in regenerative medicine, cell therapy, and tissue engineering.

Purpose: To analyze the changes in the lacrimal gland (LG) miRNAome from male nonobese diabetic (NOD) mice with autoimmune dacryoadenitis compared with LG from healthy male BALB/c and dacryoadenitis-free female NOD mice., Methods: LG from these mice were collected for small RNA sequencing to identify dysregulated miRNAs; hits were validated by RT-qPCR in male NOD and BALB/c LG. Dysregulation of validated species within immune cell-enriched cell fractions and epithelial-enriched cell fractions from LG was probed by RT-qPCR. Ingenuity pathway analysis identified putative miRNA targets, which were examined in publicly available mRNA-seq datasets. Western blotting and confocal imaging of immunofluorescence enabled validation of some molecular changes at the protein level., Results: Male NOD LG exhibited 15 and 13 significantly up- and downregulated miRNAs, respectively. Dysregulated expression of 14 of these miRNAs (9 upregulated, 5 downregulated) was validated in male NOD versus BALB/c LG by RT-qPCR. Seven of the upregulated miRNAs were increased owing to their abundance in immune cell-enriched cell fractions, whereas four downregulated miRNAs were largely expressed in epithelial-enriched cell fractions. Ingenuity pathway analysis predicted the upregulation of IL-6 and IL-6-like pathways as an outcome of miRNA dysregulation. Increased expression of several genes in these pathways was confirmed by mRNA-seq analysis, whereas immunoblotting and immunofluorescence confirmed Ingenuity pathway analysis-predicted changes for IL-6Rα and gp130/IL-6st., Conclusions: Male NOD mouse LG exhibit multiple dysregulated miRNAs owing to the presence of infiltrating immune cells, and decreased acinar cell content. The observed dysregulation may increase IL-6Rα and gp130/IL-6st on acini and IL-6Rα on specific lymphocytes, enhancing IL-6 and IL-6-like cytokine signaling.

Objective: To describe clinical, diagnostic and therapeutic characteristics of the periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) syndrome., Data Source: Literature review in the PubMed database by using specific descriptors to identify all articles published in the English language in the last three years; 38 articles were found. After performing selection of titles and abstract analysis, 13 out of the 38 articles were fully read. Relevant studies found in the references of the reviewed articles were also included., Data Synthesis: The PFAPA syndrome (Periodic Fever, Aphthous Stomatitis, Pharyngitis and cervical Adenitis) is a medical condition grouped among the periodic fever syndromes. The etiology is uncertain, but possibly multifactorial, and its symptoms are accompanied by recurrent febrile episodes although weight and height development are preserved. It is a self-limiting disease of benign course with remission of two to three years without significant interference in the patient's overall development. Treatment consists of three pillars: interruption of febrile episodes, increase in the interval between episodes, and remission., Conclusions: Despite several attempts to establish more sensitive and specific criteria, the diagnosis of PFAPA syndrome is still clinical and reached by exclusion, based on the modified Marshall's criteria. The most common pharmacological options for treatment include prednisolone and betamethasone; colchicine may be used as prophylaxis, and surgical treatment with tonsillectomy can be considered in selected cases.

Objective: The tear miRNAome of the male NOD mouse, a model of ocular symptoms of Sjögren's syndrome (SS), was analyzed to identify unique miRNAs., Methods: Male NOD mice, aged 12-14 weeks, were used to identify tear miRNAs associated with development of autoimmune dacryoadenitis. Age- and sex-matched male BALB/c mice served as healthy controls while age-matched female NOD mice that do not develop the autoimmune dacryoadenitis characteristic of SS were used as additional controls. Total RNA was isolated from stimulated tears pooled from 5 mice per sample and tear miRNAs were sequenced and analyzed. Putative miRNA hits were validated in additional mouse cohorts as well as in tears of SS patients versus patients with another form of dry eye disease, meibomian gland disease (MGD) using qRT-PCR. The pathways influenced by the validated hits were identified using Ingenuity Pathway Analysis., Results: In comparison to tears from both healthy (male BALB/c) and additional control (female NOD) mice, initial analy1sis identified 7 upregulated and 7 downregulated miRNAs in male NOD mouse tears. Of these, 8 were validated by RT-qPCR in tears from additional mouse cohorts. miRNAs previously implicated in SS pathology included mmu-miR-146a/b-5p, which were significantly downregulated, as well as mmu-miR-150-5p and mmu-miR-181a-5p, which were upregulated in male NOD mouse tears. All other validated hits including the upregulated miR-181b-5p and mmu-miR-203-3p, as well as the downregulated mmu-miR-322-5p and mmu-miR-503-5p, represent novel putative indicators of autoimmune dacryoadenitis in SS. When compared to tears from patients with MGD, miRNAs hsa-miR-203a-3p, hsa-miR-181a-5p and hsa-miR-181b-5p were also significantly increased in tears of SS patients., Conclusions: A panel of differentially expressed miRNAs were identified in tears of male NOD mice, with some preliminary validation in SS patients, including some never previously linked to SS. These may have potential utility as indicators of ocular symptoms of SS; evaluation of the pathways influenced by these dysregulated miRNAs may also provide further insights into SS pathogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kakan, Edman, Yao, Okamoto, Nguyen, Hjelm and Hamm-Alvarez.)

Sjögren's Syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration and loss of function of moisture-producing exocrine glands as well as systemic inflammation. SS diagnosis is cumbersome, subjective and complicated by manifestation of symptoms that overlap with those of other rheumatic and ocular diseases. Definitive diagnosis averages 4-5 years and this delay may lead to irreversible tissue damage. Thus, there is an urgent need for diagnostic biomarkers for earlier detection of SS. Extracellular vesicles called exosomes carry functional small non-coding RNAs which play a critical role in maintaining cellular homeostasis via transcriptional and translational regulation of mRNA. Alterations in levels of specific exosomal miRNAs may be predictive of disease status. Here, we have assessed serum exosomal RNA using next generation sequencing in a discovery cohort of the NOD mouse, a model of early-intermediate SS, to identify dysregulated miRNAs that may be indicative of SS. We found five miRNAs upregulated in serum exosomes of NOD mice with an adjusted p < 0.05-miRNA-127-3p, miRNA-409-3p, miRNA-410-3p, miRNA-541-5p, and miRNA-540-5p. miRNAs 127-3p and 541-5p were also statistically significantly upregulated in a validation cohort of NOD mice. Pathway analysis and existing literature indicates that differential expression of these miRNAs may dysregulate pathways involved in inflammation. Future studies will apply these findings in a human cohort to understand how they are correlated with manifestations of SS as well as understanding their functional role in systemic autoimmunity specific to SS., (Copyright © 2020 Kakan, Janga, Cooperman, Craig, Edman, Okamoto and Hamm-Alvarez.)

