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Refractory materials resistant to aluminum corrosion were developed by the addition of SrSORefractory materials resistant to aluminum corrosion were developed by the addition of SrSO4. Substrates of Mullite-20 %wt. SrSO4 and SiO2-20 %wt. SrSO4, were sintered at 1400 ºC for 6 h. Corrosion test by liquid aluminum were performed from 700 to 900 ºC for several periods of time (2-10 h). The samples were analyzed by SEM (Scanning electron microscopy) and XRD (X ray diffraction). Results of XRD showed the formation of SrAl2Si2O8 for the sample Mullite-SrSO4 and the formation of SrSiO3, for the sample SiO2-SrSO4 after sintering. Mullite samples without Sr addition showed a corrosion layer of about 2 mm for a corrosion test for 10 h at 900 ºC. Samples of mullite with Sr addition showed a corrosion layer of about 100 mm under the same conditions. The addition of Sr to the SiO2 was not effective to improve corrosion resistant.Se obtuvieron materiales refractarios a base de mullita resistentes a la corrosión por aluminio líquido mediante la adición de sulfato de estroncio. Se sinterizaron sustratos de mullita-20 %e.p. SrSO4 y sílice- 20%e.p. SrSO4 a 1400 ºC por 6 h. Se realizaron pruebas de corrosión con aluminio puro de 750 a 900 ºC por periodos de 2 a 10 h. Los sustratos se analizaron por Microscopía Electrónica de Barrido (MEB) y Difracción de Rayos X (DRX). Patrones de DRX mostraron la presencia de mullita y SrAl2Si2O8 en la mezcla de mullita-SrSO4. En la mezcla de sílice-SrSO4, se detectó la presencia de sílice y SrSiO3. Para las muestras de mullita sin adición de SrSO4 se encontró una capa de corrosión compuesta de alúmina con un espesor de 2 mm para una temperatura de 900 ºC por 10 h. Las muestras que contienen estroncio presentaron una capa de corrosión de aproximadamente 100 μm bajo las mismas condiciones. Por otro lado, los análisis de MEB mostraron que la adición de sulfato de estroncio a la sílice no incrementó la resistencia a la corrosión por aluminio líquido.

In Argentina, hemolytic uremic syndrome (HUS) caused by EHEC has the highest incidence in the world. EHEC infection has an endemo-epidemic behavior, causing 20-30% of acute bloody diarrhea syndrome in children under 5 years old. In the period 2016-2020, 272 new cases per year were notified to the National Health Surveillance System. Multiple factors are responsible for HUS incidence in Argentina including person-to-person transmission. In order to detect possible EHEC carriers, we carried out a preliminary study of the frequency of kindergarten teachers with anti-LPS antibodies against the most prevalent EHEC serotypes in Argentina. We analyzed 61 kindergarten teachers from 26 institutions from José C. Paz district, located in the suburban area of Buenos Aires province, Argentina. Fifty-one percent of the plasma samples had antibodies against O157, O145, O121 and O103 LPS: 6.4% of the positive samples had IgM isotype (n=2), 61.3% IgG isotype (n=19) and 32.3% IgM and IgG (n=10). Given that antibodies against LPS antigens are usually short-lived specific IgM detection may indicate a recent infection. In addition, the high percentage of positive samples may indicate a frequent exposure to EHEC strains in the cohort studied, as well as the existence of a large non-symptomatic population of adults carrying pathogenic strains that could contribute to the endemic behavior through person-to-person transmission. The improvement of continuous educational programs in kindergarten institutions could be a mandatory measure to reduce HUS cases not only in Argentina but also globally., (Copyright © 2023 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

