Affinity maturation by somatic hypermutation is thought to occur within germinal centres. Mice deficient in lymphotoxin-alpha (LT alpha-/- mice) have no lymph nodes or Peyer's patches, and fail to form germinal centres in the spleen. We tested whether germinal centres are essential for maturation of antibody responses to T-cell-dependent antigens. LT alpha-/- mice immunized with low doses of (4-hydroxy-3-nitrophenyl)acetyl-ovalbumin (NP-OVA) showed dramatically impaired production of high-affinity anti-NP IgG1. However, LT alpha-/- mice immunized with high doses of NP-OVA, even though they failed to produce germinal centres, manifested a high-affinity anti-NP IgG1 response similar to wild-type mice. Furthermore, when LT alpha-/- mice were multiply immunized with high doses of NP-OVA, the predominantly expressed anti-NP VH gene segment VH186.2 showed somatic mutations typical of affinity maturation. Thus, B-cell memory and affinity maturation are not absolutely dependent on the presence of germinal centres.
The 3,6-dideoxyhexoses are found in the lipopolysaccharides of gram-negative bacteria, where they have been shown to be the dominant antigenic determinants. Of the five 3,6-dideoxyhexoses known to A large degree of bacterial immunological diversity in gram-negative species is attributed to the portion of the lipopolysaccharides known as the 0 antigen. Exposed on the cell envelope, the 0 antigen is the dominant surface entity which is polymorphic and provides the basis for the serological classification of the family Enterobacteriaceae (4,16,26). Among the large number of monosaccharides found as components of 0-specific polysaccharides, derivatives of the 3,6-dideoxyhexoses have drawn special attention because of their highly immunogenic characteristics (22,43,45). It has been shown that only a limited number of species of gram-negative bacteria, all in the family Enterobacteraceae, are able to produce these unusual sugars, and, of the eight possible stereoisomers of 3,6-dideoxyhexoses, only five have been identified in nature. Although colitose (3, and abequose (3,6-dideoxy-D-ylo-hexose, compound I [see Fig. 1]) can be detected in the lipopolysaccharide of some Escherichia coli serotypes and Citrobacter strains, respectively, the five 3,6-dideoxyhexoses known to occur naturally are present mainly in Salmonella and Yersinia spp. as major antigenic determinants (23). For example, abequose, colitose, tyvelose (3,6-dideoxy-D-arabino-hexose, compound II), and paratose (3,6-dideoxy-D-ribo-hexose, compound III) have been found in strains of Salmonella enterica (34), while all five, including ascarylose (3,6-dideOxy-L-arabino-hexose, compound * Corresponding author. Phone: (612) 626-7541. t Present address:
Results between different clinical laboratory measurement procedures (CLMP) should be equivalent, within clinically meaningful limits, to enable optimal use of clinical guidelines for disease diagnosis and patient management. When laboratory test results are neither standardized nor harmonized, a different numeric result may be obtained for the same clinical sample. Unfortunately, some guidelines are based on test results from a specific laboratory measurement procedure without consideration of the possibility or likelihood of differences between various procedures. When this happens, aggregation of data from different clinical research investigations and development of appropriate clinical practice guidelines will be flawed. A lack of recognition that results are neither standardized nor harmonized may lead to erroneous clinical, financial, regulatory, or technical decisions. Standardization of CLMPs has been accomplished for several measurands for which primary (pure substance) reference materials exist and/or reference measurement procedures (RMPs) have been developed. However, the harmonization of clinical laboratory procedures for measurands that do not have RMPs has been problematic owing to inadequate definition of the measurand, inadequate analytical specificity for the measurand, inadequate attention to the commutability of reference materials, and lack of a systematic approach for harmonization. To address these problems, an infrastructure must be developed to enable a systematic approach for identification and prioritization of measurands to be harmonized on the basis of clinical importance and technical feasibility, and for management of the technical implementation of a harmonization process for a specific measurand.
The CDP-6-deoxy-delta 3,4-glucoseen reductase (E3) is a NADH-dependent enzyme which catalyzes the key reduction of the C-3 deoxygenation step during the formation of CDP-ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis. This highly purified enzyme is also a NADH oxidase capable of mediating the direct electron transfer from NADH to O2, forming H2O2. While previous work showed that E3 contains no common cofactor, one FAD and one plant ferredoxin type [2Fe-2S] center were found in this study to be associated with each molecule of E3. The iron-sulfur center is essential for E3 activity since bleaching of the [2Fe-2S] center leads to inactive enzyme. These results suggest that E3 employs a short electron-transport chain composed of both FAD and the iron-sulfur center to shuttle electrons from NADH to its acceptor. The order of electron flow, as indicated by EPR measurement with partially reduced E3, starts with hydride reduction of FAD by NADH. The iron-sulfur cluster, receiving electrons one at a time from the reduced flavin, relays the reducing equivalents via another iron-sulfur center in the active site of E1 to its final acceptor, the E1-bound PMP-glucoseen adduct. The participation of a one-electron-carrying iron-sulfur center in this reduction is advantageous since both electrons are dispatched from the same redox state of the prosthetic group, allowing electrons of equal energy to be delivered to the final acceptor.(ABSTRACT TRUNCATED AT 250 WORDS)
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