Production of 2-Aminophenoxazin-3-one by Staphylococcus aureus Causes False-Positive Results in β-Galactosidase Assays

Staphylococcus aureus can be distinguished from similar coagulase-positive staphylococci by its absence of β-galactosidase activity. This is commonly tested using o-nitrophenyl-β-d-galactopyranoside (ONPG) as the substrate. Unexpectedly, 111 and 58 of 123 isolates displayed apparent β-galactosidase activity in the ONPG assay and on the Vitek 2 system, respectively. Compositional analysis showed that the yellow coloration of the positive ONPG assay resulted from production of 2-aminophenoxazin-3-one. Alternative β-galactosidase substrates like X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) should be used for testing staphylococci.     

Role of α7- and β4-Containing Nicotinic Acetylcholine Receptors in the Affective and Somatic Aspects of Nicotine Withdrawal: Studies in Knockout Mice

To assess which nicotinic acetylcholine receptors (nAChRs) are involved in the aversive aspects of nicotine withdrawal, brain reward function and the somatic signs of nicotine withdrawal were assessed in mice that lack α7 and β4 nAChR subunits. Brain reward function was assessed with the intracranial self-stimulation (ICSS) procedure, in which elevations in ICSS thresholds reflect an anhedonic mood state. At 3–6 h of spontaneous nicotine/saline withdrawal, thresholds were elevated in nicotine-withdrawing α7^+/+ and β4^+/+, but not α7^−/− or β4^−/−, mice compared with saline-withdrawing mice, indicating a delay in the onset of withdrawal in the knockout mice. From 8 to 100 h of withdrawal, thresholds in α7^+/+ and α7^−/− mice were equally elevated, whereas thresholds in β4^+/+ and β4^−/− mice returned to baseline levels. Somatic signs were attenuated in nicotine-withdrawing β4^−/−, but not α7^−/−, mice. Administration of a low dose of the nAChR antagonist mecamylamine induced threshold elevations in α7^−/−, but not α7^+/+, mice, whereas the highest dose tested only elevated thresholds in α7^+/+ mice. Mecamylamine-induced threshold elevations were similar in β4^−/− and β4^+/+ mice. In conclusion, null mutation of the α7 and β4 nAChR subunits resulted in a delayed onset of the anhedonic aspects of the spontaneous nicotine withdrawal syndrome. Previous findings of attenuated somatic signs of nicotine withdrawal in β4^−/−, but not α7^−/−, mice were confirmed in the present study, indicating an important role for β4-containing nAChRs in the somatic signs of nicotine withdrawal. The mecamylamine-precipitated withdrawal data suggest that compensatory adaptations may occur in constitutive α7^−/− mice or that mecamylamine may interact with other receptors besides nAChRs in these mice. In summary, the present results indicate an important role for α7 and β4-containing nAChRs in the anhedonic or somatic signs of nicotine withdrawal.     

Discriminating Foot-and-Mouth Disease Virus-Infected and Vaccinated Animals by Use of β-Galactosidase Allosteric Biosensors