The malevolent application of neuroscience is an emerging threat to the U.S. military. At present, U.S. military medical personnel are not capable of adequately diagnosing or treating the injuries and illnesses that may result from exposure to potential neuroweapons. This fact was illustrated in 2016 when U.S. diplomats serving in Havana, Cuba reported hearing strange noises accompanied by a constellation of unexplained health effects. Similar incidents have been reported in China and Russia. Although various hypotheses have been put forward to explain these symptoms, none of them have been verified. The reported symptoms were analogous to the physiological responses that have been produced in the laboratory by exposing volunteers to pulsed microwave energy. However, these incidents of undetermined origin demonstrate that widespread neurological illness can be disruptive to U.S. government operations and that it is currently not possible to identify the cause, determine the correct treatment, or ascribe attribution to potential neuroweapon use in an overseas setting. Since it is likely that Special Operations medical personnel will be among the first to respond to neuroweapon attacks in the deployed environment, it is essential that they be made aware of this emerging threat and that efforts be made to incorporate potential directed energy neuroweapons and other neuroweapon configurations into future Chemical, Biological, Radiological, Nuclear, and high yield Explosives (CBRN-E) training modules. The intention of this article is to introduce the concept of the neuroweapon to military medical personnel and to provide a brief review of the relevant literature., (2023.)

SARS-CoV-2 is the virus responsible for the disease that is known as COVID-19. While there have been numerous studies detailing the survival rates of SARS-CoV-2 on various materials, there are currently no published data regarding whether this virus is stable on standard military uniforms. Consequently, there are no standard operating procedures for washing uniforms once exposed to the virus. This study aimed to determine whether SARS-CoV-2 could be removed from Army combat uniform material by washing with a commercially available detergent and tap water. Washing the fabric with detergent followed by a rinse step with tap water effectively removes detectable viral particles. Importantly, it was found that washing with hot water alone was not effective. Therefore, it is recommended that military personnel wash their uniforms with detergent and water as soon as possible after exposure to SARS-CoV-2; hot water should not be used as a substitute for detergent., (2023.)

Calcium ion movements between cellular stores and the cytosol govern muscle contraction, the most energy-consuming function in mammals, which confers skeletal myofibers a pivotal role in glycemia regulation. Chronic myoplasmic calcium elevation ("calcium stress"), found in malignant hyperthermia-susceptible (MHS) patients and multiple myopathies, has been suggested to underlie the progression from hyperglycemia to insulin resistance. What drives such progression remains elusive. We find that muscle cells derived from MHS patients have increased content of an activated fragment of GSK3β - a specialized kinase that inhibits glycogen synthase, impairing glucose utilization and delineating a path to hyperglycemia. We also find decreased content of junctophilin1, an essential structural protein that colocalizes in the couplon with the voltage-sensing Ca V 1.1, the calcium channel RyR1 and calpain1, accompanied by an increase in a 44 kDa junctophilin1 fragment (JPh44) that moves into nuclei. We trace these changes to activated proteolysis by calpain1, secondary to increased myoplasmic calcium. We demonstrate that a JPh44-like construct induces transcriptional changes predictive of increased glucose utilization in myoblasts, including less transcription and translation of GSK3β and decreased transcription of proteins that reduce utilization of glucose. These effects reveal a stress-adaptive response, mediated by the novel regulator of transcription JPh44., Competing Interests: ET, LF, CM, DV, NK, CI, AK, SR, ER No competing interests declared, (© 2023, Tammineni et al.)

Introduction: Female participation in the field of medicine and research has increased in recent years; however, there are still inequities in the proportion of men and women in medical leadership, especially in management positions and editorial committees of scientific journals., Objective: To identify female participation in the editorial committees of medical journals in Latin America and explore the association with editorial positions and impact indicators., Materials and Methods: We conducted a descriptive bibliometric study to determine female participation in the editorial committees of medical & journals in Latin America. We included 113 medical journals published in Latin America and indexed in Scopus, updated and current in 2020, selected from the Scimago Journal Country Rank portal. The gender of editorial committee members was identified on the web pages of each magazine., Results: Regarding editorial leadership in the 113 journals included, women represented 12.9% of 264 members; as for the functions within the editorial committee, of 1,449 members, 28.9% were women while in advisory committees, of 4,575 members 19.0% were women. The presence of women in editorial committees was higher in journals from Chile, Brazil, and Venezuela in specialties such as public health, pediatrics, and anesthesiology., Conclusions: Female participation in the editorial committees of medical journals in Latin America is low.