Bronchopulmonary dysplasia (BPD) is an abnormality that occurs in premature neonate lung development. The pathophysiology is uncertain, but the inflammatory response to lung injury may be the responsible pathway. The objective of this study is to evaluate the role of interleukins 6, 8, 10, and 17 through the anatomopathological and immunohistochemical study of the lungs of premature neonates with BPD. Thirty-two cases of neonatal autopsies from the Pathology Department of the Clinics Hospital of the Universidade Federal do Paraná, who presented between 1991 and 2005 were selected. The sample included neonates less than 34 weeks of gestational age who underwent oxygen therapy and had pulmonary formalin-fixed paraffin-embedded (FFPE) samples. Pulmonary specimens were later classified into three groups according to histopathological and morphometric changes (classic BPD, new BPD, and without BPD) and subjected to immunohistochemical analysis. The antibodies selected for the study were anti-IL-6, anti-IL-8, anti-IL-10, and anti-IL-17A monoclonal antibodies. IL-6, IL-8, and IL-10 showed no significant differences in tissue expression among the groups. IL-17A had higher tissue immunoreactivity in the group without BPD compared with the classic BPD group (1686 vs. 866 μm 2 , p = 0.029). This study showed that the involvement of interleukins 6, 8, and 10 might not be significantly different between the two types of BPD. We speculated that IL-17A could be a protective factor in this disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The extracellular matrix (ECM) is comprised of a large network of proteins that are essential for tissue development and repair. A bioactive RGD-containing peptide from laminin α1 chain, A99 (AGTFALRGDNPQG), promotes strong cell attachment and has demonstrated utility in cell culture and tissue engineering. Various materials can be utilized as a scaffold for bioactive peptides; however, it may be advantageous to design materials that use bioconjugation strategies that do not affect bioactivity, generate homogenous products, and can be produced at scale. This report is the first to compare the methods for preparing chemically conjugated and recombinant A99 to elastin-like polypeptides (ELPs) as the scaffold and characterize the biological and cell attachment activity using human dermal fibroblasts (HDFs). ELPs are biocompatible protein-polymers that are also thermo-responsive. Below a lower critical solution temperature (LCST), they are highly soluble. Above the LCST, ELPs phase separate into a polymer-rich liquid, known as a coacervate. Both chemically conjugated and recombinant fusion between A99 and an ELP (A99-ELP-R) show dose-dependent cell attachment. In addition, coating above the LCST provides better cell spreading compared to coating at 4°C. ELPs provide an excellent structural framework for deposition of bioactive peptides of the ECM, and their intrinsic biophysical properties make laminin peptide-ELPs promising biomaterials for cell culture and tissue engineering., (© 2020 Federation of American Societies for Experimental Biology.)

Protein-protein interactions are key to define the function of nucleotide binding domain (NBD) and leucine-rich repeat (LRR) family, pyrin domain (PYD)-containing protein 12 (NLRP12). cDNA encoding the human PYD + NBD of NLRP12 was used as bait in a yeast two-hybrid screen with a human leukocyte cDNA library as prey. Hematopoiesis cell kinase (HCK), a member of the c-SRC family of non-receptor tyrosine kinases, was among the top hits. The C-terminal 40 amino acids of HCK selectively bound to NLRP12's PYD + NBD, but not to that of NLRP3 and NLRP8. Amino acids F503, I506, Q507, L510, and D511 of HCK are critical for the binding of HCK's C-terminal 40 amino acids to NLRP12's PYD + NBD. Additionally, the C-terminal 30 amino acids of HCK are sufficient to bind to NLRP12's PYD + NBD, but not to its PYD alone nor to its NBD alone. In cell lines that express HCK endogenously, it was co- immunoprecipitated with stably expressed exogenous NLRP12. Also, NLRP12 co-immunoprecipitated and co-localized with HCK when both were overexpressed in 293T cells. In addition, in this overexpression system, steady-state NLRP12 protein expression levels significantly decreased when HCK was co-expressed. Bioinformatic analysis showed that HCK mRNA co-occurred with NLRP12 mRNA, but not with other NLRP mRNAs, in blood and marrow samples from acute myeloid leukemia (AML) patients. The mRNA of NLRP12 is also co-expressed with HCK in AML patient samples, and the levels of mRNA expression of each are correlated. Together these data suggest that NLRP12, through its binding to HCK, may have an effect on the pathogenesis of AML., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Tears are a known source of biomarkers for both ocular and systemic diseases with particular advantages; specifically, the noninvasiveness of sample collection and a unique and increasingly better-defined protein composition. Here, we discuss our rationale for use of tears for discovery of biomarkers for Parkinson's disease (PD). These reasons include literature supporting changes in tear flow and composition in PD, and the interconnections between the ocular surface system and neurons affected in PD. We highlight recent data on the identification of tear biomarkers including oligomeric α-synuclein, associated with neuronal degeneration in PD, in tears of PD patients and discuss possible sources for its release into tears. Challenges and next steps for advancing such biomarkers to clinical usage are highlighted.

Caveolae are membrane organelles formed by submicron invaginations in the plasma membrane, and are involved in mechanosensing, cell signaling, and endocytosis. Although implicated broadly in physiology and pathophysiology, better tools are required to elucidate the precise role of caveolar processes through selective activation and inactivation of their trafficking. Our group recently reported that thermally-responsive elastin-like polypeptides (ELPs) can trigger formation of 'genetically engineered protein microdomains (GEPMs)' functionalized with either Clathrin-light chain or the epidermal growth factor receptor. This manuscript is the first report of this strategy to modulate caveolin-1 (CAV1). By attaching different ELP sequences to CAV1, mild heating can be used to self-assemble CAV1-ELP microdomains inside of cells. The temperature of self-assembly can be controlled by tuning the ELP sequence. The formation of CAV1-ELP microdomains internalizes Cholera Toxin Subunit B, a commonly used marker of caveolae mediated endocytosis. CAV1-ELPs also colocalize with Cavin 1, an essential component of functional caveolae biogenesis. With the emerging significance of caveolae in health and disease and the lack of specific probes to rapidly and reversibly affect caveolar function, CAV1-ELP microdomains are a new tool to rapidly probe caveolae associated processes in endocytosis, cell signaling, and mechanosensing., Competing Interests: Conflict of Interest JA MacKay is an inventor on patents describing applications of elastin-like polypeptides.

Aim: Due to active engagement of sensory and afferent nerve fibers in reflex tearing which could be affected in Parkinson's disease (PD), we tested reflex tears as a source of potential PD biomarkers. Patients & methods: Reflex tears collected from 84 PD and 84 age- and sex-equivalent healthy controls (HC) were used to measure levels of oligomeric α-Syn (α-Syn Oligo ), total α-Syn (α-Syn Total ), CCL2, DJ-1, lactoferrin and MMP9. Results: α-syn Oligo (p < 0.0001), CCL2 (p = 0.003) and lactoferrin (p = 0.002) were significantly elevated in PD patient tears relative to HC tears. Tear flow was significantly lower in PD relative to HC (p = 0.001). Conclusion: Reflex tears are a potential source for detection of characteristic changes in PD patients.

Aim: Secretion of proteins into basal tears of Parkinson's disease (PD) patients may be altered by changes in nerve function. Materials & methods: Oligomeric α-Syn Oligo  and total α-Syn Total , CCL-2, DJ-1, LF and MMP-9 were measured in basal tears from 93 PD patients and 82 age- and sex-equivalent healthy controls. Results: α-Syn Total was decreased (p = 0.0043), whereas α-Syn Oligo (p < 0.0001) and the ratio of α-Syn Oligo /α-Syn Total (p < 0.0001) were increased in basal tears from PD patients compared with healthy controls. Area under receiver-operating curves of α-Syn Oligo and α-Syn Oligo /α-Syn Total contents were 0.70 (95% confidence limits: 0.621-0.774) and 0.72 (95% confidence limits: 0.642-0.792). Conclusion: PD patient basal tears may contain biomarkers that can be assayed noninvasively and inexpensively.

