Monoclonal antibodies to streptococcal group A carbohydrate. II. The VK1GAC light chain is preferentially associated with serum IgG3

A kappa-light chain variable region (V kappa) dominantly employed in the serum antibody response of A/J mice to streptococcal group A carbohydrate (GAC) has been termed VK1GAC. Examination of in vitro recombinants between the isolated heavy and light chains of VK1GAC+ and VK1GAC-anti-GAC hybridomas and non-GAC-binding myeloma proteins indicated that two antisera (anti-Id5 and anti-Id20) recognized the VK1GAC light chain when it was free in solution or paired with several heterologous heavy chains. Screening of a panel of A/J anti-GAC monoclonal antibodies with these antisera showed almost complete concordance between Id5 and Id20 expression and the presence of VK1GAC light chain as detected by its unique isoelectric focusing spectrotype. These antisera were used to examine serum expression of the VK1GAC light chain in normal and hyperimmune serum of A/J mice. Normal A/J serum contained from 20 to 100 micrograms Id5/ml serum, whereas only 1 to 10 micrograms Id20/ml serum was detected. The levels of both VK1GAC idiotypes increased dramatically 10- to 20-fold after hyperimmunization of mice with group A vaccine. When serum IgG from normal and immune mice was fractionated into the IgG subclasses (IgG1, IgG2a, and IgG3), it was found that the VK1GAC light chain does not pair randomly with heavy chains of the IgG subclasses, but rather is associated preferentially with heavy chains of the IgG3 subclass whether or not it is associated with antibodies to GAC. These results suggest that the heavy chain pairing exhibited by this VK product may not be random.     

Human antibodies to group A streptococcal carbohydrate. Ontogeny, subclass restriction, and clonal diversity

To investigate immuno-incompetence to polysaccharide Ag in young children, antibodies to the polysaccharide and protein Ag of Streptococcus pyogenes were studied. S. pyogenes was chosen because it commonly causes natural infections and has well-characterized polysaccharide and protein Ag. In children over the age of 2 yr it was found that the maturation of antibody responses to the polysaccharide Ag of S. pyogenes (A-CHO) appeared to occur in parallel with, or even earlier, than the responses to streptococcal protein Ag. When antibodies to group A carbohydrate (A-CHO) were studied in detail, qualitative differences between the antibodies of children and adults were demonstrated. Although anti-A-CHO antibodies of adults were strikingly restricted to the IgG2 subclass, those of children were found in both the IgG1 and IgG2 subclasses. In addition, the clonal diversity of IgG antibodies to A-CHO increased with age, and additional clonotypes were detectable in convalescent sera of some subjects of all ages after infection. Two cases with major additional clonotypes after group A streptococcal infection were studied in detail. In these two cases the additional clonotypes belonged to a different IgG subclass than the previously dominant clonotypes, and the expression of the additional major clonotypes occurred in both IgG1 and IgG2 subclasses.     

Induction and de novo synthesis of uricase, a nitrogen-regulated enzyme in Neurospora crassa.

Two efficient procedures are presented for the purification of the purine catabolic enzyme uricase from Neurospora crassa. A specific antiserum for uricase was prepared and used to examine the regulation of uricase expression. Even when wild-type cells are growing under full nitrogen repression conditions, they possess a considerable basal level of uricase. Induction results in a severalfold increase in the level of this enzyme and reflects de novo enzyme synthesis. Identical forms of uricase were translated in vitro from RNA isolated from control and induced cells, but, unexpectedly, induced cells contained less translatable uricase mRNA than did control cells. Although uricase is localized in peroxisomes, the enzyme subunit appears to be synthesized in mature form without any requirement for processing.     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. IV. The less frequently expressed VL are heterogeneous