Background: Previous studies have assessed the prevalence and characteristics of self-medication in COVID-19. However, no systematic review has summarized their findings., Objective: We conducted a systematic review to assess the prevalence of self-medication to prevent or manage COVID-19., Methods: We used different keywords and searched studies published in PubMed, Scopus, Web of Science, Embase, two preprint repositories, Google, and Google Scholar. We included studies that reported original data and assessed self-medication to prevent or manage COVID-19. The risk of bias was assessed using the Newcastle-Ottawa Scale (NOS) modified for cross-sectional studies., Results: We identified eight studies, all studies were cross-sectional, and only one detailed the question used to assess self-medication. The recall period was heterogeneous across studies. Of the eight studies, seven assessed self-medication without focusing on a specific symptom: four performed in the general population (self-medication prevalence ranged between <4% to 88.3%) and three in specific populations (range: 33.9% to 51.3%). In these seven studies, the most used medications varied widely, including antibiotics, chloroquine or hydroxychloroquine, acetaminophen, vitamins or supplements, ivermectin, and ibuprofen. The last study only assessed self-medication for fever due to COVID-19. Most studies had a risk of bias in the "representativeness of the sample" and "assessment of outcome" items of the NOS., Conclusions: Studies that assessed self-medication for COVID-19 found heterogeneous results regarding self-medication prevalence and medications used. More well-designed and adequately reported studies are warranted to assess this topic., Competing Interests: The authors have declared that no competing interests exist.

Most glucose is processed in muscle, for energy or glycogen stores. Malignant Hyperthermia Susceptibility (MHS) exemplifies muscle conditions that increase [Ca 2+ ] cytosol . 42% of MHS patients have hyperglycemia. We show that phosphorylated glycogen phosphorylase (GP a ), glycogen synthase (GS a ) - respectively activated and inactivated by phosphorylation - and their Ca 2+ -dependent kinase (PhK), are elevated in microsomal extracts from MHS patients' muscle. Glycogen and glucose transporter GLUT4 are decreased. [Ca 2+ ] cytosol , increased to MHS levels, promoted GP phosphorylation. Imaging at ~100 nm resolution located GP a at sarcoplasmic reticulum (SR) junctional cisternae, and apo -GP at Z disk. MHS muscle therefore has a wide-ranging alteration in glucose metabolism: high [Ca 2+ ] cytosol activates PhK, which inhibits GS, activates GP and moves it toward the SR, favoring glycogenolysis. The alterations probably cause these patients' hyperglycemia. For basic studies, MHS emerges as a variable stressor, which forces glucose pathways from the normal to the diseased range, thereby exposing novel metabolic links., Competing Interests: ET, NK, LF, CM, CI, AK, SR, ER No competing interests declared, (© 2020, Tammineni et al.)

