SNAP23 is essential for platelet and mast cell development and required in connective tissue mast cells for anaphylaxis.

Degranulation, a fundamental effector response from mast cells (MCs) and platelets, is an example of regulated exocytosis. This process is mediated by SNARE proteins and their regulators. We have previously shown that several of these proteins are essential for exocytosis in MCs and platelets. Here, we assessed the role of the SNARE protein SNAP23 using conditional knockout mice, in which SNAP23 was selectively deleted from either the megakaryocyte/platelet or connective tissue MC lineages. We found that removal of SNAP23 in platelets results in severe defects in degranulation of all three platelet secretory granule types, i.e., alpha, dense, and lysosomal granules. The mutation also induces thrombocytopenia, abnormal platelet morphology and activation, and reduction in the number of alpha granules. Therefore, the degranulation defect might not be secondary to an intrinsic failure of the machinery mediating regulated exocytosis in platelets. When we removed SNAP23 expression in MCs, there was a complete developmental failure in vitro and in vivo. The developmental defects in platelets and MCs and the abnormal translocation of membrane proteins to the surface of platelets indicate that SNAP23 is also involved in constitutive exocytosis in these cells. The MC conditional deletant animals lacked connective tissue MCs, but their mucosal MCs were normal and expanded in response to an antigenic stimulus. We used this mouse to show that connective tissue MCs are required and mucosal MCs are not sufficient for an anaphylactic response.

Munc18-2, but not Munc18-1 or Munc18-3, regulates platelet exocytosis, hemostasis, and thrombosis.

Platelet degranulation, a form of regulated exocytosis, is crucial for hemostasis and thrombosis. Exocytosis in platelets is mediated by SNARE proteins, and in most mammalian cells this process is controlled by Munc18 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 18) proteins. Platelets express all Munc18 paralogs (Munc18-1, -2, and -3), but their roles in platelet secretion and function have not been fully characterized. Using Munc18-1, -2, and -3 conditional knockout mice, here we deleted expression of these proteins in platelets and assessed granule exocytosis. We measured products secreted by each type of platelet granule and analyzed EM platelet profiles by design-based stereology. We observed that the removal of Munc18-2 ablates the release of alpha, dense, and lysosomal granules from platelets, but we found no exocytic role for Munc18-1 or -3 in platelets. In vitro, Munc18-2-deficient platelets exhibited defective aggregation at low doses of collagen and impaired thrombus formation under shear stress. In vivo, megakaryocyte-specific Munc18-2 conditional knockout mice had a severe hemostatic defect and prolonged arterial and venous bleeding times. They were also protected against arterial thrombosis in a chemically induced model of arterial injury. Taken together, our results indicate that Munc18-2, but not Munc18-1 or Munc18-3, is essential for regulated exocytosis in platelets and platelet participation in thrombosis and hemostasis.

Syntaxin 3, but not syntaxin 4, is required for mast cell-regulated exocytosis, where it plays a primary role mediating compound exocytosis.

Mast cells (MCs) participate in allergy, inflammation, and defense against pathogens. They release multiple immune mediators via exocytosis, a process that requires SNARE proteins, including syntaxins (Stxs). The identity of the Stxs involved in MC exocytosis remains controversial. Here, we studied the roles of Stx3 and -4 in fully developed MCs from conditional knockout mice by electrophysiology and EM, and found that Stx3, and not Stx4, is crucial for MC exocytosis. The main defect seen in Stx3-deficient MCs was their inability to engage multigranular compound exocytosis, while leaving most single-vesicle fusion events intact. We used this defect to show that this form of exocytosis is not only required to accelerate MC degranulation but also essential to achieve full degranulation. The exocytic defect was severe but not absolute, indicating that an Stx other than Stx3 and -4 is also required for exocytosis in MCs. The removal of Stx3 affected only regulated exocytosis, leaving other MC effector responses intact, including the secretion of cytokines via constitutive exocytosis. Our in vivo model of passive systemic anaphylaxis showed that the residual exocytic function of Stx3-deficient MCs was sufficient to drive a full anaphylactic response in mice.

