The tripeptide glutathione (GSH) is the predominant low molecular weight thiol reductant in mammalian cells. In this report, we show that at concentrations at which GSH is typically present in the intracellular milieu, GSH and the oxidized GSH derivatives GSH disulfide (GSSG) and glutathione sulfonate each irreversibly inactivate up to 100% of the activity of purified Ca 2؉ -and phosphatidylserine (PS)-dependent protein kinase C (PKC) isozymes in a concentration-dependent manner by a novel nonredox mechanism that requires neither glutathiolation of PKC nor the reduction, formation, or isomerization of disulfide bridges within PKC. Our evidence for a nonredox mechanism of PKC inactivation can be summarized as follows. GSSG antagonized the Ca 2؉ -and PS-dependent activity of purified rat brain PKC with the same efficacy (IC 50 ‫؍‬ 3 mM) whether or not the reductant dithiothreitol was present. Glutathione sulfonate, which is distinguished from GSSG and GSH by its inability to undergo disulfide/thiol exchange reactions, was as effective as GSSG in antagonizing Ca 2؉ -and PS-dependent PKC catalysis. The irreversibility of the inactivation mechanism was indicated by the stability of the inactivated form of PKC to dilution and extensive dialysis. The inactivation mechanism did not involve the nonspecific phenomena of denaturation and aggregation of PKC because it obeyed pseudo-first order kinetics and because the hinge region of PKC-␣ remained a preferential target of tryptic attack following GSH inactivation. The selectivity of GSH in the inactivation of PKC was also indicated by the lack of effect of the tripeptides Tyr-Gly-Gly and Gly-Ala-Gly on the activity of PKC. Furthermore, GSH antagonism of the Ser/ Thr kinase casein kinase 2 was by comparison weak (<25%). Inactivation of PKC-␣ was not accompanied by covalent modification of the isozyme by GSH or other irreversible binding interactions between PKC-␣ and the tripeptide, but it was associated with an increase in the susceptibility of PKC-␣ to trypsinolysis. Treatment of cultured rat fibroblast and human breast cancer cell lines with N-acetylcysteine resulted in a substantial loss of Ca 2؉ -and PS-dependent PKC activity in the cells within 30 min. These results suggest that GSH exerts negative regulation over cellular PKC isozymes that may be lost when oxidative stress depletes the cellular GSH pool.The phospholipid-dependent isozyme family protein kinase C (PKC) 1 plays an important role in diverse physiological processes, including cell growth and differentiation, muscle contraction, and neurotransmission, and pathological developments, e.g. tumor promotion and multiple drug resistance in cancer (1-4). The PKC family consists of Ca 2ϩ -dependent, phorbol ester-activated isozymes (␣, ␤ 1 , ␤ 2 , and ␥), Ca 2ϩ -independent, phorbol ester-activated isozymes (␦, ⑀, , ⌰, and ), and Ca 2ϩ -and phorbol ester-independent isozymes ( and ). Phosphatidylserine (PS) dependence is universal among PKC isozymes (1, 5). Both stimulatory and inhibitory endogenous regulators of ...

The performance of a commercially available, rapid membrane enzyme immunoassay for influenza A and B virus detection was compared to that of viral culture in 4,092 respiratory specimens collected from patients presenting with respiratory symptoms during the 2002-2003 influenza season. The test's overall sensitivity was 43.83%, lower than previously reported but similar for detection of both influenza A and B viruses (42.98 versus 44.76%). However, specificity, 99.74%, was excellent for both influenza A and B viruses (99.82 versus 99.92%). These values make this test a very good confirmatory test when clinical suspicion is high, but a less accurate screening test for large populations.Influenza viruses are important causes of morbidity and mortality in individuals of all age groups, especially the elderly and patients with chronic disabilities (5, 15). Influenza virus testing is often a part of the evaluation of febrile respiratory illness in hospital settings, as well as in the practitioner's office and emergency and urgent care centers (1,5,8,10,16). The reference method is viral culture. However, since the results of viral culture may be delayed for days to weeks, most centers perform rapid testing for influenza virus by immunofluorescence assay or enzyme immunoassay (2-6). A rapid and correct diagnosis is essential to help clinicians with decisions regarding early antiviral treatment, need for additional testing, and cohorting of the patients. Rapid response teams investigating outbreaks of severe respiratory disease also may use rapid tests to help differentiate between infection with influenza virus, biological warfare agents, such as those that cause anthrax and smallpox (2,8), and other causes of epidemic respiratory illnesses, such as coronaviruses associated with severe acute respiratory syndrome (5, 12, 13). Currently, there are several commercially available rapid influenza virus test kits that can easily be performed in less than 30 min (2, 4, 6, 9). However, these tests have different methodologies and performance characteristics and may be expensive to perform in large numbers (Table 1). Furthermore, the performance of rapid influenza virus tests and their impact on patient care should be evaluated periodically not only by manufacturers, but also by physicians and laboratories who perform the tests in large numbers of patients, in real-world clinical settings (2,3,10,11,16). MATERIALS AND METHODSThis study evaluated the performance of a relatively new rapid test for detec- Rapid tests were routinely performed by virology laboratory technicians according to the manufacturer's instructions, 24 h daily, 7 days a week, and results were reported within 2 h of specimen receipt. Briefly, the BD Directigen Flu AϩB test is a rapid membrane enzyme immunoassay test that involves extraction of influenza A or B viral antigens from the patient specimens. The extracted specimen is expelled through a filter assembly into each of two wells of a triangular plastic test device containing a membrane surface. Viral ant...