The increment of eryptosis in lead-exposed workers has been associated with oxidative stress, having as the main mediator [Ca 2+ ] i . However, other molecules could participate as signals, such as PLA 2 and SMase, which have been proposed to increase PGE 2 and ceramides, both involved in the increment of PS externalization due to osmotic stress. To study the role of these enzymes in lead intoxication, we studied 30 lead exposed workers and 27 non-lead exposed individuals. We found, compared to non-exposed subjects, lead intoxication characterized by high blood lead concentration (median = 39.1 μg/dL), and low δ-ALAD activity (median = 348 nmol of porphobilinogen/h/mL); oxidative stress with high lipid peroxidation (median = 1.31 nmol of malondialdehyde/mL) and low TAC (median = 370 mM Trolox equivalents); a higher enzymatic activity of PLA 2 (median = 518 AFU/mg) and SMase (median = 706 AFU/mg) and higher eryptosis (median = 0.92% PS externalization). Correlation and conditional probability analyses permit to associate oxidative stress and eryptosis with high PLA 2 activity. However, high SMase activity was only associated with PLA 2 activity. The role of these enzymes in the signal path to eryptosis induced by oxidative stress in lead-exposed workers is discussed., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Shiga toxin (Stx), produced by Escherichia coli , is the main pathogenic factor of diarrhea-associated hemolytic uremic syndrome (HUS), which is characterized by the obstruction of renal microvasculature by platelet-fibrin thrombi. It is well known that the oxidative imbalance generated by Stx induces platelet activation, contributing to thrombus formation. Moreover, activated platelets release soluble CD40 ligand (sCD40L), which in turn contributes to oxidative imbalance, triggering the release of reactive oxidative species (ROS) on various cellular types. The aim of this work was to determine if the interaction between the oxidative response and platelet-derived sCD40L, as consequence of Stx-induced endothelium damage, participates in the pathogenic mechanism during HUS. Activated human glomerular endothelial cells (HGEC) by Stx2 induced platelets to adhere to them. Although platelet adhesion did not contribute to endothelial damage, high levels of sCD40L were released to the medium. The release of sCD40L by activated platelets was inhibited by antioxidant treatment. Furthermore, we found increased levels of sCD40L in plasma from HUS patients, which were also able to trigger the respiratory burst in monocytes in a sCD40L-dependent manner. Thus, we concluded that platelet-derived sCD40L and the oxidative response are reciprocally stimulated during Stx2-associated HUS. This process may contribute to the evolution of glomerular occlusion and the microangiopathic lesions., Competing Interests: The authors declare that they have no conflict of interest.

Hemolytic uremic syndrome (HUS) is one of the most common causes of acute renal failure in children. The majority of cases are associated with Shiga toxin (Stx)-producing Escherichia coli (STEC). In Argentina, HUS is endemic and presents the highest incidence rate in the world. STEC strains expressing Stx type 2 (Stx2) are responsible for the most severe cases of this pathology. Subtilase cytotoxin (SubAB) is another STEC virulence factor that may contribute to HUS pathogenesis. To date, neither a licensed vaccine nor effective therapy for HUS is available for humans. Considering that Ouabain (OUA) may prevent the apoptosis process, in this study we evaluated if OUA is able to avoid the damage caused by Stx2 and SubAB on human glomerular endothelial cells (HGEC) and the human proximal tubule epithelial cell (HK-2) line. HGEC and HK-2 were pretreated with OUA and then incubated with the toxins. OUA protected the HGEC viability from Stx2 and SubAB cytotoxic effects, and also prevented the HK-2 viability from Stx2 effects. The protective action of OUA on HGEC and HK-2 was associated with a decrease in apoptosis and an increase in cell proliferation. Our data provide evidence that OUA could be considered as a therapeutic strategy to avoid the renal damage that precedes HUS., Competing Interests: The authors declare no conflict of interest.

