Vaccine-associated paralytic poliomyelitis and BCG-osis in an immigrant child with severe combined immunodeficiency syndrome - Texas, 2013.

Poliovirus transmission has been eliminated in most of the world through the use of inactivated poliovirus vaccine (IPV) and live, attenuated oral poliovirus vaccine (OPV). In the United States, use of OPV was discontinued by the year 2000 because of the potential for vaccine-associated paralytic polio (VAPP); an average of eight cases were reported each year in the United States during 1980-2000. Polio eradication efforts in other parts of the world continue to rely on OPV to take advantage of transmission of poliovirus vaccine strains to unvaccinated persons in the population, lower cost, and ease of administration. In 2013, an infant aged 7 months who recently immigrated to the United States from India was referred to a hospital in San Antonio, Texas. The infant had fever, an enlarging skin lesion in the deltoid region with axillary lymphadenopathy, decreased activity, and inability to bear weight on the left leg, progressing to paralysis of the left leg over a 6-week period. Recognition of lymphopenia on complete blood count led to immune evaluation, which revealed the presence of severe combined immunodeficiency syndrome (SCIDS), an inherited disorder. A history of OPV and bacille Calmette-Guérin (BCG) vaccination in India led to the diagnoses of VAPP and BCG-osis, which were confirmed microbiologically. This report demonstrates the importance of obtaining a comprehensive clinical history in a child who has recently immigrated to the United States, with recognition that differing vaccine practices in other countries might require additional consideration of potential etiologies.

Impact of sub-lethal residues of azinphos-methyl on the pheromone-communication systems of insecticide-susceptible and insecticide-resistant obliquebanded leafrollers Choristoneura rosaceana (Lepidoptera: Tortricidae).

The effects of sub-lethal residues of azinphos-methyl on pheromone production, calling, female attractiveness and the ability of males to locate sources of natural and synthetic pheromone were compared in azinphos-methyl-susceptible (susceptible) and azinphos-methyl-resistant (resistant) obliquebanded leafrollers, Choristoneura rosaceana (Harris). The amount of pheromone in susceptible females was reduced by 29-33% after exposure to azinphos-methyl; this treatment did not affect the pheromone content of resistant females. Azinphos-methyl-treated resistant females contained 39-43% less pheromone than azinphos-methyl-treated susceptible females. Resistant females that were not treated with azinphos-methyl contained 35-56% less pheromone than susceptible females that were not treated with insecticide. The incidence of calling was reduced by 67-100% in azinphos-methyl-treated susceptible females; the incidence of calling by resistant females was not affected by exposure to azinphos-methyl. The incidence of calling by azinphos-methyl-treated susceptible females was 58-100% lower than that of azinphos-methyl-treated resistant females. There was no difference in the incidence of calling between susceptible and resistant females that had not been treated with insecticide. In a flight tunnel, treatment with insecticide reduced the attractiveness of susceptible females by 38%; treatment with insecticide did not affect the attractiveness of resistant females. There was no difference in the proportion of males attracted to susceptible and resistant females that had, or had not been treated with insecticide. In an apple orchard, the attractiveness of susceptible and resistant females treated with azinphos-methyl was reduced by 84 and 12%, respectively. The proportion of males attracted to azinphos-methyl-treated susceptible females was 58% lower than the proportion attracted to azinphos-methyl-treated resistant females, whereas, if females were not treated with insecticide, the proportion attracted to resistant females was 57% lower than the proportion attracted to susceptible females. In a flight tunnel, azinphos-methyl did not affect the ability of susceptible or resistant males to locate a source of pheromone gland extract. Likewise, in an apple orchard, the insecticide treatment had no effect on the ability of susceptible or resistant males to locate a source of synthetic pheromone. In a flight tunnel, there was no difference in the proportion of azinphos-methyl-treated susceptible and resistant males locating a source of pheromone gland extract; however, in the orchard, 39% fewer azinphos-methyl-treated resistant males located a source of synthetic pheromone than azinphos-methyl-treated susceptible males. A similar proportion of susceptible and resistant males that had not been treated with insecticide located a source of pheromone gland extract in the flight tunnel, but in the orchard, the proportion of resistant males not treated with azinphos-methyl that located the source of synthetic pheromone was 32% lower than the proportion of susceptible males not treated with this insecticide. The implications of the differences in the effect of sub-lethal residues of azinphos-methyl on the pheromone communication system of susceptible and resistant moths are discussed in relation to the theory of the development of insecticide resistance, the detection of resistance in feral populations of moths using sex pheromone-baited traps, and the control of moths using sex pheromone-mediated mating disruption.

Five genes involved in biosynthesis of the pyruvylated Galbeta1,3-epitope in Schizosaccharomyces pombe N-linked glycans.