Disruption of PCDH10 and TNRC18 Genes due to a Balanced Translocation.

Balanced chromosomal rearrangements are usually associated with a normal phenotype, although in some individuals, phenotypic alterations are observed. In these patients, molecular characterization of the breakpoints can reveal the pathogenic mechanism, providing the annotation of disease-associated loci and a better genotype-phenotype correlation. In this study, we describe a patient with a balanced reciprocal translocation between 4q27 and 7p22 associated with neurodevelopmental delay. We performed cytogenetic evaluation, next-generation sequencing of microdissected derivative chromosomes, and Sanger sequencing of the junction points to define the translocation's breakpoints at base pair resolution. We found that the PCDH10 and TNRC18 genes were disrupted by the breakpoints at chromosomes 4 and 7, respectively, with the formation of chimeric genes at the junction points. Gene expression studies in the patient's peripheral blood showed reduced expression of TNRC18, a gene with unknown function and clinical significance. PCDH10 plays a role in the development of the nervous system and might be involved with the patient's neurodevelopmental delay. In this study, the full molecular characterization of the junction points was shown as an efficient tool for fine breakpoint mapping in balanced translocations in order to unmask gene disruptions and investigate the potential pathogenic role of the disrupted genes.

Munc18-2, but not Munc18-1 or Munc18-3, controls compound and single-vesicle-regulated exocytosis in mast cells.

Mast cells (MCs) play pivotal roles in many inflammatory conditions including infections, anaphylaxis, and asthma. MCs store immunoregulatory compounds in their large cytoplasmic granules and, upon stimulation, secrete them via regulated exocytosis. Exocytosis in many cells requires the participation of Munc18 proteins (also known as syntaxin-binding proteins), and we found that mature MCs express all three mammalian isoforms: Munc18-1, -2, and -3. To study their functions in MC effector responses and test the role of MC degranulation in anaphylaxis, we used conditional knockout (cKO) mice in which each Munc18 protein was deleted exclusively in MCs. Using recordings of plasma membrane capacitance for high-resolution analysis of exocytosis in individual MCs, we observed an almost complete absence of exocytosis in Munc18-2-deficient MCs but intact exocytosis in MCs lacking Munc18-1 or Munc18-3. Stereological analysis of EM images of stimulated MCs revealed that the deletion of Munc18-2 also abolishes the homotypic membrane fusion required for compound exocytosis. We confirmed the severe defect in regulated exocytosis in the absence of Munc18-2 by measuring the secretion of mediators stored in MC granules. Munc18-2 cKO mice had normal morphology, development, and distribution of their MCs, indicating that Munc18-2 is not essential for the migration, retention, and maturation of MC-committed progenitors. Despite that, we found that Munc18-2 cKO mice were significantly protected from anaphylaxis. In conclusion, MC-regulated exocytosis is required for the anaphylactic response, and Munc18-2 is the sole Munc18 isoform that mediates membrane fusion during MC degranulation.

Munc13 proteins control regulated exocytosis in mast cells.

Mast cells (MCs) are involved in host defenses against pathogens and inflammation. Stimulated MCs release substances stored in their granules via regulated exocytosis. In other cell types, Munc13 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 13) proteins play essential roles in regulated exocytosis. Here, we found that MCs express Munc13-2 and -4, and we studied their roles using global and conditional knock-out (KO) mice. In a model of systemic anaphylaxis, we found no difference between WT and Munc13-2 KO mice, but global and MC-specific Munc13-4 KO mice developed less hypothermia. This protection correlated with lower plasma histamine levels and with histological evidence of defective MC degranulation but not with changes in MC development, distribution, numbers, or morphology. In vitro assays revealed that the defective response in Munc13-4-deficient MCs was limited to regulated exocytosis, leaving other MC secretory effector responses intact. Single cell capacitance measurements in MCs from mouse mutants differing in Munc13-4 expression levels in their MCs revealed that as levels of Munc13-4 decrease, the rate of exocytosis declines first, and then the total amount of exocytosis decreases. A requirement for Munc13-2 in MC exocytosis was revealed only in the absence of Munc13-4. Electrophysiology and EM studies uncovered that the number of multigranular compound events (i.e. granule-to-granule homotypic fusion) was severely reduced in the absence of Munc13-4. We conclude that although Munc13-2 plays a minor role, Munc13-4 is essential for regulated exocytosis in MCs, and that this MC effector response is required for a full anaphylactic response.