Background: We describe the analytical validation of an assay panel intended to assist clinicians with the diagnosis of systemic lupus erythematosus (SLE). The multi-analyte panel includes quantitative assessment of complement activation and measurement of autoantibodies., Methods: The levels of the complement split product C4d bound to erythrocytes (EC4d) and B-lymphocytes (BC4d) (expressed as mean fluorescence intensity [MFI]) are measured by quantitative flow cytometry, while autoantibodies (inclusive of antinuclear and anti-double stranded DNA antibodies) are determined by immunoassays. Results of the multi-analyte panel are reported as positive or negative based on a 2-tiered index score. Post-phlebotomy stability of EC4d and BC4d in EDTA-anticoagulated blood is determined using specimens collected from patients with SLE and normal donors. Three-level C4 coated positive beads are run daily as controls. Analytical validity is reported using intra-day and inter-day coefficient of variation (CV)., Results: EC4d and BC4d are stable for 2days at ambient temperature and for 4days at 4°C post-phlebotomy. Median intra-day and inter-day CV range from 2.9% to 7.8% (n=30) and 7.3% to 12.4% (n=66), respectively. The 2-tiered index score is reproducible over 4 consecutive daysupon storage of blood at 4°C. A total of 2,888 three-level quality control data were collected from 6 flow cytometers with an overall failure rate below 3%. Median EC4d level is 6 net MFI (Interquartile [IQ] range 4-9 net MFI) and median BC4d is 18 net MFI (IQ range 13-27 net MFI) among 86,852 specimens submitted for testing. The incidence of 2-tiered positive test results is 13.4%., Conclusion: We have established the analytical validity of a multi-analyte assay panel for SLE., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Sore throat is common after intubation for surgery. This observational study investigated cuff pressure and a large range of clinical covariates to explore the etiology of sore throat., Methods: Approximately 24 hours after surgery six questions relating to pain, upper airway symptoms and sore throat were delivered to patients who had undergone intubation. Sore throat was correlated with demographics, anesthesia variables and cuff pressure (measured for a subset of patients)., Results: Sore throat was reported by 270/518 (52%) patients with VAS Score 45.9±25.1 (range 0-100). Sore throat patients were significantly younger, had a lower ASA status, were more frequently female, had shorter surgeries and lower nitrous oxide exposure, had a higher proportion of smaller tracheal tubes (7.5 mm internal diameter vs. 8 mm), had a higher incidence of nasogastric drainage, higher propofol doses and a higher usage of ketorolac. Decreasing age (odds ratio 0.976, 95% confidence intervals 0.961-0.992, P=0.003) and the presence of a nasogastric tube when the questionnaire was delivered (OR 1.83, 95% CI: 1.06-3.14, P=0.03) remained significant predictors of sore throat on multivariate analysis. Mean cuff pressure (measured for 160 patients) was 56.8±41.9 mmHg. Cuff pressure was similar amongst patients with and without sore throat (57±46 vs. 53±38 mmHg, P=0.58). There was no correlation between cuff pressure and severity of sore throat (r=0.004, P=0.37)., Conclusions: Only age and the presence of a nasogastric tube after surgery were significant predictors for sore throat. This result contradicts most other studies of cuff pressure where fewer covariates were measured.

The Arabidopsis thaliana central cell, the companion cell of the egg, undergoes DNA demethylation before fertilization, but the targeting preferences, mechanism, and biological significance of this process remain unclear. Here, we show that active DNA demethylation mediated by the DEMETER DNA glycosylase accounts for all of the demethylation in the central cell and preferentially targets small, AT-rich, and nucleosome-depleted euchromatic transposable elements. The vegetative cell, the companion cell of sperm, also undergoes DEMETER-dependent demethylation of similar sequences, and lack of DEMETER in vegetative cells causes reduced small RNA-directed DNA methylation of transposons in sperm. Our results demonstrate that demethylation in companion cells reinforces transposon methylation in plant gametes and likely contributes to stable silencing of transposable elements across generations.

Parent-of-origin-specific (imprinted) gene expression is regulated in Arabidopsis thaliana endosperm by cytosine demethylation of the maternal genome mediated by the DNA glycosylase DEMETER, but the extent of the methylation changes is not known. Here, we show that virtually the entire endosperm genome is demethylated, coupled with extensive local non-CG hypermethylation of small interfering RNA-targeted sequences. Mutation of DEMETER partially restores endosperm CG methylation to levels found in other tissues, indicating that CG demethylation is specific to maternal sequences. Endosperm demethylation is accompanied by CHH hypermethylation of embryo transposable elements. Our findings demonstrate extensive reconfiguration of the endosperm methylation landscape that likely reinforces transposon silencing in the embryo.