Objective: To evaluate the prevalence of neonatal dermatoses in the early neonatal period and to associate them with neonatal, demographic and obstetric variables., Methods: A cross-sectional study with neonates and their respective mothers, who were hospitalized in a public maternity hospital in Curitiba, PR, Brazil. Data collection was performed using information present in the medical records and a physical examination of the newborn during the period between April 2015 and May 2016., Results: 350 neonates were evaluated. 54.8% were male, and 94.8% (332/350) presented a dermatosis. Among them, 84.6% had, concomitantly, two or more dermatoses. A total of 23 types of dermatoses were diagnosed. The most prevalent were: sebaceous hyperplasia (66%); fluff (42.6%); and salmon patches (41.4%). The mean age of the mothers was 24.9±4.9 years old, and they were predominately white (57.7%). Vernix caseosa was associated with the female gender (p=0.034). Nonwhite mothers were associated with genital hyperpigmentation (p=0.03) and Mongolian spots (p=0.001). Physiological flaking was associated with cesarean deliveries (p=0.03) and a gestational age of over 40 weeks (p=0.054). Salmon patches was associated with primiparity (p=0.0001)., Conclusions: There was a high prevalence of neonatal dermatosis in the studied population. Each newborn had, on average, three different dermatoses. Dermatosis in neonates was associated with primiparity, nonwhites, a gestational age of over 40 weeks, and the sex of the newborn.

The dynamic manipulation of kinases remains a major obstacle to unraveling cell-signaling networks responsible for the activation of biological systems. For example, epidermal growth factor (EGF) stimulates the epidermal growth factor receptor (EGFR/ErbB1); however, EGF also recruits other kinases (HER2/ErbB2) involved with various signaling pathways. To better study EGFR we report a new strategy to selectively activate receptor tyrosine kinases fused to elastin-like polypeptides (ELPs), which can be visualized inside mammalian cells using fixed and live-cell fluorescence microscopy. ELPs are high molecular weight polypeptides that phase separate abruptly upon heating. When an EGFR-ELP fusion is heated, it clusters, initiates receptor internalization, phosphorylates, initiates downstream kinase signaling, and undergoes retrograde transport towards the cell body. Unlike other strategies to block EGFR (small molecule inhibitors, RNAi, or transcriptional regulators), EGFR-ELP clustering can be specifically switched on or off within minutes. Live-cell imaging suggests that EGFR-ELPs assemble in most cells with only a 3 °C increase in temperature. This strategy was found reversible and able to dynamically control the downstream phosphorylation/activation of the ERK1/2 pathway. For the first time, this strategy enables the rational engineering of specific temperature-sensitive receptors that may have broad applications in the study and manipulation of biological processes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The vectorial secretion and absorption of fluid and solutes by epithelial cells is dependent on the polarized expression of membrane solute transporters and channels at the apical and basolateral membranes. The establishment and maintenance of this polarized expression of transporters and channels are affected by divers protein-trafficking complexes. Moreover, regulation of the magnitude of transport is often under control of physiological stimuli, again through the interaction of transporters and channels with protein-trafficking complexes. This review highlights the value in utilizing transporters and channels as cargo to characterize core trafficking machinery by which epithelial cells establish and maintain their polarized expression, and how this machinery regulates fluid and solute transport in response to physiological stimuli., (Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.)

Liver kinase B 1 (LKB1 or STK11) and PTEN (phosphatase and tensin homologue deleted on chromosome 10) are two tumor suppressors that regulate the mTOR signaling pathway. Deletion studies show that loss of either Lkb1 ( Lkb +/- ) or Pten ( Pten loxP/loxP ; Alb-Cre + ) leads to liver injury and development of hepatocarcinoma. In this study, we investigated the crosstalk of LKB1 and PTEN loss during tumorigenesis and liver development. We show here that haplo-insufficiency of Lkb1 in the liver leads to advanced tumor development in the Pten null mice ( Pten loxP/loxP ; Lkb loxP/+ ; Alb-Cre + ). Our analysis shows that LKB1 and PTEN interacted with each other in their regulation of fatty acid synthase as well as p21 expression. The combined loss of LKB1 and PTEN ( Pten loxP/loxP ; Lkb loxP/loxP ; Alb-Cre + ) also led to the inability to form zonal structures in the liver. The lack of metabolic zonal structures is consistent with the inability of the livers to store glycogen as well as elevated plasma bilirubin and alanine aminotransferase (ALT), indicative of liver dysfunction. These structural and functional defects are associated with cytoplasm distribution of a canalicular membrane protein MRP2 (multidrug resistant protein 2) which is responsible for clearing bilirubin. This observed regulation of MRP2 by LKB1 likely contributed to the lack of cellular polarity and the early lethality phenotype associated with homozygous loss of Lkb1 alone or in combination with Pten . Finally, Pten deletion does not rescue the precocious ductal plate formation reported for Lkb1 deleted livers., Conclusion: Our study dissected the functional and molecular crosstalk of PTEN and LKB1 and elucidate key molecular targets for such interaction.

Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this study, a novel Fc-based drug carrier, single chain Fc-dimer (sc(Fc) 2 ), was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions. The resultant sc(Fc) 2 candidate preserved the neonatal Fc receptor (FcRn) binding. sc(Fc) 2 -mediated delivery was then evaluated using a therapeutic protein with a short plasma half-life, human growth hormone (hGH), as the protein drug cargo. This novel carrier protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based drug carrier. sc(Fc) 2 technology has the potential to greatly advance and expand the use of Fc-technology for improving the pharmacokinetics and bioactivity of protein therapeutics., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The mechanism responsible for the altered spectrum of tear proteins secreted by lacrimal gland acinar cells (LGAC) in patients with Sjögren's Syndrome (SS) remains unknown. We have previously identified increased cathepsin S (CTSS) activity as a unique characteristic of tears of patients with SS. Here, we investigated the role of Rab3D, Rab27a, and Rab27b proteins in the enhanced release of CTSS from LGAC. Similar to patients with SS and to the male nonobese diabetic (NOD) mouse model of SS, CTSS activity was elevated in tears of mice lacking Rab3D. Findings of lower gene expression and altered localization of Rab3D in NOD LGAC reinforce a role for Rab3D in suppressing excess CTSS release under physiological conditions. However, CTSS activity was significantly reduced in tears of mice lacking Rab27 isoforms. The reliance of CTSS secretion on Rab27 activity was supported by in vitro findings that newly synthesized CTSS was detected in and secreted from Rab27-enriched secretory vesicles and that expression of dominant negative Rab27b reduced carbachol-stimulated secretion of CTSS in cultured LGAC. High-resolution 3D-structured illumination microscopy revealed microdomains of Rab3D and Rab27 isoforms on the same secretory vesicles but present in different proportions on different vesicles, suggesting that changes in their relative association with secretory vesicles may tailor the vesicle contents. We propose that a loss of Rab3D from secretory vesicles, leading to disproportionate Rab27-to-Rab3D activity, may contribute to the enhanced release of CTSS in tears of patients with SS., (Copyright © 2016 the American Physiological Society.)

Introduction: Chronic kidney disease (CKD) compromises the health and routine of the patient. On the fifth stage of CKD, the patient becomes eligible to start renal replacement therapy: hemodialysis (HD), peritoneal dialysis (PD) or kidney transplantation. The type of CKD treatment is essential to improving quality of life of the patient., Objective: To compare the quality of life of CKD stage 5 patients who perform HD and home PD., Methods: Cross-sectional study with data collection, by convenience, through the application of socioeconomic and KDQOL SF-36 questionnaires in HD and PD patients of the Pro-Renal Foundation and satellite clinics in Curitiba-PR., Results: The sample was 338 patients, 222 HD and 116 PD. Average age: 54.4 years for HD group (± 15.28) and 58.00 for the DP group (± 13.99). The variables: work status (p < 0.05), encouragement by dialysis staff (p < 0.01) and patient satisfaction (p < 0.001) were in favor of DP; while physical functioning (p < 0.05) and emotional function (p < 0.01) were to HD., Conclusion: Objectively, PD was more favorable regarding quality of life, for the large number of items with significant results when compared to HD. However, the two variables of greatest significance found in HD (physical functioning and emotional functioning) ended up having a much greater impact on well-being and daily-life of the patient in the environment external to the clinic than those who were higher in DP, making HD the most favorable for patient quality of life.