We previously demonstrated that the human anti-Haemophilus influenzae type b polysaccharide (Hib-PS) VL repertoire is dominated by a product of the V kappa II gene, A2, and that V kappa II-A2 anti-Hib-PS antibodies have little or no somatic mutation in VL. To further study this VL repertoire, we studied non-A2 anti-Hib-PS antibodies that were identified either serologically or by amino-terminal amino acid sequence analysis. Of 15 non-A2 anti-Hib-PS antibodies from 12 vaccinated adults, we found four V lambda, five V kappa I, one non-A2 V kappa II, four V kappa III, and one V kappa IV antibodies. As expected, all but two of these subjects also produced V kappa II-A2 antibodies. Interestingly, one of these subjects lacks the A2 gene in the germ line. However, both subjects who did not produce detectable V kappa II antibody did produce normal amounts of total anti-Hib-PS antibody after vaccination. Candidate V kappa genes for the non-A2 antibodies were identified by comparison of up to 60 VL amino acid residues, including CDR1 and CDR2, with all sequenced V kappa genes. V kappa I antibodies appear to be products of three newly sequenced V kappa I genes, O8, O18, and L11, that are reported here. The O8 and O18 genes encode identical amino acid sequences. The non-A2 V kappa II antibody is a likely product of the A1 or A17 genes, the V kappa III antibodies are likely products of the A27 gene, and the V kappa IV antibody is a product of the single V kappa IV gene, B3. Unlike V kappa II-A2 antibodies, the V kappa I, V kappa III, and V kappa IV antibodies differed by one to five CDR residues from the germ line product of the candidate genes, suggesting the presence of somatic mutations. Thus, anti-Hib-PS antibodies can be divided into two types, the most frequently observed A2 antibodies with little or no somatic mutation and non-A2 antibodies that likely contain somatic mutations.     

The spatio-temporal expression pattern of cytoplasmic Cu/Zn superoxide dismutase (SOD1) mRNA during mouse embryogenesis

The cytoplasmic Cu/Zn-superoxide dismutase (SOD1) represents along with catalase and glutathione peroxidase at the first defense line against reactive oxygen species in all aerobic organisms, but little is known about its distribution in developing embryos. In this study, the expression patterns of SOD1 mRNA in mouse embryos were investigated using real-time RT-PCR and in situ hybridization analyses. Expression of SOD1 mRNA was detected in all embryos with embryonic days (EDs) 7.5–18.5. The signal showed the weakest level at ED 12.5, but the highest level at ED 15.5. SOD1 mRNA was expressed in chorion, allantois, amnion, and neural folds at ED 7.5 and in neural folds, notochord, neuromeres, gut, and primitive streak at ED 8.5. In central nervous system, SOD1 mRNA was expressed greatly in embryos of EDs 9.5–11.5, but weakly in embryos of ED 12.5. At EDs 9.5–12.5, the expression of SOD1 mRNA was high in sensory organs such as tongue, olfactory organ (nasal prominence) and eye (optic vesicle), while it was decreased in ear (otic vesicle) after ED 10.5. In developing limbs, SOD1 mRNA was greatly expressed in forelimbs at EDs 9.5–11.5 and in hindlimbs at EDs 10.5–11.5. The signal increased in liver, heart and genital tubercle after ED 11.5. In the sections of embryos after ED 13.5, SOD1 mRNA was expressed in various tissues and especially high in mucosa and metabolically active sites such as lung, kidney, stomach, and intestines and epithelial cells of skin, whisker follicles, and ear and nasal cavities. These results suggest that SOD1 may be related to organogenesis of embryos as an antioxidant enzyme.     

Ecophysiology of selected tree species in different plant communities at the periphery of the Atlantic Forest of SE—Brazil III. Three legume trees in a semi-deciduous dry forest