A 52-year-old man noted distal dominant slowly progressive muscle weakness at age 36 years. On muscle CT, the red muscles of the soleus, anterior tibial and paraspinal muscles, where type 1 fiber is known to predominate, were almost totally replaced by fat tissue while quadriceps femoris, gastrocnemius and upper extremity muscles were relatively spared. Quadriceps muscle biopsy revealed multi-minicores in addition to occasional larger cores, in about 70% of the type 1 fibers. A novel heterozygous nucleotide change c.5869T > A (p.S1957T) was identified in RYR1. Although pathogenicity was not confirmed, this nucleotide change was absent in 100 control DNA. We did not find a mutation in either multi-minicore disease-associated gene, SEPN1, or major distal myopathy-related genes, including GNE, ZASP, MYOT, exons 32-36 of MYH7, and the last exon of TTN. This is probably a unique form of distal myopathy characterized by the presence of multi-minicores with preferential involvement of type 1 fibers.

Malignant hyperthermia (MH) is an autosomal dominant disorder of skeletal muscle calcium regulation, and the rate of calcium-induced calcium release (CICR), determined by using skinned fibers of skeletal muscle, has been employed as a diagnostic test for MH susceptibility in Japan. The ryanodine receptor (RYR1), encoding the major calcium-release channel in skeletal muscle sarcoplasmic reticulum, has been shown to be mutated in a number of MH pedigrees. We experienced the detection of accelerated CICR and/or an RYR1 mutation in a patient with an MH episode and his family. Accelerated CICR and an RYR1 mutation (c.14512C>G, p.L4838V) were found in the patient and his father. The MH-causative mutation (c.14512C>G, p.L4838V) was also found in his brother and his son (resulting in the diagnosis of MH without the CICR test), but the mutation was not found in his mother or two daughters. With the detection of the family-specific mutation in other family members, the diagnosis of MH was made without the invasive CICR test.

Background: COL6 gene mutations are associated with Ullrich congenital muscular dystrophy (UCMD), which is clinically characterized by muscle weakness from early infancy, hyperlaxity of distal joints, and multiple proximal joint contractures. We previously reported that the majority of patients with UCMD have sarcolemma-specific collagen VI deficiency (SSCD). More recently, we found heterozygous COL6A1 glycine substitutions in patients with UCMD with SSCD., Objective: To elucidate how COL6A1 glycine mutation leads to SSCD., Methods: We evaluated the synthesis, formation, and binding of collagen VI to the extracellular matrix in fibroblasts with p.G284R mutation in COL6A1., Results: Collagen VI was normally secreted into the cultured medium in fibroblasts harboring p.G284R mutation. When the medium with normal collagen VI was added to collagen VI-deficient fibroblast culture, collagen VI bound surrounding the cells, while collagen VI with p.G284R mutation did not. Cell adhesion of fibroblasts with p.G284R mutation was markedly reduced similarly to that of collagen VI-deficient cells. Interestingly, this reduction in adhesion of the cells with p.G284R mutation was recovered by the addition of the medium with normal collagen VI, which would suggest a therapeutic strategy for a replacement therapy., Conclusion: Heterozygous glycine substitution in COL6A1 may cause decreased binding of collagen VI microfibrils to the extracellular matrix resulting in sarcolemma-specific collagen VI deficiency.

Interactions between normal mammary epithelial cells and extracellular matrix (ECM) are important for mammary gland homeostasis. Loss of interactions between ECM and normal mammary epithelial cells are thought to be an early event in mammary carcinogenesis. CREB-binding protein (CBP) is an important regulator of proliferation and apoptosis but the role of CBP in ECM signaling is poorly characterized. CBP was suppressed in basal-cytokeratin-positive HMECs (CK5/6+, CK14+, CK8-, CK18-, CK19-). Suppression of CBP resulted in loss of reconstituted ECM-mediated growth control and apoptosis and loss of laminin-5 alpha3-chain expression. Suppression of CBP in normal human mammary epithelial cells (HMECs) resulted in loss of CBP occupancy of the LAMA3A promoter and decreased LAMA3A promoter activity and laminin-5 alpha-3 chain expression. Exogenous expression of CBP in CBP-negative HMECs that have lost reconstituted ECM-mediated growth regulation and apoptosis resulted in (1) CBP occupancy of the LAMA3A promoter, (2) increased LAMA3A activity and laminin-5 alpha3-chain expression, and (3) enhancement of reconstituted ECM-mediated growth regulation and apoptosis. Similarly, suppression of laminin-5 alpha3-chain expression in HMECs resulted in loss of reconstituted ECM-mediated growth control and apoptosis. These observations suggest that loss of CBP in basal-cytokeratin-positive HMECs results in loss of reconstituted ECM-mediated growth control and apoptosis through loss of LAMA3A activity and laminin-5 alpha3-chain expression. Results in these studies may provide insight into early events in basal-type mammary carcinogenesis.