The N-linked galactomannans of Schizosaccharomyces pombe have pyruvylated Galbeta1,3-(PvGal) caps on a portion of the Galalpha1,2-residues in their outer chains (Gemmill, T. R., and Trimble, R. B. (1998) Glycobiology 8, 1087-1095). PvGal biosynthesis was investigated by ethyl methanesulfonate mutagenesis of S. pombe, followed by the isolation of cells devoid of negatively charged N-glycans by Q-Sepharose exclusion and failure to bind human serum amyloid P component, which acts as a lectin for terminal PvGal residues. Mutant glycans were characterized by lectin binding, saccharide composition, exoglycosidase sensitivity, and NMR spectroscopy. Restoration of the cell surface negative charge by complementation with an S. pombe genomic library led to the identification of five genes involved in PvGal biosynthesis, which we designated pvg1-pvg5. Pvg1p may be a pyruvyltransferase, since NMR of pvg1(-) mutant N-glycans revealed the absence of only the pyruvyl moiety. Pvg2p-Pvg5p are crucial for attachment of the Galbeta1,3-residue that becomes pyruvylated. Pvg3p is predicted to be a member of the beta1,3-galactosyltransferase family, and Pvg3p-green fluorescent protein labeling was consistent with Golgi localization. Predicted Pvg1p and Pvg3p functions imply that Galbeta1,3-is added to the galactomannans and is then pyruvylated in situ, rather than by an en bloc addition of PvGalbeta1,3-caps to the outer chain. Pvg4p-green fluorescent protein targeted to the nucleus, and its sequence contains a MADS-box DNA-binding and dimerization domain; however, it does not appear to solely control transcription of the other identified genes. Pvg2p and/or Pvg5p may contribute to an enzyme complex. Whereas a functional role for the PvGal epitope in S. pombe remains unclear, it is nonessential for either cell growth or mating under laboratory conditions.

Characterization of N- and O-linked glycosylation of recombinant human bile salt-stimulated lipase secreted by Pichia pastoris.

Recombinant human bile salt-stimulated lipase (hBSSL) was expressed in and secreted by Pichia pastoris, an organism exploited for the large-scale production of recombinant (glyco)proteins by bioprocessing technology. The 76.3-kDa glycoprotein was associated with 75-80 Man and a small amount of GlcNAc. hBSSL has one N-glycosylation site at Asn187, which was 38-40% occupied with a Man(10)GlcNAc(2) structure defined previously in Pichia as the oligosaccharide-lipid form of Man(9)GlcNAc(2) trimmed of the middle-arm terminal alpha 1,2-Man and elongated with Man alpha 1,2Man alpha 1,6-disaccharide attached to the lower-arm core alpha 1,3-Man (Trimble et al. [1991], J. Biol. Chem., 266, 22807-22817). The C-terminal 192 residues of hBSSL contain 16 Pro-rich 11-amino-acid repeats, which include 32 Ser/Thr residues as potential O-glycosylation sites. Using hBSSL as a platform to study Pichia's O-glycosylation capabilities, we found that nearly all of these sites were occupied by mannose-containing O-glycans, whose structures, after beta-elimination and purification, were assigned by (1)H NMR and, in some cases, by linkage-specific exoglycosidases and methylation analysis. The most abundant O-glycan was alpha 1,2-mannobiitol (55%), followed by alpha 1,2-mannotriitol (16%) and mannitol (10%) and a lesser amount was alpha 1,2-mannotetraitol. Unexpectedly, Man(5) and Man(6) O-glycans were present, which had the structure Man beta 1,2Man beta 1,2Man alpha 1,2(Man alpha 1,2)(1,2)mannitol. Also a small amount of a phosphorylated Man(6) O-glycan was characterized by MALDI-TOF MS postsource decay analysis as having the reducing-end mannitol disubstituted with a glycosidically linked phosphorylated Man and an unbranched Man(4) polymer elongated from a different mannitol carbon. This is the first report of the synthesis of beta-Man- and phosphate-containing O-linked constituents on glycoproteins synthesized by P. pastoris.

Hypoglycosylation in the alg12delta yeast mutant destabilizes protease A and causes proteolytic loss of external invertase.

The Saccharomyces cerevisiae alg12delta mutant accumulates oligosaccharide lipid with a Man(7)GlcNAc(2) oligosaccharide. To determine the N-glycan structures present on S. cerevisiae glycoproteins in the alg12delta strain, we made attempts to purify external invertase, a highly glycosylated secreted protein. These efforts revealed that, in the alg12delta background, external invertase was mildly hypoglycosylated and rapidly destroyed proteolytically. Although secreted alg9delta invertase was more severely hypoglycosylated than the alg12delta form, it was paradoxically stable during purification. The loss of periplasmic invertase was prevented by addition of pepstatin A to the cell cultures, suggesting that aspartyl proteases were active. We found that during overexpression of invertase in alg12delta yeast, sufficient protease A was mistargeted to the periplasmic space, where it hydrolyzed the invertase. Even though alg9delta invertase is underglycosylated in comparison to the alg12delta form, it is more stable because in this genetic background much less protease A is secreted compared to alg12delta cells. These observations may be relevant to studies using other extracellular proteins (e.g., mating factors, alpha-glucosidase) as probes when characterizing glycosylation defects in yeast.