Recombinant β-galactosidases accommodating one or two different peptides from the foot-and-mouth disease virus (FMDV) nonstructural protein 3B per enzyme monomer showed a drastic enzymatic activity reduction, which mainly affected proteins with double insertions. Recombinant β-galactosidases were enzymatically reactivated by 3B-specific murine monoclonal and rabbit polyclonal antibodies. Interestingly, these recombinant β-galactosidases, particularly those including one copy of each of the two 3B sequences, were efficiently reactivated by sera from infected pigs. We found reaction conditions that allowed differentiation between sera of FMDV-infected pigs, cattle, and sheep and those of naïve and conventionally vaccinated animals. These FMDV infection-specific biosensors can provide an effective and versatile alternative for the serological distinction of FMDV-infected animals.Foot-and-mouth disease (FMD) is one of the most important and highly transmissible diseases of livestock that causes severe production losses, and it is a major constraint to international trade of live animals and their products (42, 50). FMD affects extensive areas worldwide and is included in the list of diseases notifiable to the World Organization for Animal Health (http://www.oie.int/eng/en_index.htm). The etiological agent FMD virus (FMDV) infects artiodactyla, mostly cattle, swine, sheep, and goats (38, 51). FMDV is the prototypic member of the genus Aphthovirus, within the family Picornaviridae (45). The FMDV genome consists of a positive-sense RNA molecule of approximately 8,500 nucleotides that encodes a unique polyprotein, which is processed in infected cells to yield different polypeptide precursors and the mature viral proteins (2, 47). The open reading frame of FMDV encodes the capsid proteins, as well as a total of 10 additional mature, nonstructural (NS) proteins (33). In particular, the genomic organization of the region encoding FMDV proteins 3A and 3B is unique among the Picornaviridae family in that 3A extends its carboxy terminus at least 60 amino acid (aa) residues in length, and three nonidentical copies of 3B (named 3B-1, 3B-2, and 3B-3) are sequentially encoded and expressed in susceptible cells (26, 34).In areas of endemicity, FMD control is implemented by regular immunization with inactivated vaccines (14). Within the European Union, a nonvaccination, stamping-out policy—implying the slaughtering of infected and contact animals, as well as animal movement restrictions—is applied in cases of outbreaks (50); eventually, emergency ring vaccination can be applied around outbreaks (1). These restrictions extend to the importation of animals from FMD-free regions in which vaccination is implemented, due to problems associated with the serological distinction between uninfected animals and infected animals (30).FMDV shows high genetic and antigenic variabilities, which are reflected in the seven serotypes and the numerous variants described to date (15). Detection of circulating antibodies to FMDV particles has been carried out by different techniques, including enzyme-linked immunosorbent assay (ELISA) (28). These techniques are serotype specific and do not allow reliable distinction between infected and vaccinated animals, since FMDV NS proteins, mainly the 3D polymerase, formerly used as an infection-associated antigen (12, 54), can contaminate vaccine preparations (39). This has led to purification of the FMDV antigens used for vaccine production to minimize the presence of contaminating NS proteins (14). Detection of FMDV-specific antibodies is important, particularly for cattle, since they frequently develop a persistent and unapparent infection, even among vaccinated animals (41). Antibodies to other FMDV NS proteins, mainly those to the 3AB region, have been shown to be specific markers for FMDV infection (9, 10, 13, 31, 32, 36, 40, 48) and allow detection of antibodies regardless of the viral serotype causing the infection (7).In the serological diagnosis of infectious diseases, the use of allosteric biosensors, namely, hybrid enzymes that respond enzymatically to antibodies directed to foreign peptides displayed on the enzyme surface (53), is highly promising (23). We previously showed that multiple insertions of a major FMDV B-cell epitope from the VP1 capsid protein near the active site of recombinant β-galactosidases dramatically increased the enzyme responsiveness to specific antipeptide antibodies, including sera from infected animals (4, 17). In this study, we report that recombinant β-galactosidases accommodating one or two different peptides from the FMDV NS protein 3B per enzyme monomer can be reactivated by anti-3B monoclonal antibodies (MAbs). Interestingly, these recombinant β-galactosidases, particularly those including one copy of each of the two 3B sequences, could be also efficiently reactivated by sera from infected animals. We found reaction conditions that permitted differentiation between sera from infected animals and those from naïve and conventionally vaccinated pigs. These infection-specific FMDV biosensors can provide an effective and versatile alternative for the serological distinction of FMDV-infected animals.     

