Quarterly individual outpatients lifestyle counseling after initial inpatients education on type 2 diabetes: the REDIA Prev-2 randomized controlled trial in Reunion Island.

AIMS: This study aimed to describe the 1-year evolution of type 2 diabetes (T2D) patients who attended inpatients education, and to assess whether quarterly outpatients counseling visits by nurses and dietitians can improve metabolic control and health-related behaviours. METHODS: Following in-hospital educational sessions, 398 adult T2D patients were randomized to either attend quarterly individual lifestyle counseling visits by a nurse and a dietitian (intervention group), or receive the usual care (control group). Primary (HbA(1c)) and secondary endpoints (fasting blood glucose, lipids, body mass index, waist circumference, fat mass, blood pressure, diet, physical activity) were assessed at baseline and at 12 months. RESULTS: HbA(1c) changes from baseline to 12 months were -1.74±2.64% (P<0.0001) for the intervention group and -2.02±2.57% (P<0.0001) for the control group. There was no statistically significant difference between the intervention group (n=153) and the controls (n=166) for any of the clinical and biological outcomes. In both groups, total energy and fat intakes decreased significantly from baseline levels. Also, no difference was found between the groups for any dietary outcome. A slight enhancement in sports activity was observed in the intervention group, but the difference between the two groups did not reach statistical significance, and no difference was found concerning any other physical activity scores. CONCLUSION: In this study of adults with T2D, patients significantly improved their metabolic control, and dietary and exercise habits, 1 year after receiving intensive inpatients education, whereas subsequent quarterly outpatients counseling visits with nurses and dietitians have not demonstrated any superiority compared with the usual care.

Microarray analysis reveals influence of the sesquiterpene lactone parthenolide on gene transcription profiles in human epithelial cells.

Sesquiterpene lactones are known for their anti-inflammatory activity which has been proven in various assays on DNA, mRNA and protein level. Here we report on the change in the gene expression profile in TNF-alpha stimulated human 293 cells after treatment with parthenolide using a cDNA microarray analysis. Twenty-one of 7028 genes were found to be up- and 18 down-regulated. They encode for chemoattractants, immune system proteins, glycoproteins, metabolism, serine proteinases, and transcription factors. Confirmatory analyses were carried out using quantitative real-time RT-PCR (TaqMan). Additional studies with selected genes revealed the concentration-dependent influence of parthenolide on the expression of these genes.

[Is HbA1c appropriate for the screening of diabetes in general pratice?]. / L'HbA1c peut-elle être utilisée par le praticien pour le dépistage du diabète?

The measurement of glycated haemoglobin (HbA(1c)) is a practical and more sensitive tool than fasting plasma glucose (FPG) in screening type 2 diabetes in current practice. Its use has been limited so far by the variability of the analytical methods. The standardization process is going on, and many laboratories are currently using valid methods. Our study is consistent with the results of other groups who recommended this measurement to identify undiagnosed diabetic patients, that are about 25% to 30% in the French population. The demonstration was provided through a survey including a screening step by both HbA(1c) and G0, and a second exam with a 2 hr OGTT in a sample of positive screenees according to at least one criterion (HbA(1c) >=6% or G0 >=1.26 g/L), as well as in a sample of negative screenees. We showed that nine confirmed diabetic subjects out of ten had HbA(1c) >=6% at the screening step, while only a half had G0 >=1.26 g/L. Conversely, 22% of the positive screenees according to HbA(1c) were not confirmed as diabetic by the OGTT, including however more than half with abnormal glucose values. A chart for practical use is proposed to define patients at risk, the process of screening, and the patient follow-up according to the results of the tests.

Modulation of interleukin production by ascorbic acid.