Soluble expression of an amebic cysteine protease in the cytoplasm of Escherichia coli SHuffle Express cells and purification of active enzyme.

BACKGROUND: Recombinant production of amebic cysteine proteases using Escherichia coli cells as the bacterial system has become a challenging effort, with protein insolubility being the most common issue. Since many of these enzymes need a native conformation stabilized by disulfide bonds, an elaborate process of oxidative folding is usually demanded to get a functional protein. The cytoplasm of E. coli SHuffle Express cells owns an enhanced ability to properly fold proteins with disulfide bonds. Because of this cellular feature, it was possible to assume that this strain represents a reliable expression system and worthwhile been considered as an efficient bacterial host for the recombinant production of amebic cysteine proteases. RESULTS: Using E. coli SHuffle Express cells as the bacterial system, we efficiently produce soluble recombinant EhCP1protein. Enzymatic and inhibition analyses revealed that it exhibits proper catalytic abilities, proceeds effectively over the substrate (following an apparent Michaelis-Menten kinetics), and displays a typical inhibition profile. CONCLUSIONS: We report the first feasibility study of the recombinant production of amebic cysteine proteases using E. coli SHuffle Express as the bacterial host. We present a simple protocol for the recombinant expression and purification of fully soluble and active EhCP1 enzyme. We confirm the suitability of recombinant EhCP1 as a therapeutic target. We propose an approachable bacterial system for the recombinant production of amebic proteins, particularly for those with a need for proper oxidative folding.

Platelet Munc13-4 regulates hemostasis, thrombosis and airway inflammation.

Platelet degranulation is crucial for hemostasis and may participate in inflammation. Exocytosis in platelets is mediated by SNARE proteins and should be controlled by Munc13 proteins. We found that platelets express Munc13-2 and -4. We assessed platelet granule exocytosis in Munc13-2 and -4 global and conditional knockout (KO) mice, and observed that deletion of Munc13-4 ablates dense granule release and indirectly impairs alpha granule exocytosis. We found no exocytic role for Munc13-2 in platelets, not even in the absence of Munc13-4. In vitro, Munc13-4-deficient platelets exhibited defective aggregation at low doses of collagen. In a flow chamber assay, we observed that Munc13-4 acted as a rate-limiting factor in the formation of thrombi. In vivo, we observed a dose-dependency between Munc13-4 expression in platelets and both venous bleeding time and time to arterial thrombosis. Finally, in a model of allergic airway inflammation, we found that platelet-specific Munc13-4 KO mice had a reduction in airway hyper-responsiveness and eosinophilic inflammation. Taken together, our results indicate that Munc13-4-dependent platelet dense granule release plays essential roles in hemostasis, thrombosis and allergic inflammation.

Mistrust in Medicine: The Rise and Fall of America's First Vaccine Institute.

In 1813, the American government passed An Act to Encourage Vaccination, the first federal endorsement of a medical practice in American history. The law tasked a federal agent with maintaining a supply of the smallpox vaccine and distributing it nationwide. James Smith, a well-respected physician and proponent of vaccination, was appointed as vaccine agent. Smith was skeptical of claims that only well-trained physicians should be allowed to perform vaccination; he felt it was a simple procedure that should be available to all American citizens. In 1822, he made a tragic error that caused several deaths and left him vulnerable to criticism from political opponents and his medical peers. This ended Smith's professional career and led to the repeal of the act itself. In this article, we use the rise and fall of James Smith to provide a historical perspective on contemporary debates surrounding delayed vaccination schedules. We explain how physicians-in the 19th century and today-have worked to build public trust in vaccination in an American culture suspicious of medical expertise.