A ubiquitous approach to study protein function is to knock down activity (gene deletions, siRNA, small molecule inhibitors, etc) and study the cellular effects. Using a new methodology, this manuscript describes how to rapidly and specifically switch off cellular pathways using thermally responsive protein polymers. A small increase in temperature stimulates cytosolic elastin-like polypeptides (ELPs) to assemble microdomains. We hypothesize that ELPs fused to a key effector in a target macromolecular complex will sequester the complex within these microdomains, which will bring the pathway to a halt. To test this hypothesis, we fused ELPs to clathrin-light chain (CLC), a protein associated with clathrin-mediated endocytosis. Prior to thermal stimulation, the ELP fusion is soluble and clathrin-mediated endocytosis remains 'on.' Increasing the temperature induces the assembly of ELP fusion proteins into organelle-sized microdomains that switches clathrin-mediated endocytosis 'off.' These microdomains can be thermally activated and inactivated within minutes, are reversible, do not require exogenous chemical stimulation, and are specific for components trafficked within the clathrin-mediated endocytosis pathway. This temperature-triggered cell switch system represents a new platform for the temporal manipulation of trafficking mechanisms in normal and disease cell models and has applications for manipulating other intracellular pathways.

Selective inhibitors of myosin or actin function and confocal microscopy were used to test the role of an actomyosin complex in controlling morphology, trafficking, and fusion of tubulovesicles (TV) containing H-K-ATPase with the apical secretory canaliculus (ASC) of primary-cultured rabbit gastric parietal cells. In resting cells, myosin IIB and IIC, ezrin, and F-actin were associated with ASC, whereas H-K-ATPase localized to intracellular TV. Histamine caused fusion of TV with ASC and subsequent expansion resulting from HCl and water secretion; F-actin and ezrin remained associated with ASC whereas myosin IIB and IIC appeared to dissociate from ASC and relocalize to the cytoplasm. ML-7 (inhibits myosin light chain kinase) caused ASC of resting cells to collapse and most myosin IIB, F-actin, and ezrin to dissociate from ASC. TV were unaffected by ML-7. Jasplakinolide (stabilizes F-actin) caused ASC to develop large blebs to which actin, myosin II, and ezrin, as well as tubulin, were prominently localized. When added prior to stimulation, ML-7 and jasplakinolide prevented normal histamine-stimulated transformations of ASC/TV and the cytoskeleton, but they did not affect cells that had been previously stimulated with histamine. These results indicate that dynamic pools of actomyosin are required for maintenance of ASC structure in resting cells and for trafficking of TV to ASC during histamine stimulation. However, the dynamic pools of actomyosin are not required once the histamine-stimulated transformation of TV/ASC and cytoskeleton has occurred. These results also show that vesicle trafficking in parietal cells shares mechanisms with similar processes in renal collecting duct cells, neuronal synapses, and skeletal muscle.

The polymeric immunoglobulin receptor (pIgR) mediates transcytosis of dimeric immunoglobulin A (dIgA) and its release into mucosal secretions. The present study reveals the complexity of the trafficking of pIgR to the apical plasma membrane in epithelial cells with exocrine secretory functions; in rabbit lacrimal gland acinar cells (LGACs), trafficking of pIgR involves both the transcytotic pathway and one arm of the regulated secretory pathway. By specifically tracking pIgR endocytosed from the basolateral membrane, we show here that the Rab11a-regulated transcytotic pathway mediates the basal-to-apical transport of pIgR, and that pIgR sorted into the transcytotic pathway does not access the regulated secretory pathway. However, previous work in LGACs expanded in the present study has shown that some pIgR is localized to Rab3D-enriched mature secretory vesicles (SVs). Myosin Vb and myosin Vc motors modulate release of proteins from the Rab11a-regulated transcytotic pathway and the Rab3D-enriched secretory pathway in LGACs, respectively. Confocal fluorescence microscopy and biochemical assays showed that inhibition of myosin Vb and myosin Vc activity by overexpression of their dominant-negative mutants each significantly but differentially impaired aspects of apically targeted pIgR trafficking and secretory component release, suggesting that these motors function to regulate pIgR trafficking in both the transcytotic and exocytotic pathways. Intriguingly, a second mature SV population enriched in Rab27b was devoid of pIgR cargo, suggesting the specialization of Rab3D-enriched mature SVs to carry a particular subset of cargo proteins from the trans-Golgi network to the apical plasma membrane.

Objectives: To examine changes in preprofessional pharmacy curricular requirements and trends, and determine rationales for and implications of modifications., Methods: Prerequisite curricular requirements compiled between 2006 and 2011 from all doctor of pharmacy (PharmD) programs approved by the Accreditation Council of Pharmacy Education were reviewed to ascertain trends over the past 5 years. An online survey was conducted of 20 programs that required either 3 years of prerequisite courses or a bachelor's degree, and a random sample of 20 programs that required 2 years of prerequisites. Standardized telephone interviews were then conducted with representatives of 9 programs., Results: In 2006, 4 programs required 3 years of prerequisite courses and none required a bachelor's degree; by 2011, these increased to 18 programs and 7 programs, respectively. Of 40 programs surveyed, responses were received from 28 (70%), 9 (32%) of which reported having increased the number of prerequisite courses since 2006. Reasons given for changes included desire to raise the level of academic achievement of students entering the PharmD program, desire to increase incoming student maturity, and desire to add clinical sciences and experiential coursework to the pharmacy curriculum. Some colleges and schools experienced a temporary decrease in applicants., Conclusions: The preprofessional curriculum continues to evolve, with many programs increasing the number of course prerequisites. The implications of increasing prerequisites were variable and included a perceived increase in maturity and quality of applicants and, for some schools, a temporary decrease in the number of applicants.

Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real-time PCR, we show that the expression of clathrin and adaptor protein subunits are upregulated in maturation stage rodent enamel organ cells. AP complex 2 (AP-2) is the most upregulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts, with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin-dependent endocytosis, thus implying the likelihood of specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also upregulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1); cluster of differentiation 63 and 68 (Cd63 and Cd68); ATPase, H(+) transporting, lysosomal V0 subunit D2 (Atp6v0d2); ATPase, H(+) transporting, lysosomal V1 subunit B2 (Atp6v1b2); chloride channel, voltage-sensitive 7 (Clcn7); and cathepsin K (Ctsk). Immunohistologic data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain showed upregulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor-regulated pathway for the endocytosis of enamel matrix proteins. These data together define an endocytotic pathway likely used by ameloblasts to remove the enamel matrix during enamel maturation., (Copyright © 2013 American Society for Bone and Mineral Research.)

Targeting of drugs and their carrier systems by using receptor-mediated endocytotic pathways was in its nascent stages 25 years ago. In the intervening years, an explosion of knowledge focused on design and synthesis of nanoparticulate delivery systems as well as elucidation of the cellular complexity of what was previously-termed receptor-mediated endocytosis has now created a situation when it has become possible to design and test the feasibility of delivery of highly specific nanoparticle drug carriers to specific cells and tissue. This review outlines the mechanisms governing the major modes of receptor-mediated endocytosis used in drug delivery and highlights recent approaches using these as targets for in vivo drug delivery of nanoparticles. The review also discusses some of the inherent complexity associated with the simple shift from a ligand-drug conjugate versus a ligand-nanoparticle conjugate, in terms of ligand valency and its relationship to the mode of receptor-mediated internalization., (Copyright © 2012 Elsevier B.V. All rights reserved.)