Potassium channels are widely distributed. To serve their physiological functions, such as neuronal signaling, control of insulin release, and regulation of heart rate and blood flow, it is essential that K+ channels allow K+ but not the smaller and more abundant Na+ ions to go through. The narrowest part of the channel pore, the selectivity filter formed by backbone carbonyls of the GYG-containing K+ channel signature sequence, approximates the hydration shell of K+ ions. However, the K+ channel signature sequence is not sufficient for K+ selectivity. To identify structural elements important for K+ selectivity, we randomly mutagenized the G protein-coupled inwardly rectifying potassium channel 3.2 (GIRK2) bearing the S177W mutation on the second transmembrane segment. This mutation confers constitutive channel activity but abolishes K+ selectivity and hence the channel's ability to complement the K+ transport deficiency of Deltatrk1Deltatrk2 mutant yeast. S177W-containing GIRK2 mutants that support yeast growth in low-K+ medium contain multiple suppressors, each partially restoring K+ selectivity to S177W-containing double mutants. These suppressors include mutations in the first transmembrane segment and the pore helix, likely exerting long-range actions to restore K+ selectivity, as well as a mutation of a second transmembrane segment residue facing the cytoplasmic half of the pore, below the selectivity filter. Some of these suppressors also affected channel gating (channel open time and opening frequency determined in single-channel analyses), revealing intriguing interplay between ion permeation and channel gating.

The binding of plasminogen activator inhibitor-1 (PAI-1) to serine proteinases, such as tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), is mediated by the exosite interactions between the surface-exposed variable region-1, or 37-loop, of the proteinase and the distal reactive center loop (RCL) of PAI-1. Although the contribution of such interactions to the inhibitory activity of PAI-1 has been established, the specific mechanistic steps affected by interactions at the distal RCL remain unknown. We have used protein engineering, stopped-flow fluorimetry, and rapid acid quenching techniques to elucidate the role of exosite interactions in the neutralization of tPA, uPA, and beta-trypsin by PAI-1. Alanine substitutions at the distal P4' (Glu-350) and P5' (Glu-351) residues of PAI-1 reduced the rates of Michaelis complex formation (k(a)) and overall inhibition (k(app)) with tPA by 13.4- and 4.7-fold, respectively, whereas the rate of loop insertion or final acyl-enzyme formation (k(lim)) increased by 3.3-fold. The effects of double mutations on k(a), k(lim), and k(app) were small with uPA and nonexistent with beta-trypsin. We provide the first kinetic evidence that the removal of exosite interactions significantly alters the formation of the noncovalent Michaelis complex, facilitating the release of the primed side of the distal loop from the active-site pocket of tPA and the subsequent insertion of the cleaved reactive center loop into beta-sheet A. Moreover, mutational analysis indicates that the P5' residue contributes more to the mechanism of tPA inhibition, notably by promoting the formation of a final Michaelis complex.

