Intestinal gamma delta T cells develop in mice lacking thymus, all lymph nodes, Peyer's patches, and isolated lymphoid follicles

Through analysis of athymic (nu/nu) mice carrying a transgenic gene encoding GFP instead of RAG-2 product, it has recently been reported that, in the absence of thymopoiesis, mesenteric lymph nodes and Peyer's patches (PP) but not gut cryptopatches are pivotal birthplace of mature T cells such as the thymus-independent intestinal intraepithelial T cells (IEL). To explore and evaluate this important issue, we generated nu/nu mice lacking all lymph nodes (LN) and PP by administration of lymphotoxin-beta receptor-Ig and TNF receptor 55-Ig fusion proteins into the timed pregnant nu/+ mice that had been mated with male nu/nu mice (nu/nu LNP- mice). We also generated nu/nu aly/aly (aly, alymphoplasia) double-mutant mice that inherently lacked all LN, PP, and isolated lymphoid follicles. Although gammadelta-IEL were slightly smaller in number than those in nu/nu mice, substantial colonization of gammadelta-IEL was found to take place in the intestinal epithelia of nu/nu LNP- and nu/nu aly/aly mice. Notably, the population size of a major CD8alphaalpha+ gammadelta-IEL subset was maintained, the use of TCR-gamma-chain variable gene segments by these gammadelta-IEL was unaltered, and the development of cryptopatches remained intact in these nu/nu LNP- and nu/nu aly/aly mice. These findings indicate that all LN, including mesenteric LN, PP, and isolated lymphoid follicles, are not an absolute requirement for the development of gammadelta-IEL in athymic nu/nu mice.     

Cloning and characterization of integrin alpha subunits from the solitary ascidian, Halocynthia roretzi

Recent molecular and biochemical analysis has revealed the presence of an opsonic complement system in the solitary ascidian, Halocynthia roretzi, composed of at least C3, two mannan binding protein-associated serine proteases, and factor B. To elucidate further the structure and function of this apparently primitive complement system in the urochordates, we looked for the ascidian complement receptor type 3 (CR3), or type 4 (CR4), which are members of the leukocyte integrin family in mammals. Using degenerate primers, we isolated two integrin alpha subunits (alpha(Hr1) and alpha(Hr2)) from the hemocyte mRNA of H. roretzi, by RT-PCR, and the entire coding sequence of alpha(Hr1) was determined from cDNA clones. alpha(Hr1) contains an I domain, the inserted domain characteristic of a subset of mammalian alpha subunits, including the leukocyte integrin family. A phylogenetic tree constructed for the alpha subunits also supports the ancestral position of alpha(Hr1) in the monophyletic cluster of I domain-containing alpha integrins. The alpha(Hr1) gene shows hemocyte-specific expression on Northern blot analysis. Western blot analysis and immunocytochemical staining of the hemocytes of H. roretzi using anti-alpha(Hr1) Ab showed that alpha(Hr1) subunits exist on the surface of a subpopulation of phagocytic hemocytes. Furthermore, anti-alpha(Hr1) Ab inhibited C3-dependent phagocytosis, but not basic phagocytosis, of yeast cells by ascidian hemocytes. These observations strongly suggest that alpha(Hr1) constitutes an integrin molecule on the hemocytes of H. roretzi that functions as an ancestral form of CR3 and CR4 and mediates phagocytosis in the primitive complement system of the ascidian.     

Ex vivo survival of peripheral blood mononuclear cells in sheep induced by bovine leukemia virus (BLV) mainly occurs in CD5- B cells that express BLV

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis (EBL). In a previous report, we found that in a sheep model, only CD5(-) B cells proliferated clonally, while CD5(+) B cells rapidly decreased when the disease progressed to the lymphoma stage. We demonstrate here that, although both CD5(+) and CD5(-) B cells, but not CD4(+) T, CD8(+) T and gammadeltaTCR(+)T cells, are protected from spontaneous ex vivo apoptosis in sheep infected with wild-type and a mutant BLV that encodes a mutant Tax D247G protein with elevated trans-activation activity, only CD5(-) B cells become the main target for ex vivo survival when the disease proceeds to the persistent lymphocytotic stage, which showed an increased expansion of the CD5(-) B cells. In addition, we identified, by four-color flow cytometric analysis, that in CD5(-) B cells, the apoptotic rates of cells that expressed wild-type and mutant BLV were greatly decreased compared with those of BLV-negative cells. There was only a slight reduction in the apoptotic rates in BLV-positive cells from CD5(+) B cells. In addition, supernatants from peripheral blood mononuclear cell (PBMC) cultures from wild-type- and mutant BLV-infected sheep mainly protected CD5(-) B cells from spontaneous apoptosis. Our results suggest that, although BLV can protect both CD5(+) and CD5(-) B cells from ex vivo apoptosis, the mechanisms accounting for the ex vivo survival between these two B-cell subsets differ. Therefore, it appears that the phenotypic changes in cells that express CD5 at the lymphoma stage could result from a difference in susceptibility to apoptosis in CD5(+) and CD5(-) B cells in BLV-infected sheep.     