From mitochondria to the nuclear envelope, the controlled assembly of micro- and nanostructures is essential for life; however, the level at which we can deliberately engineer the assembly of microstructures within intracellular environments remains primitive. To overcome this obstacle, we present a platform to reversibly assemble genetically engineered protein microdomains (GEPMs) on the time scale of minutes within living cells. Biologically inspired from the human protein tropoelastin, these protein polymers form a secondary aqueous phase above a tunable transition temperature. This assembly process is easily manipulated to occur at or near physiological temperature by adjusting molecular weight and hydrophobicity. We fused protein polymers to green fluorescent protein (GFP) to visualize their behavior within the cytoplasm. While soluble, these polymers have a similar intracellular diffusion constant as cytosolic proteins at 7.4 μm(2)/s; however, above their phase transition temperature, the proteins form distinct microdomains (0.1-2 μm) with a reduced diffusion coefficient of 1.1 μm(2)/s. Microdomain assembly and disassembly are both rapid processes with half-lives of 3.8 and 1.0 min, respectively. Via selection of the protein polymer, the assembly temperature is tunable between 20 and 40 °C. This approach may be useful to control intracellular formation of genetically engineered proteins and protein complexes into concentrated microdomains.

Objectives: To learn about the profile of fungal colonization and related risk factors in premature newborns., Methods: Prospective cohort, from 04/01/2010 to 04/31/2011, with 44 patients admitted to the neonatal intensive care unit, born at the hospital maternity, weighing less than 1,500 g. On admission, data were collected on pre-natal care and childbirth. Clinical and laboratory information, nasal and rectal swabs, and peripheral blood cultures were collected on days 1, 7, 10, and 14 of stay in neonatal intensive care unit and then every 7 days until discharge or death. For statistical analysis, we used the chi-square test, Fisher exact test, Kaplan-Meier and logistic regression model., Results: The incidence of colonization was 13.5/1,000 patients/day. The incidence of candidemia was 0.9/1,000 patients/day. The average hospitalization time was 30.5 days (± 20.27), and the onset of colonization occurred, in average, at 11.13 days (± 8.82). Vaginal delivery was found to be an independent risk factor for the development of fungal colonization during hospitalization (p = 0.042, odds ratio = 4.38, 95% confidence interval [95%CI] = 1,13-16,99). Likewise, leukocytosis (> 30,000/mm3) on admission was an indicator for the simultaneous presence of fungal colonization (p = 0.048). The presence of bronchopulmonary dysplasia tends to be a factor of higher probability for the development of colonization (p = 0.067). The most affected colonization site was the rectal mucosa: 89.09 versus 10.9% of the nasal mucosa., Conclusions: Vaginal delivery and leukocytosis over 30,000/mm3 on admission were found to be risk factors for fungal colonization during hospitalization.

Background & Aims: Rabs are monomeric guanosine triphosphatases that regulate membrane trafficking and acid secretion in gastric parietal cells. Using a proteomics approach, we identified a new Rab, Rab27b, in tubulovesicle membranes and determined its role in parietal cell activation., Methods: We used mass spectrometry (MS) to identify Rab27b in purified tubulovesicular membrane fractions and used immunoblot and immunofluorescence analyses to study its expression. Wild-type, constitutively active (Rab27bQ78L), and dominant negative (Rab27bN133I) forms of Rab27b were tagged with yellow fluorescent protein (YFP) and expressed in parietal cells using adenoviral constructs to study localization and function. Localization was visualized by fluorescence microscopy in resting and stimulated cells. Acid secretion in primary cell cultures was measured by aminopyrine accumulation., Results: A tandem MS peptide mass fingerprint was matched to 7 peptides of Rab27b. Rab27b localized to tubulovesicle membranes, based on immunoblot and immunocytochemical analyses. Endogenous Rab27b, YFP/wild-type Rab27b, Rab27bQ78L, and Rab27bN133I all distributed throughout the cytoplasm of resting parietal cells. After stimulation, wild-type Rab27b and YFP-Rab27bQ78L translocated to the apical membrane, but YFPR-ab27bN133I did not. Expression of wild-type YFP-Rab27b or YFP-Rab27bQ78L did not affect acid secretion, whereas expression of Rab27bN133I almost completely inhibited acid secretion., Conclusions: Rab27b is associated with tubulovesicle membranes in the parietal cell and Rab27b may play a role in stimulation-associated membrane recruitment and gastric acid secretion., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The lacrimal gland is responsible for tear production, and a major protein found in tears is secretory component (SC), the proteolytically cleaved fragment of the extracellular domain of the polymeric Ig receptor (pIgR), which is the receptor mediating the basal-to-apical transcytosis of polymeric immunoglobulins across epithelial cells. Immunofluorescent labeling of rabbit lacrimal gland acinar cells (LGACs) revealed that the small GTPase Rab3D, a regulated secretory vesicle marker, and the pIgR are colocalized in subapical membrane vesicles. In addition, the secretion of SC from primary cultures of LGACs was stimulated by the cholinergic agonist carbachol (CCH), and its release rate was very similar to that of other regulated secretory proteins in LGACs. In pull-down assays from resting LGACs, recombinant wild-type Rab3D (Rab3DWT) or the GDP-locked mutant Rab3DT36N both pulled down pIgR, but the GTP-locked mutant Rab3DQ81L did not. When the pull-down assays were performed in the presence of guanosine-5'-(gamma-thio)-triphosphate, GTP, or guanosine-5'-O-(2-thiodiphosphate), binding of Rab3DWT to pIgR was inhibited. In blot overlays, recombinant Rab3DWT bound to immunoprecipitated pIgR, suggesting that Rab3D and pIgR may interact directly. Adenovirus-mediated overexpression of mutant Rab3DT36N in LGACs inhibited CCH-stimulated SC release, and, in CCH-stimulated LGACs, pull down of pIgR with Rab3DWT and colocalization of pIgR with endogenous Rab3D were decreased relative to resting cells, suggesting that the pIgR-Rab3D interaction may be modulated by secretagogues. These data suggest that the novel localization of pIgR to the regulated secretory pathway of LGACs and its secretion therefrom may be affected by its novel interaction with Rab3D.

The enamel matrix protein amelogenin is secreted by ameloblasts into the extracellular space to guide the formation of highly ordered hydroxyapatite mineral crystallites, and, subsequently, is almost completely removed during mineral maturation. Amelogenin interacts with the transmembrane proteins CD63 and LAMP (lysosome-associated membrane protein) 1, which are involved in endocytosis. Exogenously added amelogenin has been observed to move rapidly into CD63/LAMP1-positive vesicles in cultured cells. In the present study, we demonstrate the protein region defined by amino acid residues 103-205 for CD63 interacts not only with amelogenin, but also with other enamel matrix proteins (ameloblastin and enamelin). A detailed characterization of binding regions in amelogenin, CD63 and LAMP1 reveals that the amelogenin region defined by residues PLSPILPELPLEAW is responsible for the interaction with CD63 through residues 165-205, with LAMP1 through residues 226-251, and with the related LAMP2 protein through residues 227-259. We predict that the amelogenin binding region is: (i) hydrophobic; (ii) largely disordered; and (iii) accessible to the external environment. In contrast, the binding region of CD63 is likely to be organized in a '7' shape within the mushroom-like structure of CD63 EC2 (extracellular domain 2). In vivo, the protein interactions between the secreted enamel matrix proteins with the membrane-bound proteins are likely to occur at the specialized secretory surfaces of ameloblast cells called Tomes' processes. Such protein-protein interactions may be required to establish short-term order of the forming matrix and/or to mediate feedback signals to the transcriptional machinery of ameloblasts and/or to remove matrix protein debris during enamel biomineralization.