Prevalence of Burnout Among Physicians: A Systematic Review.

Importance: Burnout is a self-reported job-related syndrome increasingly recognized as a critical factor affecting physicians and their patients. An accurate estimate of burnout prevalence among physicians would have important health policy implications, but the overall prevalence is unknown. Objective: To characterize the methods used to assess burnout and provide an estimate of the prevalence of physician burnout. Data Sources and Study Selection: Systematic search of EMBASE, ERIC, MEDLINE/PubMed, psycARTICLES, and psycINFO for studies on the prevalence of burnout in practicing physicians (ie, excluding physicians in training) published before June 1, 2018. Data Extraction and Synthesis: Burnout prevalence and study characteristics were extracted independently by 3 investigators. Although meta-analytic pooling was planned, variation in study designs and burnout ascertainment methods, as well as statistical heterogeneity, made quantitative pooling inappropriate. Therefore, studies were summarized descriptively and assessed qualitatively. Main Outcomes and Measures: Point or period prevalence of burnout assessed by questionnaire. Results: Burnout prevalence data were extracted from 182 studies involving 109 628 individuals in 45 countries published between 1991 and 2018. In all, 85.7% (156/182) of studies used a version of the Maslach Burnout Inventory (MBI) to assess burnout. Studies variably reported prevalence estimates of overall burnout or burnout subcomponents: 67.0% (122/182) on overall burnout, 72.0% (131/182) on emotional exhaustion, 68.1% (124/182) on depersonalization, and 63.2% (115/182) on low personal accomplishment. Studies used at least 142 unique definitions for meeting overall burnout or burnout subscale criteria, indicating substantial disagreement in the literature on what constituted burnout. Studies variably defined burnout based on predefined cutoff scores or sample quantiles and used markedly different cutoff definitions. Among studies using instruments based on the MBI, there were at least 47 distinct definitions of overall burnout prevalence and 29, 26, and 26 definitions of emotional exhaustion, depersonalization, and low personal accomplishment prevalence, respectively. Overall burnout prevalence ranged from 0% to 80.5%. Emotional exhaustion, depersonalization, and low personal accomplishment prevalence ranged from 0% to 86.2%, 0% to 89.9%, and 0% to 87.1%, respectively. Because of inconsistencies in definitions of and assessment methods for burnout across studies, associations between burnout and sex, age, geography, time, specialty, and depressive symptoms could not be reliably determined. Conclusions and Relevance: In this systematic review, there was substantial variability in prevalence estimates of burnout among practicing physicians and marked variation in burnout definitions, assessment methods, and study quality. These findings preclude definitive conclusions about the prevalence of burnout and highlight the importance of developing a consensus definition of burnout and of standardizing measurement tools to assess the effects of chronic occupational stress on physicians.

Prevalence of Depression, Depressive Symptoms, and Suicidal Ideation Among Medical Students: A Systematic Review and Meta-Analysis.