Progression to Adrenocortical Tumorigenesis in Mice and Humans through Insulin-Like Growth Factor 2 and β-Catenin

Dysregulation of the WNT and insulin-like growth factor 2 (IGF2) signaling pathways has been implicated in sporadic and syndromic forms of adrenocortical carcinoma (ACC). Abnormal β-catenin staining and CTNNB1 mutations are reported to be common in both adrenocortical adenoma and ACC, whereas elevated IGF2 expression is associated primarily with ACC. To better understand the contribution of these pathways in the tumorigenesis of ACC, we examined clinicopathological and molecular data and used mouse models. Evaluation of adrenal tumors from 118 adult patients demonstrated an increase in CTNNB1 mutations and abnormal β-catenin accumulation in both adrenocortical adenoma and ACC. In ACC, these features were adversely associated with survival. Mice with stabilized β-catenin exhibited a temporal progression of increased adrenocortical hyperplasia, with subsequent microscopic and macroscopic adenoma formation. Elevated Igf2 expression alone did not cause hyperplasia. With the combination of stabilized β-catenin and elevated Igf2 expression, adrenal glands were larger, displayed earlier onset of hyperplasia, and developed more frequent macroscopic adenomas (as well as one carcinoma). Our results are consistent with a model in which dysregulation of one pathway may result in adrenal hyperplasia, but accumulation of a second or multiple alterations is necessary for tumorigenesis.     

Characterization of Novel β-Galactosidase Activity That Contributes to Glycoprotein Degradation and Virulence in Streptococcus pneumoniae