The performance of a new rapid lateral-flow chromatographic membrane immunoassay test kit for detection of influenza virus was evaluated and compared to that of viral culture in respiratory secretions collected from 400 adults and children seen at three large university hospitals during the recent 2003 influenza season. The rapid test provided results in 15 min, with excellent overall performance statistics (sensitivity, 94.4%; specificity, 100%; positive predictive value, 100%; negative predictive value, 97.5%). Both influenza A and B type viruses were reliably detected, with no significant difference in performance statistics noted by influenza virus type or by the center performing the test.Influenza virus is a major cause of respiratory infection in both adults and children and is a common cause of hospitalization, especially in young children, elderly adults, and persons with chronic diseases (17,20). Influenza epidemics also account for over 47,000 deaths annually in the United States. Furthermore, three global pandemics during the 20th century caused over 50 million deaths (22). Readily available, rapid, and accurate detection methods for influenza virus allow for prompt administration of appropriate antiviral therapy and judicious use of antibiotics, assist in isolation of patients in hospitals and emergency centers to reduce health care-associated spread of infection, and identify local epidemics of influenza in a timely manner (10, 13-15, 19, 21, 22). Rapid detection of influenza virus also is currently important because of increased concern for pandemic influenza caused by either naturally occurring strains, such as avian H5N1, or altered strains that may be used in an act of bioterrorism (7,8,18). Also, the diagnosis of influenza based on clinical grounds alone may be inaccurate, because the presenting symptoms of influenza are similar to those caused by other infectious agents (13). Furthermore, the rapid and accurate determination that a severe respiratory or flu-like illness is caused by influenza virus rather than severe acute respiratory syndrome-associated coronavirus or a bioterrorism agent, such as smallpox or tularemia, is helpful not only to the individual patient but also from the public health perspective (6).Currently, there are at least seven different test kits approved by the Food and Drug Administration for detection of influenza virus in respiratory samples (2-4, 9, 12, 16, 21, 23). However, not all available methods distinguish the type of influenza virus present in the sample, and those that do distinguish type A and B influenza viruses have not shown consistently reliable performance for both types of virus (3,4,9,12). This recent multicenter study documented promising performance of a new rapid lateral-flow chromatographic immunoassay for both detection and differentiation of influenza A and B type viruses in respiratory samples. . Most (239 of 400, or 59.75%) specimens were nasal washes, 122 of 400 (30.5%) were nasopharyngeal swabs, 30 of 400 (7.5%) were throat swabs, 4 of 400 ...

Clostridioides difficile toxins TcdA and TcdB are large clostridial glucosyltransferases which are the main pathogenicity factors in C. difficile-associated diseases. Four highly conserved cysteines are present in all large clostridial glucosyltransferases. In this study we focused on the conserved cysteine 2232 within the combined repetitive oligopeptide domain of TcdB from reference strain VPI10463 (clade I). Cysteine 2232 is not present in TcdB from hypervirulent strain R20291 (clade II), where a tyrosine is found instead. Replacement of cysteine 2232 by tyrosine in TcdBV PI10463 reduced binding to the soluble fragments of the two known TcdB receptors, frizzled-2 (FZD2) and poliovirus receptor-like protein-3/nectin-3 (PVRL3). In line with this, TcdBR20291 showed weak binding to PVRL3 in pull-down assays which was increased when tyrosine 2232 was exchanged for cysteine. Surprisingly, we did not observe binding of TcdBR20291 to FZD2, indicating that this receptor is less important for this toxinotype. Competition assay with the receptor binding fragments (aa 1101–1836) of TcdBV PI10463 and TcdBR20291, as well as antibodies newly developed by antibody phage display, revealed different characteristics of the yet poorly described delivery domain of TcdB harboring the second receptor binding region. In summary, we found that conserved Cys-2232 in TcdB indirectly contributes to toxin–receptor interaction.

Summary. -Enterovirus 70 (EV70) is the causative agent of acute hemorrhagic conjunctivitis (AHC), for which no effective vaccine is available. This study revealed a high reactivity of the N-terminal region of EV70 VP1 (VP1-1) with an anti-EV70 mouse serum. The analysis of overlapping synthetic peptides of VP1-1 identified a B-cell epitope in this region. The E-peptide (14-ANTVESEIKAELGVI-28) showing the highest reactivity with the anti-EV70 serum induced neutralizing antibodies in mice and reduced the virus titer in the eyes, suggesting that it is a candidate vaccine against AHC caused by EV70.Keywords: enterovirus 70; AHC; B-cell epitope; peptide vaccine * Corresponding author. E-mail: jhnam@catholic.ac.kr; phone: +82-2-2164-4917. # These authors contributed equally to this work. Abbreviations: EV70 = enterovirus 70; AHC = acute hemorrhagic conjunctivitis; VP1-1 = virus capsid protein VP1 amino-terminus; VP1-2 = virus capsid protein VP1 middle; VP1-3 = virus capsid protein carboxy-terminus