We studied the influence of ascorbate (vitamin C) on peripheral blood mononuclear cells (PBMC) of pigs with hereditary deficiency in ascorbate synthesis. Groups of animals were depleted of, or supplemented with dietary ascorbate for up to 5 weeks. B lymphocytes and T lymphocyte subsets differed in the two experimental groups only marginally and transiently as determined by analysis of cell surface markers. The proliferative response of PBMC to B and T lymphocyte mitogens was lower in depleted as compared to supplemented animals. Interleukin (IL)-2 and IL-6 were determined by bioassays and were secreted within few hours after mitogenic activation of PBMC which contained normal physiological concentrations of ascorbate. IL-2 production peaked at about 24 h of in vitro culture after Con A activation, but it lasted for 2-3 days after PWM activation. The production of IL-2 and IL-6 were compared during systemic depletion and supplementation with ascorbate. Depleted PBMC produced IL-2 which accumulated in cultures instead of being rapidly consumed by IL-2 dependent cell growth. This suggests that cellular ascorbate influences the production of IL-2. Secretion of IL-6 by mitogen activated PBMC was also affected by prolonged dietary ascorbate depletion. The results suggest that ascorbate levels exert an early effect on immune homeostasis via reactive oxygen intermediates (ROI)-dependent expression of interleukin genes, since the transcription factor NF-kappa B is sensitive to ROI and regulates the expression of interleukin genes.

Relation between the heavy chain complementarity region 3 characteristics and rheumatoid factor binding properties.

Among the rheumatoid factors (RFs), monospecific and polyspecific types can be distinguished. However the molecular basis responsible for their different specificity is not well understood. In a previous report, we have shown that the binding of the majority of the polyspecific antibodies is salt-sensitive. No binding to IgG was observed under high ionic strength (0.3-0.5 M NaCl). This salt-sensitivity was only observed for 18% of the monospecific RFs. Here, we have analyzed 14 RFs representing the 3 different groups (6 salt-insensitive monospecific, 4 salt-sensitive monospecific and 4 salt-sensitive polyspecific RFs). By analysis of the amino acid composition and the distribution of polar and non-polar residues of their heavy chain complementarity-determining region 3 (H-CDR3) in relation to mono/polyspecificity, salt-sensitivity and reactivity against human IgG subclasses, we have identified common structural features responsible for their different binding properties. Salt-sensitive RFs (mono as well as polyspecific antibodies) were characterized by long H-CDR3's (15.3+/-2.7) that contained large numbers of hydrophilic residues such as arginine and serine, while salt-insensitive RFs had more hydrophobic H-CDR3's of smaller length (11.3+/-2.4). In addition, for the monospecific RFs, remarkably similar hydrophilicity H-CDR3 profiles were found that were correlated with their specificity for IgG subclasses. These observations confirm the importance of the H-CDR3 for the binding of RFs to IgG. Furthermore, on the basis of their shorter H-CDR3's and their rather unique H-CDR3 hydrophilicity profiles, it is likely that the majority of the monospecific RFs should be considered as a group of RFs that is independent of the polyspecific RF repertoire.

Dependence of growth, bone metabolism and functions of polymorphonuclear leukocytes on ascorbic acid in pigs.

Pigs with hereditary ascorbate deficiency (OD pigs) were depleted of, or supplemented with, ascorbic acid by respective diets. Depletion of young (i.e. 5-7 weeks old) animals for at least three weeks had a negative effect on growth, body temperature and levels of bone alkaline phosphatase and induced symptoms of scurvy. Doses of 5 mg ascorbic acid kg-1 body weight day-1 were sufficient to reverse these effects. The level of ascorbic acid sharply decreased in plasma within one week of depletion, whereas in leukocytes it declined more slowly and to a lower extent. Bone alkaline phosphatase levels substantially declined in ascorbic acid depleted animals. Supplementation with > 100 mg ascorbic acid kg-1 body weight day-1 did not improve growth. Dietary ascorbic acid was absorbed from the intestinal lumen into the blood within less than 1 hour and reached a peak 5-6 hours after the meal. The extent of this absorption depended on the systemic ascorbic acid level. Ascorbic acid influenced leukocyte function, since the production of reactive oxygen intermediates by polymorphonuclear leukocytes decreased in supplemented animals. Thus, this animal model permits to establish the level of dietary ascorbic acid that is critical for growth of pigs as well as to study its absorption into the blood and the associated alterations in polymorphonuclear leukocytes and bone metabolism.