Importance: Medical students are at high risk for depression and suicidal ideation. However, the prevalence estimates of these disorders vary between studies. Objective: To estimate the prevalence of depression, depressive symptoms, and suicidal ideation in medical students. Data Sources and Study Selection: Systematic search of EMBASE, ERIC, MEDLINE, psycARTICLES, and psycINFO without language restriction for studies on the prevalence of depression, depressive symptoms, or suicidal ideation in medical students published before September 17, 2016. Studies that were published in the peer-reviewed literature and used validated assessment methods were included. Data Extraction and Synthesis: Information on study characteristics; prevalence of depression or depressive symptoms and suicidal ideation; and whether students who screened positive for depression sought treatment was extracted independently by 3 investigators. Estimates were pooled using random-effects meta-analysis. Differences by study-level characteristics were estimated using stratified meta-analysis and meta-regression. Main Outcomes and Measures: Point or period prevalence of depression, depressive symptoms, or suicidal ideation as assessed by validated questionnaire or structured interview. Results: Depression or depressive symptom prevalence data were extracted from 167 cross-sectional studies (n = 116 628) and 16 longitudinal studies (n = 5728) from 43 countries. All but 1 study used self-report instruments. The overall pooled crude prevalence of depression or depressive symptoms was 27.2% (37 933/122 356 individuals; 95% CI, 24.7% to 29.9%, I2 = 98.9%). Summary prevalence estimates ranged across assessment modalities from 9.3% to 55.9%. Depressive symptom prevalence remained relatively constant over the period studied (baseline survey year range of 1982-2015; slope, 0.2% increase per year [95% CI, -0.2% to 0.7%]). In the 9 longitudinal studies that assessed depressive symptoms before and during medical school (n = 2432), the median absolute increase in symptoms was 13.5% (range, 0.6% to 35.3%). Prevalence estimates did not significantly differ between studies of only preclinical students and studies of only clinical students (23.7% [95% CI, 19.5% to 28.5%] vs 22.4% [95% CI, 17.6% to 28.2%]; P = .72). The percentage of medical students screening positive for depression who sought psychiatric treatment was 15.7% (110/954 individuals; 95% CI, 10.2% to 23.4%, I2 = 70.1%). Suicidal ideation prevalence data were extracted from 24 cross-sectional studies (n = 21 002) from 15 countries. All but 1 study used self-report instruments. The overall pooled crude prevalence of suicidal ideation was 11.1% (2043/21 002 individuals; 95% CI, 9.0% to 13.7%, I2 = 95.8%). Summary prevalence estimates ranged across assessment modalities from 7.4% to 24.2%. Conclusions and Relevance: In this systematic review, the summary estimate of the prevalence of depression or depressive symptoms among medical students was 27.2% and that of suicidal ideation was 11.1%. Further research is needed to identify strategies for preventing and treating these disorders in this population.

Prevalence of Depression and Depressive Symptoms Among Resident Physicians: A Systematic Review and Meta-analysis.

IMPORTANCE: Physicians in training are at high risk for depression. However, the estimated prevalence of this disorder varies substantially between studies. OBJECTIVE: To provide a summary estimate of depression or depressive symptom prevalence among resident physicians. DATA SOURCES AND STUDY SELECTION: Systematic search of EMBASE, ERIC, MEDLINE, and PsycINFO for studies with information on the prevalence of depression or depressive symptoms among resident physicians published between January 1963 and September 2015. Studies were eligible for inclusion if they were published in the peer-reviewed literature and used a validated method to assess for depression or depressive symptoms. DATA EXTRACTION AND SYNTHESIS: Information on study characteristics and depression or depressive symptom prevalence was extracted independently by 2 trained investigators. Estimates were pooled using random-effects meta-analysis. Differences by study-level characteristics were estimated using meta-regression. MAIN OUTCOMES AND MEASURES: Point or period prevalence of depression or depressive symptoms as assessed by structured interview or validated questionnaire. RESULTS: Data were extracted from 31 cross-sectional studies (9447 individuals) and 23 longitudinal studies (8113 individuals). Three studies used clinical interviews and 51 used self-report instruments. The overall pooled prevalence of depression or depressive symptoms was 28.8% (4969/17,560 individuals, 95% CI, 25.3%-32.5%), with high between-study heterogeneity (Q = 1247, τ2 = 0.39, I2 = 95.8%, P < .001). Prevalence estimates ranged from 20.9% for the 9-item Patient Health Questionnaire with a cutoff of 10 or more (741/3577 individuals, 95% CI, 17.5%-24.7%, Q = 14.4, τ2 = 0.04, I2 = 79.2%) to 43.2% for the 2-item PRIME-MD (1349/2891 individuals, 95% CI, 37.6%-49.0%, Q = 45.6, τ2 = 0.09, I2 = 84.6%). There was an increased prevalence with increasing calendar year (slope = 0.5% increase per year, adjusted for assessment modality; 95% CI, 0.03%-0.9%, P = .04). In a secondary analysis of 7 longitudinal studies, the median absolute increase in depressive symptoms with the onset of residency training was 15.8% (range, 0.3%-26.3%; relative risk, 4.5). No statistically significant differences were observed between cross-sectional vs longitudinal studies, studies of only interns vs only upper-level residents, or studies of nonsurgical vs both nonsurgical and surgical residents. CONCLUSIONS AND RELEVANCE: In this systematic review, the summary estimate of the prevalence of depression or depressive symptoms among resident physicians was 28.8%, ranging from 20.9% to 43.2% depending on the instrument used, and increased with calendar year. Further research is needed to identify effective strategies for preventing and treating depression among physicians in training.