The pneumococcus obtains its energy from the metabolism of host glycosides. Therefore, efficient degradation of host glycoproteins is integral to pneumococcal virulence. In search of novel pneumococcal glycosidases, we characterized the Streptococcus pneumoniae strain D39 protein encoded by SPD_0065 and found that this gene encodes a β-galactosidase. The SPD_0065 recombinant protein released galactose from desialylated fetuin, which was used here as a model of glycoproteins found in vivo. A pneumococcal mutant with a mutation in SPD_0065 showed diminished β-galactosidase activity, exhibited an extended lag period in mucin-containing defined medium, and cleaved significantly less galactose than the parental strain during growth on mucin. As pneumococcal β-galactosidase activity had been previously attributed solely to SPD_0562 (bgaA), we evaluated the contribution of SPD_0065 and SPD_0562 to total β-galactosidase activity. Mutation of either gene significantly reduced enzymatic activity, but β-galactosidase activity in the double mutant, although significantly less than that in either of the single mutants, was not completely abolished. The expression of SPD_0065 in S. pneumoniae grown in mucin-containing medium or tissues harvested from infected animals was significantly upregulated compared to that in pneumococci from glucose-containing medium. The SPD_0065 mutant strain was found to be attenuated in virulence in a manner specific to the host tissue.Streptococcus pneumoniae is the leading cause of bacterial pneumonia, otitis media, bacterial meningitis, and septicemia (21). Furthermore, a high percentage of the population carries the bacterium in the nasopharynx, asymptomatically or as a prelude to disease (53). The upsurge in antibiotic-resistant strains and the search for new vaccines highlight the need to understand more completely the nature of pneumococcal virulence. A survey of pneumococcal virulence studies indicates a clear asymmetry in favor of research focusing on factors involved in attachment and damage to host tissues and in immune evasion (2, 11, 15, 22, 38). Although these studies have revealed useful information about pneumococcal virulence, there is a severe lack of knowledge on the basic physiology of S. pneumoniae, such that little information is available on how the pneumococcus fulfils its nutritional requirements for the generation of energy.Carbohydrates are the principal energy sources for the pneumococcus, and these must be obtained exclusively from its host (16). Although the pneumococcus is known to utilize a variety of free sugars through at least 14 sugar utilization operons (10), in the upper respiratory tract the concentration of free sugars is low (39). However, monosaccharides are copious within the structures of O- and N-linked oligosaccharides of glycoproteins, which are found predominantly in the structures of mucins (Fig. ​(Fig.1)1) and in circulatory glycoproteins, respectively. Previous studies have shown that the sequential deglycosylation of N-linked glycan structures by pneumococcal glycosidases mediates in vitro growth of S. pneumoniae (5).Open in a separate windowFIG. 1.Schematic representation of the oligosaccharide structure of mucins. The carbohydrate chain is O-glycosidically linked to the apomucin through N-acetylgalactosamine (GalNAc). Galactose (Gal), N-acetylglucosamine (GlcNAc), sialic acid (Neu5Ac), sulfate (SO₃²⁻), and fucose (Fuc) residues are indicated. The structure of 9-O-acetylated sialic acid is shown as a structural formula (constructed from data presented in reference 49).The apical surfaces of the respiratory tract epithelium are covered with mucus, which plays a vital role in the removal of pathogens and environmental toxins via the mucocilliary escalator (42). Paradoxically, mucus is a potentially rich source of carbon and nitrogen for bacteria with a sufficient array of glycosidase activities (43). Mucin is the major macromolecular compound of mucus, and the heavily glycosylated side chains of this glycoprotein may be utilized by bacteria as a carbohydrate source. Although the detailed structure of mucins in different organs exhibits variation, the majority of mucin glycans contain N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose, sulfated sugars, and sialic acids in various proportions (Fig. ​(Fig.1)1) (41, 43). The acetylation of terminal sialic acids and sulfation of other monosaccharides, to a certain degree, protect carbohydrate side chains against the cleavage by microbial glycosidases (7).Pneumococci degrade mucin for growth, and NanA is essential for the mucin degradation ability of the pathogen (52), presumably for removal of terminal sialic acid residues to expose the carbohydrate core of oligosaccharides to further degradation by other glycosidases. Recently it was also shown that NanB plays a role in the deglycosylation of human glycoconjugates by some pneumococcal strains (5). It has been demonstrated that mucin stimulates expression of the neuraminidase in the pneumococcus (49), thus enhancing its own degradation. Using isogenic nanA and nanB mutant strains, the involvement of neuraminidases in the pathology of pneumococcal otitis media, meningitis, and pneumonia has been demonstrated by us and others (30, 48). In addition to neuraminidases NanA (SPD_1504) and NanB (SPD_1499), BgaA (SPD_0562) and StrH (SPD0063), which catalyze the hydrolysis of terminal nonreducing β-d-galactose and N-acetyl-β-d-glucosamine residues, respectively, have been shown to be involved in the deglycosylation of human glycoconjugates and to contribute to pneumococcal colonization and pathogenesis of disease (24).The roles of other glycosidases remain unknown, yet at least one-third of the pneumococcal genome encodes proteins involved in sugar transport, degradation, and processing, suggesting that carbohydrate metabolism has a central impact on pneumococcal biology (16, 27, 47). In the sequenced pneumococcal genome, several genes are annotated as sugar hydrolases (otherwise known as glycosidases); however, the importance of these in glycoprotein degradation is not known. In this study we explored the role in glycoprotein degradation and virulence of SPD_0065, annotated as β-galactosidase 3 in the D39 genome (27). During preparation of this paper, Jeong et al. (20) reported some properties of SPD_0065. Because pneumococcal β-galactosidase activity previously had been attributed exclusively to SPD_0562 (54), we investigated the contribution of this newly identified galactosidase, SPD_0065, to total β-galactosidase activity by constructing a pneumococcal strain with both genes mutated. Using an isogenic mutant, it was shown that this glycosidase is important in mucin degradation. The involvement of SPD_0065 in pneumococcal virulence was also tested using this mutant strain in a mouse model of pneumonia and bacteremia, where it was found that SPD_0065 is important primarily for survival in the nasopharynx.     

Meningitis Caused by Escherichia coli Producing TEM-52 Extended-Spectrum β-Lactamase within an Extensive Outbreak in a Neonatal Ward: Epidemiological Investigation and Characterization of the Strain