The Saccharomyces cerevisiae alg12delta mutant reveals a role for the middle-arm alpha1,2Man- and upper-arm alpha1,2Manalpha1,6Man- residues of Glc3Man9GlcNAc2-PP-Dol in regulating glycoprotein glycan processing in the endoplasmic reticulum and Golgi apparatus.

N-glycosylation in nearly all eukaryotes proceeds in the endoplasmic reticulum (ER) by transfer of the precursor Glc(3)Man(9)GlcNAc(2) from dolichyl pyrophosphate (PP-Dol) to consensus Asn residues in nascent proteins. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide lipid properly, and the alg12 mutant accumulates a Man(7)GlcNAc(2)-PP-Dol intermediate. We show that the Man(7)GlcNAc(2) released from alg12Delta-secreted invertase is Manalpha1,2Manalpha1,2Manalpha1,3(Manalpha1,2Manalpha1,3Manalpha1,6)-Manbeta1,4-GlcNAcbeta1-4GlcNAcalpha/beta, confirming that the Man(7)GlcNAc(2) is the product of the middle-arm terminal alpha1,2-mannoslytransferase encoded by the ALG9 gene. Although the ER glucose addition and trimming events are similar in alg12Delta and wild-type cells, the central-arm alpha1,2-linked Man residue normally removed in the ER by Mns1p persists in the alg12Delta background. This confirms in vivo earlier in vitro experiments showing that the upper-arm Manalpha1,2Manalpha1,6-disaccharide moiety, missing in alg12Delta Man(7)GlcNAc(2), is recognized and required by Mns1p for optimum mannosidase activity. The presence of this Man influences downstream glycan processing by reducing the efficiency of Ochlp, the cis-Golgi alpha1,6-mannosyltransferase responsible for initiating outer-chain mannan synthesis, leading to hypoglycosylation of external invertase and vacuolar protease A.

Calmodulin kinase II and arrhythmias in a mouse model of cardiac hypertrophy.

BACKGROUND: Calmodulin kinase (CaMK) II is linked to arrhythmia mechanisms in cellular models where repolarization is prolonged. CaMKII upregulation and prolonged repolarization are general features of cardiomyopathy, but the role of CaMKII in arrhythmias in cardiomyopathy is unknown. METHODS AND RESULTS: We studied a mouse model of cardiac hypertrophy attributable to transgenic (TG) overexpression of a constitutively active form of CaMKIV that also has increased endogenous CaMKII activity. ECG-telemetered TG mice had significantly more arrhythmias than wild-type (WT) littermate controls at baseline, and arrhythmias were additionally increased by isoproterenol. Arrhythmias were significantly suppressed by an inhibitory agent targeting endogenous CaMKII. TG mice had longer QT intervals and action potential durations than WT mice, and TG cardiomyocytes had frequent early afterdepolarizations (EADs), a hypothesized mechanism for triggering arrhythmias. EADs were absent in WT cells before and after isoproterenol, whereas EAD frequency was unaffected by isoproterenol in TG mice. L-type Ca2+ channels (LTTCs) can activate EADs, and LTCC opening probability (Po) was significantly higher in TG than WT cardiomyocytes before and after isoproterenol. A CaMKII inhibitory peptide equalized TG and WT LTCC Po and eliminated EADs, whereas a peptide antagonist of the Na+/Ca2+ exchanger current, also hypothesized to support EADs, was ineffective. CONCLUSIONS: These findings support the hypothesis that CaMKII is a proarrhythmic signaling molecule in cardiac hypertrophy in vivo. Cellular studies point to EADs as a triggering mechanism for arrhythmias but suggest that the increase in arrhythmias after beta-adrenergic stimulation is independent of enhanced EAD frequency.

Calmodulin inhibitor W-7 unmasks a novel electrocardiographic parameter that predicts initiation of torsade de pointes.

BACKGROUND: We have shown that the calmodulin inhibitor W-7 suppresses torsade de pointes (TdP) without shortening the QT interval, which is consistent with other findings that QT prolongation, per se, is insufficient to generate TdP. ECGs were analyzed from a well-characterized animal model of TdP to identify more reliable predictors of this life-threatening ventricular arrhythmia. METHODS AND RESULTS: TdP was induced using methoxamine and clofilium in 12 of 14 rabbits pretreated with vehicle control, whereas pretreatment with W-7 (50 micromol/kg), an inhibitor of the intracellular Ca2+-binding protein calmodulin, significantly suppressed TdP induction (1 of 11 rabbits with TdP, P<0.001). W-7 did not affect heart rate, increases in QT intervals, or dispersion compared with measurements in vehicle-treated control animals. However, a progressive and significant increase in the ratio of U-wave to T-wave amplitude (UTA) occurred before TdP onset in control animals, and this was prevented by W-7. CONCLUSIONS: Selective suppression of TdP inducibility by W-7, without shortening the duration of cardiac repolarization, allowed identification of the UTA ratio as a new electrocardiographic index for predicting TdP onset. These findings are consistent with the idea that prolonged repolarization is not the proximate cause of arrhythmia initiation, and they suggest that an increased UTA ratio reflects activation of intracellular Ca2+/calmodulin-dependent processes that are required for triggering TdP in this model.