Microsites for immunoglobulin switch recombination breakpoints from Xenopus to mammals.

Immunoglobulin (Ig) heavy chain class switch recombination has been studied at the DNA level in a non-mammalian vertebrate, the amphibian Xenopus. A switch (S) region of about 5 kb has been identified in the JH-C mu intron of the Ig heavy chain locus in Xenopus. S mu contains 23 repeats approximately 150 bp long. Each repeat consists of internal shorter repeats and palindromic sequences, such as AGCT, which they share with mammalian switch regions. A deletion of the mu gene and the joining of the S regions of mu and chi occurs in B cells expressing IgX, one of the two non-mu isotypes in Xenopus. S chi shows no sequence homology with S mu and is characterized by 16 and 121 bp repeats and a high frequency of CATG, AGCA and TGCA palindromes. Both IgM and IgX S regions are AT rich and not GC rich like mammalian S regions. Recombination occurs, most of the time, at positions (microsites) where a single-stranded DNA folding program predicts the transition from a stem to a loop structure. This feature is conserved in most mammalian switch junctions which points to the general existence and involvement of microsites at one step of the determination of the recombination break-point. The recombinogenic nature of the switch regions is therefore linked to its structure rather than to its base composition, the repetitive occurrence of palindromes being essential at creating many microsites.

Maturation of the mitogen responsiveness, and IL2 and IL6 production by neonatal swine leukocytes.

The maturation of the immune system of neonatal piglets was studied by following changes in the phenotypic composition and function of blood-borne leukocytes. The proportion of mature T and B lymphocytes decreased in the first week of birth and the circulating cells had poorly developed capacities to respond to mitogens and to secrete interleukins. From the end of the first week, however, there was a steady increase in the proportion of mature T cells (CD4+ and CD8+) and B cells in blood until 6-7 weeks after birth, when the study was ended. By 3-4 weeks, the relative proportions of different lymphocyte subsets resembled an adult-type composition. As they increased in prevalence, lymphocytes also developed capacities to proliferate and secrete interleukins. Proliferative responses to T-cell and B-cell mitogens reached adult levels within 2 weeks and 4-5 weeks, respectively. Blood leukocytes produced large quantities of IL6 by 1-2 weeks after birth and IL2 by 2-3 weeks. In contrast to lymphocyte patterns, the myeloid and granulocyte lineages were dominant at birth but then declined steadily. Unlike lymphocytes, the monocytes, macrophages and granulocytes appeared to be fully functional from the time of birth and exhibited a strong oxidative burst after appropriate stimulations. The magnitude of this response remained constant over the first 6-7 weeks. These results indicate that the first 3-4 weeks of post-natal life are a particularly susceptible interval for newborn piglets because constitutive and functional components necessary for specific cellular immune responses remain immature. This deficit may be offset by non-specific cellular mechanisms and maternally derived antibodies.

Influence of ascorbic acid on the response to mitogens and interleukin production of porcine lymphocytes.

The influence of ascorbic acid (AA) on lymphocyte functions was examined in vitro and ex vivo in peripheral blood mononuclear cells (PBMC) of vitamin C-deficient pigs, which are unable to synthesise ascorbic acid. AA is accumulated to physiological levels in PBMC in vitro. The cell proliferation induced by T lymphocyte mitogens was unaltered at all AA concentrations tested (0-400 micrograms/ml, i.e., 0-2.3 mM). Conversely, the response to pokeweed mitogen (PWM) which activates T and B lymphocytes was significantly reduced with increasing intracellular and extracellular AA concentrations. The response to lipopolysaccharide (LPS) showed a tendency to increase at low (9 microM) and was significantly reduced at high AA concentrations (> 36 microM). The IL2 production induced by PWM (but not by concanavalin A (Con A) or phytohemagglutinin (PHA)) decreased at high AA (> 142 microM). In contrast, IL6 production induced by mitogens was not dependent on AA concentrations. In concordance with these results, AA-depleted PBMC which were obtained from pigs that were fed an AA-free diet, displayed an increasing response to LPS and PWM. Collectively, the data indicate that ascorbic acid selectively influences the proliferation of B lymphocytes and negatively acts on IL2 production by T lymphocytes when a threshold of saturation is exceeded.