Outbreaks caused by Enterobacteriaceae isolates producing extended-spectrum β-lactamases (ESBL) in neonatal wards can be difficult to control. We report here an extensive outbreak in a neonatal ward with a case of meningitis caused by an ESBL-producing Escherichia coli strain. Between 24 March and 29 April 2009, among the 59 neonates present in the ward, 26 neonates with ESBL-producing E. coli rectal colonization were detected (44%). One of the colonized neonates developed meningitis with a favorable outcome after treatment combining imipenem, gentamicin, and ciprofloxacin. Despite strict intensification of hygiene and isolation procedures for more than 1 month, ward closure to new admissions was necessary to control the outbreak. Randomly amplified polymorphic DNA and pulsed-field gel electrophoresis analysis performed on 31 isolates recovered from 26 neonates and two mother''s milk samples showed a clonal strain. ESBL PCR assays indicated that the strain harbored a TEM-52 ESBL encoded by an IncI1 replicon. Phylogenetic analysis by multilocus sequence typing showed that the strain belonged to rare phylogenetic group C, which is closely related to group B1 but appears as group A by the triplex PCR phylogrouping method. The strain harbored the virulence genes fuyA, aer, and iroN and was virulent in a mouse model of septicemia. This work indicates the high potential of colonization, transmission, and virulence of some ESBL-producing E. coli clones.With Streptococcus agalactiae, Escherichia coli is one of the two most common bacterial species frequently responsible of neonatal infections (34). E. coli is the second leading cause of neonatal bacterial meningitis in industrial countries, and recent studies suggest that extraintestinal pathogenic E. coli isolates belong mainly to phylogenetic group B2 (2, 6). An increased rate of E. coli ampicillin resistance after the initiation of intrapartum antimicrobial prophylaxis was previously reported (5, 34). However, most of the E. coli isolates remained susceptible to extended-spectrum cephalosporins (ESC), but currently, extended-spectrum β-lactamases (ESBL) are becoming an increasingly important cause of resistance to ESC in E. coli, frequently involving the CTX-M-type enzymes (29). In 2008, Boyer-Mariotte et al. reported one case of fatal neonatal meningitis caused by a CTX-M-15-producing E. coli strain (7). Emergence of ESBL-producing Enterobacteriaceae is a major problem, since the choice of drugs for antimicrobial treatment is limited; moreover, such strains have been increasingly implicated in nosocomial outbreaks in neonatal intensive care units (11, 23, 33, 35). While the spread of CTX-M-type ESBL in the family Enterobacteriaceae, especially in E. coli, has been described worldwide (11, 13, 15, 17), the spread of TEM-type ESBL has been less frequently reported (8, 9). We report here a case of neonatal meningitis caused by a TEM-52 ESBL-producing E. coli strain within a serious outbreak among neonates with rectal colonization and the characteristics of the strain.     

Modulation of Nav1.5 by β1- and β3-subunit co-expression in mammalian cells

Cardiac sodium channels (Na_v1.5) comprise a pore-forming -subunit and auxiliary -subunits that modulate channel function. In the heart, 1 is expressed throughout the atria and ventricles, whilst 3 is present only in the ventricles and Purkinje fibers. In view of this expression pattern, we determined the effects of 3 and 1 co-expression alone, and in combination, on Na_v1.5 stably expressed in Chinese hamster ovary cells. The current/voltage relationship was shifted –5 mV with either 1 or 3 co-expression alone and –10 mV with co-expression of both 1 and 3. In addition, 3 and 1/3 co-expression accelerated macroscopic current decay. There were significant hyperpolarizing shifts in equilibrium gating relationships with co-expression of 1 and 3 alone and in combination. Co-expression of 1/3 together resulted in a greater hyperpolarizing shift in channel availability, and an increase in the slopes of equilibrium gating relationships. Co-expression of 3 and 1/3, but not 1, slowed recovery from inactivation at –90 mV. Development of inactivation at –70 and –50 mV was accelerated by -subunit co-expression alone and in combination. -Subunit co-expression also reduced the late Na current measured at 200 ms. In conclusion, -subunits modulate Na_v1.5 gating with important differences between co-expression of 1 and 3 alone and 1/3 together.     