Rheumatoid arthritis associated vasculitis: a rare entity; case and review.

We present a case of a 60-year-old male with known seropositive rheumatoid arthritis and cerebral vasculitis who presented to the emergency room with abrupt onset lower back and abdominal pain. The patient developed peritonitis which led to an abdominal laparotomy where jejunal ischemia, necrosis, and perforation were found, requiring bowel resection. On pathology examination, the patient had mesenteric vessel intramural inflammation indicative of vasculitis. He developed an anastomotic leak on postoperative Day 4 and elected hospice care. A high index of suspicion for mesenteric vasculitis should be considered in patients presenting with abdominal pain in the setting of known rheumatoid arthritis associated vasculitis, especially patients with long-standing rheumatoid arthritis. The high mortality represented by gastrointestinal involvement in rheumatoid arthritis associated vasculitis warrants investigation in high-risk patients, despite its low prevalence. Treatment may consist of high-dose corticosteroids, immunosuppressive agents, biologic therapies that target the underlying autoimmune process, and in severe cases, bowel resection.

Selective Transfection of a Transferrin Receptor-Expressing Cell Line with DNA-Lipid Nanoparticles.

Despite considerable progress in using lipid nanoparticle (LNP) vehicles for gene delivery, achieving selective transfection of specific cell types remains a significant challenge, hindering the advancement of new gene or gene-editing therapies. Although LNPs have been equipped with ligands aimed at targeting specific cellular receptors, achieving complete selectivity continues to be elusive. The exact reasons for this limited selectivity are not fully understood, as cell targeting involves a complex interplay of various cellular factors. Assessing how much ligand/receptor binding contributes to selectivity is challenging due to these additional influencing factors. Nonetheless, such data are important for developing new nanocarriers and setting realistic expectations for selectivity. Here, we have quantified the selective, targeted transfection using two uniquely engineered cell lines that eliminate unpredictable and interfering cellular influences. We have compared the targeted transfection of Chinese ovary hamster (CHO) cells engineered to express the human transferrin receptor 1 (hTfR1), CHO-TRVb-hTfR1, with CHO cells that completely lack any transferrin receptor, CHO-TRVb-neo cells (negative control). Thus, the two cell lines differ only in the presence/absence of hTfR1. The transfection was performed with pDNA-encapsulating LNPs equipped with the DT7 peptide ligand that specifically binds to hTfR1 and enables targeted transfection. The LNP's pDNA encoded for the monomeric GreenLantern (mGL) reporter protein, whose fluorescence was used to quantify transfection. We report a novel LNP composition designed to achieve an optimal particle size and ζ-potential, efficient pDNA encapsulation, hTfR1-targeting capability, and sufficient polyethylene glycol sheltering to minimize random cell targeting. The transfection efficiency was quantified in both cell lines separately through flow cytometry based on the expression of the fluorescent gene product. Our results demonstrated an LNP dose-dependent mGL expression, with a 5-fold preference for the CHO-TRVb-hTfR1 when compared to CHO-TRVb-neo. In another experiment, when both cell lines were mixed at a 1:1 ratio, the DT7-decorated LNP achieved a 3-fold higher transfection of the CHO-TRVb-hTfR1 over the CHO-TRVb-neo cells. Based on the low-level transfection of the CHO-TRVb-neo cells in both experiments, our results suggest that 17-25% of the transfection occurred in a nonspecific manner. The observed transfection selectivity for the CHO-TRVb-hTfR1 cells was based entirely on the hTfR1/DT7 interaction. This work showed that the platform of two engineered cell lines which differ only in the hTfR1 can greatly facilitate the development of LNPs with hTfR1-targeting ligands.