Clostridium perfringens type D produces enterotoxaemia in sheep, goats and other animals. The disease is caused by C. perfringens epsilon toxin, and while enterotoxaemia in goats is usually characterized by enterocolitis, the disease in sheep is characterized by systemic lesions (such as lung and brain oedema) with minor and inconsistent changes observed in the intestine. A possible explanation for these differences is that epsilon toxin is more promptly absorbed by sheep than goat intestine. In an attempt to clarify this, we examined the in vitro effects of epsilon toxin on sheep and goat intestine. Pieces of intestinal mucosa from recently slaughtered animals were mounted in a modified Ussing-type chamber where net water flux (J(w)), short-circuit current (I(sc)) and tissue conductance (G(t)) were simultaneously recorded. After 70 min of incubation with epsilon toxin a reduction in absorptive J(w) and an increase in I(sc) and G(t) were observed in colonic tissues of both sheep and goats, but no alterations were registered in the ileum of either species. These in vitro results show that epsilon toxin affects the transport function of the colonic mucosa but it does not seem to produce any transport alteration in the ileum mucosa.

The pKa of the catalytic Tyr-9 in glutathione S-transferase (GST) A1-1 is lowered from 10.3 to approximately 8.1 in the apoenzyme and approximately 9.0 with a GSH conjugate bound at the active site. However, a clear functional role for the unusual Tyr-9 pKa has not been elucidated. GSTA1-1 also includes a dynamic C terminus that undergoes a ligand-dependent disorder-to-order transition. Previous studies suggest a functional link between Tyr-9 ionization and C-terminal dynamics. Here we directly probe the role of Tyr-9 ionization in ligand binding and C-terminal conformation. An engineered mutant of rGSTA1-1, W21F/F222W, which contains a single Trp at the C terminus, was used as a fluorescent reporter of pH-dependent C-terminal dynamics. This mutant exhibited a pH-dependent change in Trp-222 emission properties consistent with changes in C-terminal solvation or conformation. The apparent pKa values for the conformational transition were 7.9 +/- 0.1 and 9.3 +/- 0.1 for the apoenzyme and ligand-bound enzyme, respectively, in excellent agreement with the pKa for Tyr-9 in these states. The Y9F/W21F/F222W mutant, however, exhibited no such pH-dependent changes. Time-resolved fluorescence anisotropy studies revealed a ligand-dependent, Tyr-9-dependent, change in the order parameter of Trp-222. However, no pH dependence was observed. In equilibrium and pre-steady-state ligand binding studies, product conjugate had a decreased equilibrium binding affinity (KD), concomitant with increased binding and dissociation rates, at higher pH values. Furthermore, the recovered pKa values for the pH-dependent microscopic rate constants ranged from 7.7 to 8.4, also in agreement with the pKa of Tyr-9. In contrast, the Y9F/W21F/F222W mutant had no pH-dependent transition in KD or rate constants for ligand binding or dissociation. The combined results indicate that the macroscopic populations of "open" and "closed" states of the C terminus are not determined solely by the ionization state of Tyr-9. However, the rates of transition between these states are faster for the ionized Tyr-9. The ionized Tyr-9 states provide a parallel pathway for product dissociation, which is kinetically and thermodynamically favored. In silico kinetic models further support the functional role for the parallel dissociation pathway provided by ionized Tyr-9.

A remote flow cell based on a single strand of fused-silica fiber optic was built for UV absorbance detection with a packed capillary HPLC system, using commercially available pumps, detection electronics (Shimadzu) and fittings. This 'off-column' flow cell design is applicable to both pressure and electro-osmotically driven systems. The goals were to minimize the linearity and light leakage problems that often limit the performance of UV absorbance detection with capillary chromatography. A linear dynamic range of 10(3) (reserpine, lambda = 220 nm), and a concentration detection limit of 5.1 x 10(-8) mol l-1 were observed. Baseline noise was measured at 3.5 x 10(-5) absorbance units (AU), with a standard deviation of 1.7 x 10(-5) AU. The illuminated volume of approximately 3 nl was optimized for capillaries with inner diameters in the range 50-100 microns, and flow rates from 100 nl min-1 to 1 microliter min-1. These modifications of readily available instrumentation have allowed the construction of a practical system for fractionating complex mixtures of peptides in small amounts, prior to mass spectrometry or additional wet chemistry steps.