Comparison of rheumatoid factors of rheumatoid arthritis patients, of individuals with mycobacterial infections and of normal controls: evidence for maturation in the absence of an autoimmune response.

We analyzed the rheumatoid factors (RF) produced by Epstein-Barr virus-transformed monoclonal B cells established from four patients with rheumatoid arthritis (RA), three individuals with a history of Mycobacterium tuberculosis (TB) and four normal controls (NI). Fifty-eight RF were analyzed for specific activity (international units-RF/microgram) for the Fc part of IgG and their interaction with tetanus toxoid (TT) and DNA (polyspecificity). Furthermore, we sequenced the V-D-J heavy chain region of 16 (9TB-/7RA-) RF. Significant differences were observed between the NI-RF and the TB- and RA-RF. While the RF repertoire of normal individuals comprised of low-avidity RF of which the majority (15/17) were polyspecific, more than half of the TB- and RA-RF were monoreactive. Furthermore, the monospecific TB- and RA-RF were of significantly higher avidity than the NI-RF (RA > TB > > NI). With respect to polyspecificity specificity, the RF in the three groups were comparable: the interaction with DNA, TT as well as with Fc was inhibited either by an increase of the ionic strength to 0.3-0.5 M NaCl or by addition of the polyanion dextran sulfate, indicating that the antibodies interacted with similar anionic epitopes shared by the three antigens. Analysis of the V-D-J heavy chain regions showed significant differences between the respective RF. The salt-sensitive binding was highly correlated with the presence of arginine in the complementarity-determining region 3 (CDR3). Furthermore, whereas the polyspecific RF consisted predominantly of germ-line encoded antibodies, the genes of the monospecific RA/TB-RF were somatically mutated (RA > TB). It is therefore likely that maturation of RF can be initiated by chronic infections and that monospecific, somatically mutated RF are not a unique characteristic of autoimmune diseases.

[Clinical, biological and developmental aspects of alcoholic ketoacidosis]. / Aspects cliniques, biologiques et évolutifs de l'acidocétose alcoolique.

OBJECTIVES: To review cases of alcoholic ketoacidosis in order to better ascertain therapeutic management. METHODS: The medical files of 32 alcoholic patients with ketoacidosis hospitalized in the Saint-Pierre general hospital of the Reunion island from January 1, 1991 through 31 August 1994 were analyzed. RESULTS: There were 18 women and 14 men, mean age 47 years. The first clinical signs were predominated by digestive (n = 22) or neurological disorders (n = 10). Acidosis was severe (mean pH = 7.12) and always associated with a wide anion gap (mean anion gap = 35). There were 3 types of glycemic status: hypoglycemia 10 cases, normal or subnormal glycemia in 19 cases (mean glycemia = 9.3 mmol/l) and hyperglycemia above 20 mmol/l in 3 cases. Hypophosphatemia, elevated serum lactate levels and cytolytic hepatitis were the main abnormalities associated. CONCLUSION: Short-term outcome was favorable in all cases after rehydration. The use of insulin may be dangerous and needs to be avoided.

The chicken beta 2-microglobulin gene is located on a non-major histocompatibility complex microchromosome: a small, G+C-rich gene with X and Y boxes in the promoter.

beta 2-Microglobulin is an essential subunit of major histocompatibility complex (Mhc) class I molecules, which present antigenic peptides to T lymphocytes. We sequenced a number of cDNAs and two genomic clones corresponding to chicken beta 2-microglobulin. The chicken beta 2-microglobulin gene has a similar genomic organization but smaller introns and higher G+C content than mammalian beta 2-microglobulin genes. The promoter region is particularly G+C-rich and contains, in addition to interferon regulatory elements, potential S/W, X, and Y boxes that were originally described for mammalian class II but not class I alpha or beta 2-microglobulin genes. There is a single chicken beta 2-microglobulin gene that has little polymorphism in the coding region. Restriction fragment length polymorphisms from Mhc homozygous lines, Mhc congenic lines, and backcross families, as well as in situ hybridization, show that the beta 2-microglobulin gene is located on a microchromosome different from the one that contains the chicken Mhc. We propose that the structural similarities between the beta 2-microglobulin and Mhc genes in the chicken are due to their presence on microchromosomes and suggest that these features and the microchromosomes appeared by deletion of DNA in the lineage leading to the birds.