Neonatal Maternal Separation Alters Immune,Endocrine, and Behavioral Responses to Acute Theiler’s Virus Infection in Adult Mice

Previous studies have established a link between adverse early life events and subsequent disease vulnerability. The present study assessed the long-term effects of neonatal maternal separation on the response to Theiler’s murine encephalomyelitis virus infection, a model of multiple sclerosis. Balb/cJ mouse pups were separated from their dam for 180-min/day (180-min MS), 15-min/day (15-min MS), or left undisturbed from postnatal days 2–14. During adolescence, mice were infected with Theiler’s virus and sacrificed at days 14, 21, or 35 post-infection. Prolonged 180-min MS increased viral load and delayed viral clearance in the spinal cords of males and females, whereas brief 15-min MS increased the rate of viral clearance in females. The 15-min and 180-min MS mice exhibited blunted corticosterone responses during infection, suggesting that reduced HPA sensitivity may have altered the immune response to infection. These findings demonstrate that early life events alter vulnerability to CNS infection later in life. Therefore, this model could be used to study gene-environment interactions that contribute to individual differences in susceptibility to infectious and autoimmune diseases of the CNS.     

Disruption of the Sleep-Wake Cycle and Diurnal Fluctuation of β-Amyloid in Mice with Alzheimer's Disease Pathology

Aggregation of β-amyloid (Aβ) in the brain begins to occur years before the clinical onset of Alzheimer's disease (AD). Before Aβ aggregation, concentrations of extracellular soluble Aβ in the interstitial fluid (ISF) space of the brain, which are regulated by neuronal activity and the sleep-wake cycle, correlate with the amount of Aβ deposition in the brain seen later. The amount and quality of sleep decline with normal aging and to a greater extent in AD patients. How sleep quality as well as the diurnal fluctuation in Aβ change with age and Aβ aggregation is not well understood. We report a normal sleep-wake cycle and diurnal fluctuation in ISF Aβ in the brain of the APPswe/PS1δE9 mouse model of AD before Aβ plaque formation. After plaque formation, the sleep-wake cycle markedly deteriorated and diurnal fluctuation of ISF Aβ dissipated. As in mice, diurnal fluctuation of cerebrospinal fluid Aβ in young adult humans with presenilin mutations was also markedly attenuated after Aβ plaque formation. Virtual elimination of Aβ deposits in the mouse brain by active immunization with Aβ(42) normalized the sleep-wake cycle and the diurnal fluctuation of ISF Aβ. These data suggest that Aβ aggregation disrupts the sleep-wake cycle and diurnal fluctuation of Aβ. Sleep-wake behavior and diurnal fluctuation of Aβ in the central nervous system may be functional and biochemical indicators, respectively, of Aβ-associated pathology.     

Kakkalide and Its Metabolite Irisolidone Ameliorate Carrageenan-Induced Inflammation in Mice by Inhibiting NF-κB Pathway

The anti-inflammatory activities of kakkalide, a major constituent of the flower of Pueraria thunbergiana, and irisolidone, a metabolite of kakkalide produced by intestinal microflora, against carrageenan-induced inflammation in air pouches on the backs of mice and in lipopolysaccharide (LPS)-stimulated peritoneal macrophages were investigated. Kakkalide and irisolidone down-regulated the gene expression of cytokines [tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β)] and cyclooxygenase-2 (COX-2) and the production of pro-inflammatory cytokines, TNF-α and IL-1β, and inflammatory mediators, NO and prostaglandin E₂ (PGE₂), in LPS-stimulated peritoneal macrophages. These agents also inhibited the phosphorylation of IκB-α and the nuclear translocation of nuclear factor-kappa B (NF-κB). Orally administered kakkalide and irisolidone significantly reduced carrageenan-induced inflammatory markers, leukocyte number, and protein amount in the exudates of the air pouch. These constituents also inhibited PGE₂ production and COX-2 inducible nitric oxide synthase, IL-1β, and TNF-α expression. These agents also inhibited NF-κB activation. The anti-inflammatory effects of irisolidone were more potent than those of kakkalide. Based on these findings, kakkalide and irisolidone may inhibit inflammatory reactions via NF-κB pathway, and irisolidone, a metabolite of kakkalide, may more potently inhibit these inflammatory reactions.