Proteins of the developing enamel matrix include amelogenin, ameloblastin and enamelin. Of these three proteins amelogenin predominates. Protein-protein interactions are likely to occur at the ameloblast Tomes' processes between membrane-bound proteins and secreted enamel matrix proteins. Such protein-protein interactions could be associated with cell signaling or endocytosis. CD63 and Lamp1 are ubiquitously expressed, are lysosomal integral membrane proteins, and localize to the plasma membrane. CD63 and Lamp1 interact with amelogenin in vitro. In this study our objective was to study the molecular events of intercellular trafficking of an exogenous source of amelogenin, and related this movement to the spatiotemporal expression of CD63 and Lamp1 using various cell lineages. Exogenously added amelogenin moves rapidly into the cell into established Lamp1-positive vesicles that subsequently localize to the perinuclear region. These data indicate a possible mechanism by which amelogenin, or degraded amelogenin peptides, are removed from the extracellular matrix during enamel formation and maturation.

The mechanism in which small cationic oligopeptides are able to reach the cytosol of cells is controversial. Macropinocytosis has been recently suggested as a major mechanism for internalization of these peptides. In this report, the involvement of macropinocytosis on cytosolic localization of oligoarginine was quantitatively investigated in HeLa cells. Using a method which allows for the separate measurement of cytosolic versus vesicular oligopeptide, the results show that neither macropinosome nor filopodia formation correlates with cytosolic delivery of oligoarginine. Additionally, unlike macropinocytosis, the cytosolic delivery of oligoarginine was not inhibited by incubation at 16 degrees C, or by treatment with amiloride. Oligoarginine treatment does not contribute to leakage from endocytic vesicles, indicating the lack of endosomolytic properties. Finally, the amount of oligoarginine found in the cytosol was not substantially increased after coincubation with EGF, a known stimulator of macropinocytosis. Taken together, these data indicate that membrane transduction of oligoarginine occurs separately from macropinocytosis in HeLa cells.

Purpose: To investigate the involvement of carrier-mediated transport mechanisms in brimonidine transport in retinal pigment epithelium (RPE)., Methods: The transport of [3H]-brimonidine in bovine RPE-choroid explants was evaluated in a modified Ussing chamber. The uptake of [3H]brimonidine was evaluated in differentiated ARPE-19 cells cultured on permeable transwell filters., Results: The transport of brimonidine into (choroid-to-retina transport [inward]) and out of (retina-to-choroid transport [outward]) the eye in bovine RPE-choroid explants was temperature dependent. Both inward and outward brimonidine transport decreased at 5 microM compared with 10 nM. The melanin pigmentation of RPE did not significantly affect tissue permeability at either brimonidine dose. A saturable component was identified for the inward transport with the apparent Michaelis-Menten constant and a maximum transport rate of 51 microM and 148 pmol/(cm2 x h), respectively. Both apical (representing retina-to-choroid transport) and basolateral (representing choroid-to-retina transport) brimonidine uptake in ARPE-19 cells showed temperature dependence. Apical uptake was higher than basolateral uptake at 37 degrees C and was decreased to 70% in the presence of NaN3 or in the absence of extracellular Na+. Besides alpha2-agonists, apical uptake was inhibited by verapamil, desipramine, and quinidine, but not by MPP+ (1-methyl-4-phenylpyridinium), TEA (tetraethylammonium), decynium-22, carnitine, PHA (p-aminohippurate), alanine, or inosine. Basolateral brimonidine uptake increased by 35% at extracellular pH of 6 and decreased by 50% under cell-depolarized conditions of high medium K+ and 1 microM valinomycin. Temperature-dependent components of basolateral uptake were not saturated at doses up to 2 mM., Conclusions: A carrier-mediated transport process for brimonidine in RPE was demonstrated in bovine RPE-choroid explants and polarized ARPE-19 cells. This transport system may play a significant role in modulating the movement of brimonidine into and out of the eye.

We investigated the involvement of PKC-epsilon in apical actin remodeling in carbachol-stimulated exocytosis in reconstituted rabbit lacrimal acinar cells. Lacrimal acinar PKC-epsilon cosedimented with actin filaments in an actin filament binding assay. Stimulation of acini with carbachol (100 microM, 2-15 min) significantly (P < or = 0.05) increased PKC-epsilon recovery with actin filaments in two distinct biochemical assays, and confocal fluorescence microscopy showed a significant increase in PKC-epsilon association with apical actin in stimulated acini as evidenced by quantitative colocalization analysis. Overexpression of dominant-negative (DN) PKC-epsilon in lacrimal acini with replication-defective adenovirus (Ad) resulted in profound alterations in apical and basolateral actin filaments while significantly inhibiting carbachol-stimulated secretion of bulk protein and beta-hexosaminidase. The chemical inhibitor GF-109203X (10 microM, 3 h), which inhibits PKC-alpha, -beta, -delta, and -epsilon, also elicited more potent inhibition of carbachol-stimulated secretion relative to Gö-6976 (10 microM, 3 h), which inhibits only PKC-alpha and -beta. Transduction of lacrimal acini with Ad encoding syncollin-green fluorescent protein (GFP) resulted in labeling of secretory vesicles that were discharged in response to carbachol stimulation, whereas cotransduction of acini with Ad-DN-PKC-epsilon significantly inhibited carbachol-stimulated release of syncollin-GFP. Carbachol also increased the recovery of secretory component in culture medium, whereas Ad-DN-PKC-epsilon transduction suppressed its carbachol-stimulated release. We propose that DN-PKC-epsilon alters lacrimal acinar apical actin remodeling, leading to inhibition of stimulated exocytosis and transcytosis.

Developmentally regulated brain proteins (drebrins) are highly expressed in brain where they may regulate actin filament formation in dendritic spines. Recently, the drebrin E2 isoform was detected in certain epithelial cell types including the gastric parietal cell. In gastric parietal cells, activation of HCl secretion is correlated with actin filament formation and elongation within intracellular canaliculi, which are the sites of acid secretion. The aim of this study was to define the pattern of drebrin expression in gland units in the intact rabbit oxyntic gastric mucosa and to initiate approaches to define the functions of this protein in parietal cells. Drebrin E2 expression was limited entirely or almost entirely to parietal cells and depended upon the localization of parietal cells along the gland axis. Rabbit drebrin E2 was cloned and found to share 86% identity with human drebrin 1a and to possess a number of cross-species conserved protein-protein interaction and phosphorylation consensus sites. Two-dimensional Western blot and phosphoaffinity column analyses confirmed that drebrin is phosphorylated in parietal cells, and several candidate phosphorylation sites were identified by mass spectrometry. Overexpression of epitope-tagged drebrin E2 led to the formation of microspikes and F-actin-rich ring-like structures in cultured parietal cells and suppressed cAMP-dependent acid secretory responses. In Madin-Darby canine kidney cells, coexpression of epitope-tagged drebrin and the Rho family GTPase Cdc42, which induces filopodial extension, produced an additive increase in the length of microspike projections. Coexpression of dominant negative Cdc42 with drebrin E2 did not prevent drebrin-induced microspike formation. These findings suggest that 1) drebrin can induce the formation of F-actin-rich membrane projections by Cdc42-dependent and -independent mechanisms; and that 2) drebrin plays an active role in directing the secretagogue-dependent formation of F-actin-rich filaments on the parietal cell canalicular membrane. Finally, the differential distribution of drebrin in parietal cells along the gland axis suggests that drebrin E2 may be an important marker of parietal cell differentiation and functionality.