A second immunoglobulin light chain isotype in the rainbow trout.

A novel immunoglobulin (Ig) light chain isotype, termed IgL2, has been isolated from trout lymphoid tissues both by reverse transcription - polymerase chain reaction (PCR) and screening of cDNA libraries. The CL domain of the new isotype shares only 29% residues with a recently cloned trout IgL isotype, termed IgL1, which has some similarities to Ckappa and Clambda isotype domains of several vertebrate species. Using anchored PCR, a VL element rearranged to CL2 was isolated. It is a member of a new VL family (VL2) of which four members were sequenced. These differ in the sequence of CDR1 and CDR2 but are remarkably similar in CDR3, i. e., at the junction between VL and JL segments. VL elements are rearranged to novel JL elements which differ from those described for VL1-CL1 rearrangements. Two cDNA clones contained JL-CL2 segments but no VL segments. The JL segments were preceded by typical rearrangements signal sequences [RSS, nonamer-23 base pair (bp) spacer-heptamer]. Further upstream of RSS were located two to three near identical 53 bp repeats, each of which included a 16 bp sequence similar to KI and KII sequences located at similar places in human and mouse Jk1 genes. These sequences are believed to act as binding sites for the protein KLP, which could be a transcriptional factor involved in the synthesis of germline Jk transcripts. Their phylogenic conservation in vertebrates suggests that they have an important role in B-cell differentiation. Remarkably, an RNA species of about 0.7 kilobase is the predominant IgL mRNA in trout spleen and coincides in size with JLCL2 transcripts. Genomic DNA blot analysis indicates that the trout L2 locus has a cluster-like organization similar to the trout L1 locus and the IgL locus of several teleost fish. A phylogenic analysis of VL2 and CL2 corroborates their low similarity to other vertebrate IgL chains and suggests an ancient diversification of the IgL locus.

Ontogeny of TCR V beta 1 expression revealed novel invariant alternative transcripts.

Analysis of TCR-beta gene recombination and expression was performed by quantitative PCR amplification technique throughout chicken embryogenesis and development. Our data demonstrated that TCR V beta 1 promoters were turned on by day 10 of embryogenesis, 2 days before detection of TCR-beta gene recombination. The V to D recombination step was first detected by day 11 of embryogenesis whereas DJ and V(D)J rearranged genes were detected 1 day later, on day 12 of embryogenesis. Thus, transcription of unrearranged TCR-beta genes in chickens precedes the expression of V(D)J recombinase activity as in mammals. In contrast, although TCR-beta rearrangement starts with the D to J recombination step in mammals, it can start either by the VD or the DJ step in chickens. Furthermore, reverse transcriptase-PCR amplification of TCR-beta transcripts revealed the presence of two kinds of alternative transcripts. These novel alternatively spliced products appeared in thymocytes from embryonic thymus during colonization periods and were absent in transformed T cell lines. Splicing sites are located in the middle of V beta 1 segments and lead to delta V beta 1-C beta and delta V beta 1-D beta-J beta-C beta transcripts. delta V beta 1-C beta transcripts might lead to synthesis of invariant truncated TCR beta-chains containing the aminoterminal portion of the V beta 1 region followed by the C beta region. Because this type of splicing can be generated by using all known V beta 1 members, these invariant forms could play a role in thymocyte development.

The third component of Xenopus complement: cDNA cloning, structural and functional analysis, and evidence for an alternate C3 transcript.

