Efficiency of Radiolabeling Eggs Before and After Microwave Cooking for Gastric Emptying Scintigraphy Studies.

The accuracy and reproducibility of nuclear medicine gastric emptying scintigraphy (GES) require strict adherence to the Society of Nuclear Medicine and Molecular Imaging standardized protocol, which contains precise instructions for meal ingredients and preparation. Previous research demonstrated that many laboratories were using whole eggs in the test meal as opposed to the guideline-recommended liquid egg whites and that some laboratories were attempting to radiolabel the egg by adding the radiotracer after cooking. This study aimed to document the labeling efficiency of 99mTc-sulfur colloid (SC) added to whole eggs before and after microwave cooking. Methods: Whole eggs were mixed with 99mTc-SC before and after microwave cooking. The radiolabeling stability of the eggs was tested after 2 and 4 h of incubation in gastric fluid simulated using just hydrochloric acid (HCl) and using HCl with pepsin. Results: The experiment showed that no matter what the testing condition, radiolabeling by adding 99mTc-SC to whole eggs before cooking resulted in a significantly higher labeling efficiency than radiolabeling by squirting the 99mTc-SC on eggs after cooking. This finding persisted over time, with the precooking method still showing significantly higher radiolabeling at 2 and 4 h after the egg was placed in the incubation medium for both gastric fluid mediums. For simulated gastric fluid with pepsin at 2 h, the labeling was significantly higher, at 73.3%, when the radiotracer was added before cooking than the 43.3% when added after cooking (P < 0.001). The results of this study further showed that when egg labeling efficiency was tested in HCl without pepsin, the labeling was less stable than when tested in HCl with pepsin. In the HCl-only medium, the labeling efficiency decreased significantly between 2 and 4 h for both radiolabeling methods. Conclusion: The results of this study demonstrated that the addition of 99mTc-SC to whole eggs after cooking resulted in considerably inferior binding of the radiotracer to the eggs and that binding deteriorated significantly over time. The study further demonstrated that the results of radiolabeling efficiency varied depending on whether HCl or HCl with pepsin was used to simulate gastric fluid. Radiolabeling stability decreased over time when HCl without pepsin was used. The findings emphasize the criticality of adhering to the standardized meal and preparation, as alternate cooking methods have different radiolabeling efficiencies.

An unbiased systems genetics approach to mapping genetic loci modulating susceptibility to severe streptococcal sepsis.

Striking individual differences in severity of group A streptococcal (GAS) sepsis have been noted, even among patients infected with the same bacterial strain. We had provided evidence that HLA class II allelic variation contributes significantly to differences in systemic disease severity by modulating host responses to streptococcal superantigens. Inasmuch as the bacteria produce additional virulence factors that participate in the pathogenesis of this complex disease, we sought to identify additional gene networks modulating GAS sepsis. Accordingly, we applied a systems genetics approach using a panel of advanced recombinant inbred mice. By analyzing disease phenotypes in the context of mice genotypes we identified a highly significant quantitative trait locus (QTL) on Chromosome 2 between 22 and 34 Mb that strongly predicts disease severity, accounting for 25%-30% of variance. This QTL harbors several polymorphic genes known to regulate immune responses to bacterial infections. We evaluated candidate genes within this QTL using multiple parameters that included linkage, gene ontology, variation in gene expression, cocitation networks, and biological relevance, and identified interleukin1 alpha and prostaglandin E synthases pathways as key networks involved in modulating GAS sepsis severity. The association of GAS sepsis with multiple pathways underscores the complexity of traits modulating GAS sepsis and provides a powerful approach for analyzing interactive traits affecting outcomes of other infectious diseases.

Structural features of the ligand binding site on human complement protein C8gamma: a member of the lipocalin family.

Human C8 is one of five components of the cytolytic membrane attack complex of complement. It contains three subunits (C8alpha, C8beta, C8gamma) arranged as a disulfide-linked C8alpha-gamma heterodimer that is noncovalently associated with C8beta. C8gamma has the distinction of being the only lipocalin in the complement system. Lipocalins have a core beta-barrel structure forming a calyx with a binding site for a small hydrophobic ligand. A natural ligand for C8gamma has not been identified; however previous structural studies indicate C8gamma has a typical lipocalin fold that is suggestive of a ligand-binding capability. A distinctive feature of C8gamma is the division of its putative ligand binding pocket into a hydrophilic upper portion and a large hydrophobic lower cavity. Access to the latter is restricted by the close proximity of two tyrosine side chains (Y83 and Y131). In the present study, binding experiments were performed using lauric acid as a pseudoligand to investigate the potential accessibility of the lower cavity. The crystal structure of a C8gamma.laurate complex revealed that Y83 and Y131 can move to allow penetration of the hydrocarbon chain of laurate into the lower cavity. Introducing a Y83W mutation blocked access but had no effect on the ability of C8gamma to enhance C8 cytolytic activity. Together, these results indicate that the lower cavity in C8gamma could accommodate a ligand if such a ligand has a narrow hydrophobic moiety at one end. Entry of that moiety into the lower cavity would require movement of Y83 and Y131, which act as a gate at the cavity entrance.

Incorporation of human complement C8 into the membrane attack complex is mediated by a binding site located within the C8beta MACPF domain.

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the membrane attack complex (MAC). C8 is an oligomeric protein composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. C8alpha and C8beta are homologous; both contain N- and C-terminal modules and an intervening approximately 40 kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. C8beta participates in at least two binding interactions. It has a high affinity binding site for C8alpha, which facilitates its interaction with C8alpha-gamma. C8beta also mediates incorporation of C8 into the MAC by binding to C5b-7, an intermediate in the MAC assembly pathway. Little is known about the location or properties of the respective binding sites on C8beta. In this study, the MACPF domain of C8beta (betaMACPF) was expressed in Escherichia coli and its role in binding C8alpha and C5b-7 examined. Recombinant betaMACPF was shown to bind C8alpha-gamma in solution and form a noncovalent complex (betaMACPF*C8alpha-gamma) that exhibited C8 hemolytic activity. betaMACPF was also capable of binding independently to erythrocytes carrying C5b-7. Subsequent addition of C8alpha-gamma and C9 to these cells produced a hemolytically active MAC. The ability to produce a soluble, recombinant betaMACPF that retains the binding functions of C8beta suggests this segment of C8beta is an independently folded domain. Furthermore, results indicate the principal binding sites for C8alpha and C5b-7 are located within this domain, and that C8beta binding specificity is not determined by the N- and C-terminal modules.