Three legume tree species (Fabaceae) occurring abundantly in a semi-deciduous tropical dry forest of the Atlantic forest complex in southeastern Brazil were subjected to a comparative ecophysiological study at the end of the dry season/beginning of the wet season. The trees chosen were morphologically very similar: Caesalpinia echinata Lam. and Caesalpinia ferrea Mart. ex. Tul., both 10–20 m of height, of the sub-family Caesalpinioideae, and the somewhat smaller, 2–4 m tall, Machaerium obovatum Kuhlm. & Hoehne of the sub-family Faboideae. Despite their similarities with respect to their geographic distribution restricted to Brazilian dry forests, their comparable abundance in the study site and their phylogenetic proximity, the three species display distinctly different ecophysiological behaviour. Compared to the other two species, C. ferrea had the highest photosynthetic capacity (maximum apparent photosynthetic electron transport rate, ETR_max) and higher saturation light-intensity, was less subject to photoinhibition as indicated by potential quantum yield of photosystem II (F _v/F _m) and had the lowest bulk N content of which soluble non-protein N compounds were only 1.5%. It showed stronger sun plant characteristics. C. echinata had lower photosynthetic capacity, was under chronic photoinhibition and had high bulk N content of which 6.1% were soluble N compounds with high concentrations of proline. In addition to proline, high concentrations of sugars may serve as osmoprotectants. M. obovatum also showed lower photosynthetic capacity and was under chronic photoinhibition. Here, arginine may have a function as osmoprotectant. The ecophysiological differences between the three species are not related to local abundance. However, the observations presented highlight a contrasting behaviour of the otherwise very similar compatriot species.     

Peptide mimotopes as prototypic templates of broad-spectrum surrogates of carbohydrate antigens.

Peptide mimetics of carbohydrate antigens can function as templates to exploit immune mechanisms to augment vaccine design strategies as they are T cell dependent antigens. In this study we evaluate a peptide mimetic (peptide 105) of the Pneumococcal capsular polysaccharide type 14 (Pn14) as a model antigen to explore differences in antigenicity and immunogenicity of peptide mimotopes. The multiple antigenic peptide (MAP) form, by ELISA, competes with native Pn14 in a concentration-dependent manner for binding to an anti-Pn14 monoclonal antibody. It was observed that peptide priming with a conjugated form (105-BSA) and boosting with Pn14 produced higher levels of Pn14-reactive IgG1, IgG2a, IgG2b and IgG3 than priming and boosting with Pn14. This serum also displayed reactivity with multiple serotypes, as assessed by ELISA. However, when compared with serum from humans immunized with the 23-valent pneumococcal vaccines, mimetic-induced mouse serum did not display a significant ability to mediate opsonophagocytic killing of pneumococci. These results suggest the feasibility of designing mimotopes to render effective humoral responses not only to a single serotype of Streptococcus pneumoniae, but to multiple serotypes at once. Such peptides would simplify currently available vaccine approaches, yet highlights the requirement of more extensive polymerization to fully emulate native antigen.     

Growth retardation and death of rice plants irradiated with carbon ion beams is preceded by very early dose- and time-dependent gene expression changes

The carbon-ion beam (CIB) generated by the heavy-ion medical accelerator in Chiba (HIMAC) was targeted to 7-day-old rice. Physiological parameters such as growth, and gene expression profiles were examined immediately after CIB irradiation. Dose-dependent growth suppression was seen three days post-irradiation (PI), and all the irradiated plants died by 15 days PI. Microarray (Agilent rice 22K) analysis of the plants immediately after irradiation (iai) revealed effects on gene expression at 270 Gy; 353 genes were up-regulated and 87 down-regulated. Exactly the same set of genes was affected at 90 Gy. Among the highly induced genes were genes involved in information storage and processing, cellular processes and signaling, and metabolism. RT-PCR analysis confirmed the microarray data.     

An interspecific (Capsicum annuum ×C. chinese) F2 linkage map in pepper using RFLP and AFLP markers

We have constructed a molecular linkage map of pepper (Capsicum spp.) in an interspecific F₂ population of 107 plants with 150 RFLP and 430 AFLP markers. The resulting linkage map consists of 11 large (206–60.3 cM) and 5 small (32.6–10.3 cM) linkage groups covering 1,320 cM with an average map distance between framework markers of 7.5 cM. Most (80%) of the RFLP markers were pepper-derived clones, and these markers were evenly distributed across the genome. By using 30 primer combinations, we were able to generate 444 AFLP markers in the F₂ population. The majority of the AFLP markers clustered in each linkage group, although PstI/MseI markers were more evenly distributed than EcoRI/MseI markers within the linkage groups. Genes for the biosynthesis of carotenoids and capsaicinoids were mapped on our linkage map. This map will provide the basis of studying secondary metabolites in pepper. Received: 20 October 1999 / Accepted: 3 July 2000