Although the third component of complement has been purified from two amphibian species, Xenopus laevis and the axolotl, only limited information is available about its primary structure in these species. We now present (a) 95% of the cDNA sequence encoding C3 from a Xenopus laevis/Xenopus gilli (Xenopus LG) hybrid (b) an analysis of the C3 convertase and factor I cleavage sites in Xenopus C3, and (c) evidence for an alternative form of C3. The Xenopus LG sequence has a 57% nucleotide and 52% amino acid sequence identity to human C3 and contains one potential N-glycosylation site in the beta-chain. The deduced amino acid sequence showed that the C3 convertase and factor I cleavage sites (Arg-Ser) are conserved in Xenopus C3 and protein sequencing of Xenopus C3 fragments fixed on zymosan during complement activation demonstrated that Xenopus C3 is indeed cleaved by C3 convertase and factor I at these sites. Our screening of a liver cDNA library identified an unusual C3 clone with a deletion of 2502 bp, suggesting the presence of a novel C3 transcript in Xenopus LG liver. The presence of this C3 transcript was confirmed by reverse transcription polymerase chain reaction using Xenopus LG liver mRNA and specific oligonucleotide probes. This transcript encoded a putative 102-kDa protein comprising the beta-chain of C3, together with the first 59 residues and the last 103 residues of the alpha-chain; it would therefore lack many of the ligand binding sites found in the intact alpha-chain. However, the molecule may be an analog of a truncated C3 molecule that is found in the serum of allergic dermatitis patients and acts as an inhibitor of eosinophil cytotoxicity and neutrophil adherence.

T cell migration during development: homing is not related to TCR V beta 1 repertoire selection.

T cell precursors enter the chick thymus in three waves during embryonic life. Each wave of thymocyte precursors colonizing the thymus gave rise to a similar TCR V beta repertoire in thymus, spleen and intestine both in terms of V beta 1 and J beta usage as well as in the length of V beta-D beta-J beta junctions. Seventeen V beta 1s were utilized, and a new J beta segment was found. In the progeny of the third wave, more nucleotides were deleted at the 5' end of the J beta segment, but the overall size of the CDR3 was conserved by a concomitant increase of N nucleotide addition at the V beta-D beta-J beta junctions during rearrangement. This CDR3 modification was observed in the spleen but not in the intestine, implying that progeny of the third wave migrate preferentially to the spleen, a possibility that was confirmed by adoptive cell transfers into congenic chickens. Very low frequencies of non-productive rearrangements in the intestine suggested that negative selection may occur in this organ. The present analysis indicates that V beta 1+ T cells in spleen and intestine are primarily of thymic origin, this colonization of both organs occurs in waves and is not characterized by preselection of the TCR V beta 1 repertoire.

Phylogeny of immunoglobulin heavy chain isotypes: structure of the constant region of Ambystoma mexicanum upsilon chain deduced from cDNA sequence.

An RNA polymerase chain reaction strategy was used to amplify and clone a cDNA segment encoding for the complete constant part of the axolotl IgY heavy (C upsilon) chain. C upsilon is 433 amino acids long and organized into four domains (C upsilon 1-C upsilon 4); each has the typical internal disulfide bond and invariant tryptophane residues. Axolotl C upsilon is most closely related to Xenopus C upsilon (40% identical amino acid residues) and C upsilon 1 shares 46.4% amino acid residues among these species. The presence of additional cysteines in C upsilon 1 and C upsilon 2 domains is consistent with an additional intradomain S-S bond similar to that suggested for Xenopus C upsilon and C chi, and for the avian C upsilon and the human C epsilon. C upsilon 4 ends with the Gly-Lys dipeptide characteristic of secreted mammalian C gamma 3, human C epsilon 4, and avian and anuran C upsilon 4, and contains the consensus [G/GT(AA)] nucleotide splice signal sequence for joining C upsilon 4 to the transmembrane region. These results are consistent with the hypothesis of an ancestral structural relationship between amphibian, avian upsilon chains, and mammalian epsilon chains. However, these molecules have different biological properties: axolotl IgY is secretory Ig, anuran and avian IgY behave like mammalian IgG, and mammalian IgE is implicated in anaphylactic reactions.