Drugs in context: a historical perspective on theories of psychopharmaceutical efficacy.

This article demonstrates that psychoanalysis and socially oriented psychiatry were crucial to the understanding and adoption of the first effective psychopharmaceuticals in North American psychiatry. In the 1950s and the early 1960s, psychoanalysts, socially oriented psychiatrists, and biologists collaborated, debated, and organized interdisciplinary conferences to situate the biochemistry of new psychopharmaceuticals, such as chlorpromazine, in the broader psychosocial context of patients' lives. Psychoanalytical and sociological perspectives not only helped American psychiatrists explain the mechanism of drug action in research but also established the professional authority of psychiatrists over the new pharmaceuticals. As modern pharmacology narrows its focus to microscopic targets in the body, I argue that this early drug research illustrates the present-day need for holistic and interdisciplinary approaches to drug response that acknowledge the psychosocial significance of psychiatric medication in the lives of individuals.

Hydrophobic weak acid polymers as controlled release carriers.

Poly(carboxyalkyl methacrylates) were studied as a cationic-drug delivery system, at pH 6.8 and 8.0. Different polymer/drug complexes were used to prepare compressed tablets. By kinetics experiments, we have found that drug release is dependent on both the hydrophobicity of the whole complex and the pH of the environment. Furthermore, a mechanism of dissociation/erosion clearly describes the drug release from a complex formed by a polymer soluble at target pH; otherwise, a mechanism of dissolution/diffusion is depicted. Additionally, we have observed that hydrophilic fillers increase the drug release rate. Since our results using different polymer/drug complexes exhibit pH-sensitive drug release, we propose that the poly(carboxyalkyl methacrylates) have potential as a colon-specific drug-delivery system.

Entamoeba histolytica: biochemical characterization of a protein disulfide isomerase.

Protein disulfide isomerase (PDI) enzymes are eukaryotic oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. Here, we report the biochemical characterization of a PDI enzyme from the protozoan parasite Entamoeba histolytica (EhPDI). Our results show that EhPDI behaves mainly as an oxidase/isomerase and can be inhibited by bacitracin, a known PDI inhibitor; moreover, it exhibits chaperone-like activity. Albeit its physiological role in the life style of the parasite (including virulence and survival) remains to be studied, EhPDI could represent a potential drug target for anti-amebic therapy.

Acid-denatured Green Fluorescent Protein (GFP) as model substrate to study the chaperone activity of protein disulfide isomerase.

Green fluorescent protein (GFP) has been widely used in several molecular and cellular biology applications, since it is remarkably stable in vitro and in vivo. Interestingly, native GFP is resistant to the most common chemical denaturants; however, a low fluorescence signal has been observed after acid-induced denaturation. Furthermore, this acid-denatured GFP has been used as substrate in studies of the folding activity of some bacterial chaperones and other chaperone-like molecules. Protein disulfide isomerase enzymes, a family of eukaryotic oxidoreductases that catalyze the oxidation and isomerization of disulfide bonds in nascent polypeptides, play a key role in protein folding and it could display chaperone activity. However, contrasting results have been reported using different proteins as model substrates. Here, we report the further application of GFP as a model substrate to study the chaperone activity of protein disulfide isomerase (PDI) enzymes. Since refolding of acid-denatured GFP can be easily and directly monitored, a simple micro-assay was used to study the effect of the molecular participants in protein refolding assisted by PDI. Additionally, the effect of a well-known inhibitor of PDI chaperone activity was also analyzed. Because of the diversity their functional activities, PDI enzymes are potentially interesting drug targets. Since PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of cancer chemotherapy; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduces the infectivity of the human immunodeficiency virus. Although several high-throughput screening (HTS) assays to test PDI reductase activity have been described, we report here a novel and simple micro-assay to test the chaperone activity of PDI enzymes, which is amenable for HTS of PDI inhibitors.