Tuftelin-interacting protein (TFIP11) was first identified in a yeast two-hybrid screening as a protein interacting with tuftelin. The ubiquitous expression of TFIP11 suggested that it might have other functions in non-dental tissues. TFIP11 contains a G-patch, a protein domain believed to be involved in RNA binding. Using a green fluorescence protein tag, TFIP11 was found to locate in a novel subnuclear structure that we refer to as the TFIP body. An in vivo splicing assay demonstrated that TFIP11 is a novel splicing factor. TFIP11 diffuses from the TFIP body following RNase A treatment, suggesting that the retention of TFIP11 is RNA dependent. RNA polymerase II inhibitor (-amanitin and actinomycin D) treatment causes enlargement in size and decrease in number of TFIP bodies, suggesting that TFIP bodies perform a storage function rather than an active splicing function. The TFIP body may therefore represent a new subnuclear storage compartment for splicing components.

IQGAPs, GTPase-activating proteins with an IQ motif, are thought to regulate many actin cytoskeleton-based activities through interactions with Cdc42 and Rac. Recently, Cdc42 was implicated in regulation of gastric parietal cell HCl secretion, and IQGAP2 was immunolocalized with Cdc42 to F-actin-rich intracellular canalicular membranes of isolated gastric parietal cells in primary culture. Here we sought to define distribution and localization of IQGAP1 and IQGAP2 in major oxyntic (acid-secreting) gastric mucosal cell types and to determine whether secretory agonists modulate these proteins. Differential staining protocols were used to identify different cell populations (parietal, chief, surface/pit, and mucous neck cells) in semi-intact glands isolated from rabbit gastric mucosae and to characterize these same cells after dispersion and fractionation on isopycnic density gradients with simultaneous staining for F-actin, H+-K+-ATPase, and GSII lectin-binding sites. There was a pronounced increase in intracellular F-actin staining in dispersed chief cells, apparently from internalization of F-actin-rich apical membranes that normally abut the gland lumen. Therefore, other membrane-associated proteins might also be redistributed by disruption of cell-cell contacts. Western blot analyses were used to quantitate relative concentrations of IQGAPs in defined mucosal cell fractions, and gastric glands were used for in situ localizations. We detected uniform levels of IQGAP2 expression in oxyntic mucosal cells with predominant targeting to regions of cell-cell contact and nuclei of all cell types. IQGAP2 was not detected in parietal cell intracellular canaliculi. IQGAP1 expression was variable and targeted predominantly to the cortex of chief and mucous neck cells. Parietal cells expressed little or no IQGAP1 vs. other mucosal cell types. Phosphoprotein affinity chromatography, isoelectric focusing, and phosphorylation site analyses indicated that both IQGAP1 and IQGAP2 are phosphoproteins potentially regulated by [Ca2+]i/PKC and cAMP signaling pathways, respectively. Stimulation of glands with carbachol, which elevates [Ca2+]i and activates PKC, induced apparent translocation of IQGAP1, but not IQGAP2, to apical poles of chief (zymogen) and mucous neck cells. This response was mimicked by PMA but not by ionomycin or by elevation of [cAMP]i with forskolin. Our observations support a novel, PKC-dependent role for IQGAP1 in regulated exocytosis and suggest that IQGAP2 may play a more general role in regulating cell-cell interactions and possibly migration within the gastric mucosa.

Purpose: Establish and characterize a MDCK cell line that stably expressed PepT1., Methods: MDCK cells stably transfected with N-terminal HA-tagged hPepT1 were examined by immunostaining using antibodies to HA and hPepT1. HA-hPepT1 expression was verified by immunoprecipitation and Western blotting from cell lysates. The function of the expressed HA-hPepT1 was evaluated by glycyl-sarcosine uptake. The half-life of PepT1 on the cell surface was measured by biotinylation and chase-labeling of the cells, followed by immunoprecipitation with the anti-PepT1 antibody and blotting with HRP-streptavidin., Results: Immunolabeling with antibodies to HA and PepT1 showed fluorescence colocalization. Immunoprecipitation with the anti-HA antibody and Western blotting with the anti-PepT1 antibody showed a broad 90-105 kDa band, and vice versa when the antibodies were utilized in the reverse order. Glycyl-sarcosine uptake was increased in the stably transfected cells by 2-5 fold over untransfected control cells. The Km value of 375 microM accurately reflected the characteristic low affinity of PepT1. Finally, the half-life of the surface biotinylated HA-hPepT1 was measured to be 22 hrs., Conclusions: A MDCK cell line stably expressed intact and functional HA-tagged hPepT1 on its cell surface. This cell line represents an alternative to the use of Caco-2 cells to evaluate PepT1 function.

The aim of this review is to introduce the advances made over the past several years regarding the participation of actin and actin-associated proteins in clathrin-mediated endocytosis in simple cell models, and then to consider the evidence for the involvement of these effectors in apical clathrin-mediated endocytosis in epithelial cells. Basic mechanisms of clathrin-mediated endocytosis are initially addressed, followed by a detailed description of the actin cytoskeleton: its organization, function and, most importantly, the essential role played by proteins and signaling pathways responsible for the regulation of actin filament dynamics. Our focus then shifts to the GTPase, dynamin and its pivotal role as a bridge between various components of the clathrin endocytic machinery and the actin cytoskeleton. Mechanisms and effectors of dynamin-dependent endocytosis are then described, with a particular emphasis on novel proteins, which link dynamin to actin filaments. We consider additional effectors proposed to interact with actin to facilitate clathrin-mediated endocytosis in a dynamin-independent manner. The multiple roles which actin filaments are thought to play in endocytosis are addressed followed by a more detailed characterization of actin filament participation specifically in apical endocytosis. We conclude by discussing how these concepts may be integrated to improve drug internalization at the apical plasma membrane of epithelial cells.

In this article, we investigate the contributions of actin filaments and accessory proteins to apical clathrin-mediated endocytosis in primary rabbit lacrimal acini. Confocal fluorescence and electron microscopy revealed that cytochalasin D promoted apical accumulation of clathrin, alpha-adaptin, dynamin, and F-actin and increased the amounts of coated pits and vesicles at the apical plasma membrane. Sorbitol density gradient analysis of membrane compartments showed that cytochalasin D increased [14C]dextran association with apical membranes from stimulated acini, consistent with functional inhibition of apical endocytosis. Recombinant syndapin SH3 domains interacted with lacrimal acinar dynamin, neuronal Wiskott-Aldrich Syndrome protein (N-WASP), and synaptojanin; their introduction by electroporation elicited remarkable accumulation of clathrin, accessory proteins, and coated pits at the apical plasma membrane. These SH3 domains also significantly (p

The objective of this study was to develop a tissue culture system which closely mimics the in situ lacrimal gland for improved study of lacrimal acinar cell physiology. Highly purified preparations of lacrimal acinar cells from adult female New Zealand White rabbits were isolated and grown in suspension culture in the form of Matrigel 'rafts', i.e., aggregates of acinar cells enclosed within a Matrigel coating. The rafts were seeded onto Matrigel-coated culture plates and their growth was followed for up to 28 days. Immunohistochemistry was used to demonstrate the cellular sites of prolactin (PRL), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2), secretory component (SC) and major histocompatibility complex class-II molecules (MHC-II) within the acinar cells. By 3 days the cultures contained numerous, well-formed acini enclosed within the Matrigel. The acinar epithelial cells demonstrated histotypic polarity, with large, pale-staining, secretory granules aggregated adjacent to the lumen, and exocytotic release of secretory material into the lumen. From 5-10 days the pale-staining secretory granules decreased in number, while the lumenal contents of the acini increased in staining density. Throughout the culturing period as the pale-staining, secretory granules decreased in number, smaller more densely stained, secretory granules increased in number. The number of cells and size of acinar clusters increased steadily throughout the culturing period, and acini frequently achieved dimensions in excess of 0.5 mm. Increases in the size of acinar clusters were often accompanied by an increase in the size of the lumen. Frequently the lumen and its contents bulged asymmetrically towards one edge of the acinus. Immunhistochemistry demonstrated PRL and EGF within the lumens and within the apical cytoplasm of the acinar cells. Acini were strongly immunopositive for SC throughout the 28 day culture period, whereas immunopositivity for MHC-II molecules was strong initially, but diminished dramatically by 21 days. Immunostaining for FGF-2 was most intense on days 1 and 3, with staining throughout the cytoplasm, but became progressively more localized to the periphery of the acini as the culture period lengthened. In cultures of 1-28 days duration, Western blots of cell lysates demonstrated a major band (approximately 40 kDa) for PRL in 3-28 day preparations; a major band (approximately 80 kDa) for SC in 3 day and 7 day preparations that decreased in intensity in 14-28 day preparations; and a major band (approximately 23 kDa) for MHC-II protein in 1-21 day preparations that decreased in intensity in 28 day preparations. Lysosomes increased in number with time in culture, becoming a dominant cytoplasmic feature in 21 and 28 day cultures. Carbachol stimulation of 4 day rafts resulted in increased release of beta-hexosaminidase and SC from the rafts. The authors conclude that Matrigel rafts containing purified lacrimal gland acinar cells offer a highly advantageous system for study of lacrimal acinar cell function and one that correlates well with the in situ gland., (Copyright 2002 Elsevier Science Ltd.)