Evolution of vertebrate IgM: complete amino acid sequence of the constant region of Ambystoma mexicanum mu chain deduced from cDNA sequence.

cDNA clones coding for the constant region of the Mexican axolotl (Ambystoma mexicanum) mu heavy immunoglobulin chain were selected from total spleen RNA, using a cDNA polymerase chain reaction technique. The specific 5'-end primer was an oligonucleotide homologous to the JH segment of Xenopus laevis mu chain. One of the clones, JHA/3, corresponded to the complete constant region of the axolotl mu chain, consisting of a 1362-nucleotide sequence coding for a polypeptide of 454 amino acids followed in 3' direction by a 179-nucleotide untranslated region and a polyA+ tail. The axolotl C mu is divided into four typical domains (C mu 1-C mu 4) and can be aligned with the Xenopus C mu with an overall identity of 56% at the nucleotide level. Percent identities were particularly high between C mu 1 (59%) and C mu 4 (71%). The C-terminal 20-amino acid segment which constitutes the secretory part of the mu chain is strongly homologous to the equivalent sequences of chondrichthyans and of other tetrapods, including a conserved N-linked oligosaccharide, the penultimate cysteine and the C-terminal lysine. The four C mu domains of 13 vertebrate species ranging from chondrichthyans to mammals were aligned and compared at the amino acid level. The significant number of mu-specific residues which are conserved into each of the four C mu domains argues for a continuous line of evolution of the vertebrate mu chain. This notion was confirmed by the ability to reconstitute a consistent vertebrate evolution tree based on the phylogenic parsimony analysis of the C mu 4 sequences.

The ontogeny of diversification at the immunoglobulin heavy chain locus in Xenopus.

Since the larval and adult antibody responses are distinct and restricted in the clawed toad Xenopus, it offers a near ideal model for studying the ontogeny of antibody repertoires and the mechanisms involved. Immunoglobulin heavy chain (IgH) cDNA clones and B cell IgH DNA clones from various larval and adult libraries have been analysed in isogenic Xenopus. Some features are similar in adults and tadpoles, while others differ and explain the particularities observed previously at the protein level. Among the similarities we found are: (i) the mode of rearrangements (there are approximately 50% abortive events in B cells from both stages), (ii) VH family usage (10 of 11 known VH families are expressed proportionally to the number of VH elements per family), and (iii) JH usage (of the eight to nine Xenopus JH elements, two are used in approximately 70% of the VH regions in both stages of development). We found that there is relatively higher membrane exon expression in tadpoles compared with adults; and that most of the differences come from the diversification of CDR3 through DH usage and N diversification. Unlike in mammals, Xenopus DH elements are used with a remarkable flexibility with inversion, fusions and usage in different reading frames, but tadpoles show a strong bias for the usage of only a few DH elements and of a preferred reading frame. There is N diversification, which further increases CDR3 heterogeneity, in adult Xenopus but virtually none in tadpoles. These observations can account for the fact that larval antibody responses are less heterogeneous than those of adults.

Sequence of C region of L chains from Xenopus laevis Ig.

A cDNA expression library, prepared from Xenopus laevis splenocytes, was screened with antibodies to Xenopus Ig. One clone, lambda XIg23, reacted with antibodies to IgY and to IgM; the insert hybridized to approximately 1.3-kb RNA from spleen, the approximate size expected for L chain mRNA. An additional clone, lambda XIg31, was identified by cross-hybridization. The inserts of lambda XIg23 and lambda XIg31 begin in the third framework region of the V region and extend through the C region to the poly(A) tail. Except for a single nucleotide difference, the two C region sequences are identical. The amino acid sequence of the C region was compared with the sequences of a variety of C kappa and C lambda, as well as to C region sequences of L chains from Rana catesbeiana and from two species of shark. The Xenopus C region resembles mouse and human C kappa slightly more than C lambda. The similarity of the Xenopus and Rana C regions to each other is approximately the same as that of either amphibian sequence to mammalian CL. The data are discussed in terms of the evolution of kappa and lambda C regions.