The characterization of endocytotic and post-endocytotic trafficking pathways at the apical membrane of epithelial cells presents a potential avenue for the identification of targets to modulate the initial stages of absorption and transepithelial transport of macromolecules. In addition, it is becoming increasingly clear that the activity of a number of apical membrane transporters is acutely regulated by vesicular trafficking. The gastric HCl-secreting parietal (oxyntic) cell is a model system to characterize an apical membrane vesicular trafficking pathway and its relationship to the regulation of the function of the gastric proton pump. The subapical tubulovesicular compartment of the parietal cell is highly enriched in the H,K-ATPase and is a key endosomal-like system in the apical membrane recycling pathway. In the process of cataloging the proteins that interact with the H,K-ATPase and tubulovesicles, we have identified novel components that may regulate protein sorting through this compartment and candidate linker proteins between the vesicular trafficking machinery and the cytoskeleton. One protein associated with H,K-ATPase-rich tubulovesicles is the nonreceptor tyrosine kinase c-src, identified by a screen for dynamin-binding proteins. The tyrosine kinase is active, as it can tyrosine-phosphorylate tubulovesicular proteins in vitro. One of the tyrosine-phosphorylated proteins of M(r) 100 kDa may be the H,K-ATPase itself, or a protein in a complex with the H,K-ATPase that is stable to dissociation by nonionic detergents. By virtue of its association with tubulovesicular membranes, c-src may regulate the trafficking and/or activity of the H,K-ATPase. A second protein identified by a screen for dynamin-binding proteins is the protein lasp-1. Lasp-1, through its modular protein structure, may bind to dynamin and to the actin cytoskeleton, thus linking the vesicular trafficking machinery with the cytoskeleton. These two examples illustrate the utility of the parietal cell in the biochemical characterization of components potentially involved in the regulation of apical membrane trafficking pathways.

Gastric HCl secretion by the parietal cell involves the secretagogue-regulated re-cycling of the H+-K+-ATPase at the apical membrane. The trafficking of the H+-K+-ATPase and the remodelling of the apical membrane during this process are likely to involve the co-ordination of the function of vesicular trafficking machinery and the cytoskeleton. This review summarizes the progress made in the identification and characterization of components of the vesicular trafficking machinery that are associated with the H+-K+-ATPase and of components of the actin-based cytoskeleton that are associated with the apical membrane of the parietal cell. Since many of these proteins are also expressed at the apical pole of other epithelial cells, the parietal cell may represent a model system to characterize the protein- protein interactions that regulate apical membrane trafficking in many other epithelial cells.

Clathrin from H-K-ATPase-rich membranes derived from the tubulovesicular compartment of rabbit and hog gastric acid secretory (parietal) cells was characterized biochemically, and the subcellular localization of membrane-associated clathrin in parietal cells was characterized by immunofluorescence, electron microscopy, and immunoelectron microscopy. Clathrin from H-K- ATPase-rich membranes was determined to be comprised of conventional clathrin heavy chain and a predominance of clathrin light chain A. Clathrin and adaptors could be induced to polymerize quantitatively in vitro, forming 120-nm-diameter basketlike structures. In digitonin-permeabilized resting parietal cells, the intracellular distribution of immunofluorescently labeled clathrin was suggestive of labeling of the tubulovesicular compartment. Clathrin was also unexpectedly localized to canalicular (apical) membranes, as were alpha-adaptin and dynamin, suggesting that this membrane domain of resting parietal cells is endocytotically active. At the ultrastructural level, clathrin was immunolocalized to canalicular and tubulovesicular membranes. H-K-ATPase was immunolocalized to the same membrane domains as clathrin but did not appear to be enriched at the specific subdomains that were enriched in clathrin. Finally, in immunofluorescently labeled primary cultures of parietal cells, in contrast to the H-K-ATPase, intracellular clathrin was found not to translocate to the apical membrane on secretagogue stimulation. Taken together, these biochemical and morphological data provide a framework for characterizing the role of clathrin in the regulation of membrane trafficking from tubulovesicles and at the canalicular membrane in parietal cells.

Subconfluent cultures of Madin-Darby canine kidney (MDCK) and CV-1 cells were immunostained with two monoclonal antibodies (MAbs), MAb X-22 and MAb 23, against clathrin heavy chain and with polyclonal antiserum against a conserved region of all mammalian clathrin light chains. In interphase MDCK and CV-1 cells, staining by all three antibodies resulted in the characteristic intracellular punctate vesicular and perinuclear staining pattern. In mitotic cells, all three anti-clathrin antibodies strongly stained the mitotic spindle. Staining of clathrin in the mitotic spindle was colocalized with anti-tubulin staining of microtubular arrays in the spindle. Staining of the mitotic spindle was evident in mitotic cells from prometaphase to telophase and in spindles in mitotic cells released from a thymidine-nocodazole block. In CV-1 cells, staining of clathrin in the mitotic spindle was not affected by brefeldin A. On Western blots, clathrin was detected, but not enriched, in isolated spindles. The immunodetection of clathrin in the mitotic spindle may suggest a novel role for clathrin in mitosis. Alternatively, the recruitment of clathrin to the spindle may suggest a novel regulatory mechanism for localization of clathrin in mitotic cells.

Enamel biomineralization is a complex process that involves interactions between extracellular matrix proteins. To identify proteins interacting with tuftelin, a potential nucleator of enamel crystallites, the yeast two-hybrid system was applied to a mouse tooth expression library and a tuftelin-interacting protein (TIP) was isolated for further characterization. Polyclonal antibodies were prepared against two recombinant variants of this protein. Both antibodies identified a major protein product in tooth organs at 39 kDa, and this protein has been called TIP39. Northern analysis showed TIP39 messenger RNA in multiple organs, a pattern similar to that of tuftelin messenger RNA. In situ hybridization of mandibles of 1-day-old mice detected TIP39 RNA in secretory ameloblasts and odontoblasts. Immunolocalization of TIP39 and tuftelin in cultured ameloblast-like cells showed that these two proteins colocalize. Within the developing tooth organ, TIP39 and tuftelin immunolocalized to the apical pole of secretory ameloblasts (Tomes' processes) and to the newly secreted extracellular enamel matrix. TIP39 amino acid sequence appears to be highly conserved with similarities to proteins in species as diverse as yeast and primates. Available sequence data and the findings reported here suggest a role for TIP39 in the secretory pathway of extracellular proteins.