Senescent B lymphopoiesis is balanced in suppressive homeostasis: decrease in interleukin-7 and transforming growth factor-beta levels in stromal cells of senescence-accelerated mice

The suppression of the B cell population during senescence has been considered to be due to the suppression of interleukin-7 (IL-7) production and responsiveness to IL-7; however, the upregulation of transforming growth factor-beta (TGF-beta) was found to contribute to B cell suppression.To investigate the mechanism of this suppression based on the interrelationship between IL-7 and TGF-beta during senescence, senescence-accelerated mice (SAMs), the mouse model of aging, were used in this study to elucidate the mechanisms of B lymphopoietic suppression during aging. Similar to regular senescent mice, SAMs showed a decrease in the number of IL-7-responding B cell progenitors (i.e., colony-forming unit pre-B [CFU-pre-B] cells in the femoral bone marrow [BM]). A co-culture system of B lymphocytes and stromal cells that the authors established showed a significantly lower number of CFU-pre-B cells harvested when BM cells were co-cultured with senescent stromal cells than when they were co-cultured with young stromal cells. Interestingly, cells harvested from a senescent stroma and those from the control culture without stromal cells were higher in number than those harvested from a young stroma, thereby implying that an altered senescent stromal cell is unable to maintain self-renewal of the stem cell compartment. Because TGF-beta is supposed to suppress the proliferative capacity of pro-B/pre-B cells, we added a neutralizing anti-TGF-beta antibody to the co-culture system with a pro-B/pre-B cell-rich population to determine whether such suppression may be rescued. However, unexpectedly, any rescue was not observed and the number of CFU-pre-B cells remained unchanged when BM cells were co-cultured with senescent stromal cells compared with the co-culture with young stromal cells, which essentially showed an increase in the number of CFU-pre-B cells (P < 0.001 in 5 microg/ml). Furthermore, TGF-beta protein level in the supernatant of cultured senescent stroma cells was evaluated by enzyme-linked immunoabsorbent assay, but surprisingly, it was found that TGF-beta concentration was significantly lower than that of cultured young stromal cells. Thus, TGF-beta activity was assumed to decline particularly in a senescent stroma, which means a distinct difference between the senescent suppression of B lymphopoiesis and secondary B lymphocytopenia. Concerning proliferative signaling, on the other hand, the level of IL-7 gene expression in cells from freshly isolated BM decreased significantly with age. Therefore, the acceleration of proliferative signaling and the deceleration of suppressive signaling may both be altered and weakened in a senescent stroma (i.e., homeosuppression).     

ang is a novel gene expressed in early neuroectoderm, but its null mutant exhibits no obvious phenotype

To find genes that play roles in initial regionalization of anterior neuroectoderm, 15 novel genes were isolated that are expressed in anterior neuroectoderm at E8.0-E8.5. Moreover, to assess their functions by generation of mutant mice a conventional targeting strategy was designed, exploiting the availability of accurate long amplification PCR and BAC library that is coupled with genome information, in C57BL/6 strain. The ang is one of such genes; it has no known functional domains or no cognates, but is conserved not only in vertebrates, but also in Drosophila. Its expression was initially found throughout neuroectoderm at E7.5; subsequently the expression became high in rostral brain and caudal neuropore regions and low in hindbrain and spinal cord regions. At E12.5 the expression was found in undifferentiated neuroepithelium in ventricular zone, dorsal root ganglia and several non-neural tissues. However, ang null mutant was live-born without any apparent defects.     

Molecular chaperones facilitate the soluble expression of <Emphasis Type="Italic">N</Emphasis>-acyl-<Emphasis Type="SmallCaps">d</Emphasis>-amino acid amidohydrolases in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The overproduction of d-aminoacylase (d-ANase, 233.8 U/mg), N-acyl-d-glutamate amidohydrolase (d-AGase, 38.1 U/mg) or N-acyl-d-aspartate amidohydrolase (d-AAase, 6.2 U/mg) in Escherichia coli is accompanied by aggregation of the overproduced protein. To facilitate the expression of active enzymes, the molecular chaperones GroEL-GroES (GroELS), DnaK-DnaJ-GrpE (DnaKJE), trigger factor (TF), GroELS and DnaKJE or GroELS and TF were coexpressed with the enzymes. d-ANase (313.3 U/mg) and d-AGase (95.8 U/mg) were overproduced in an active form at levels 1.3- and 1.8-fold higher, respectively, upon co-expression of GroELS and TF. An E. coli strain expressing the d-AAase gene simultaneously with the TF gene exhibited a 4.3-fold enhancement in d-AAase activity (32.0 U/mg) compared with control E. coli expressing the d-AAase gene alone.     

Genetic variation in populations of the morphologically and ecologically variable fern Stegnogramma pozoi subsp. mollissima (Thelypteridaceae) in Japan

In Japanese Stegnogramma pozoi subsp. mollissima (Fisher ex Kunze) K. Iwats. there is the intrasubspecific variation among rbcL sequences. Northern and southern plants are genetically differentiated for maternally inherited cpDNA. In the present study we examined allozyme polymorphisms to test the hypothesis that northern and southern plants may be separate species. Based on allozyme data, the degree of gene flow among populations was estimated to be large. The artificial crossing experiments between cpDNA haplotypes also suggested that isolation has not developed among these cpDNA haplotypes. However, interpopulation genetic differentiation in cpDNA was observed even in the small area at the foot of Mt. Hakone, and the cpDNA haplotypes appear to have different habitat preferences.     

Cryptic species in the fern Ceratopteris thalictroides (L.) Brongn. (Parkeriaceae). I. Molecular analyses and crossing tests

For the taxonomic revision of the problematic species Ceratopteris thalictroides, molecular analyses and crossing tests were conducted for 16 sources in the world. An analysis of allozyme composition of five enzymes revealed the presence of three intraspecific entities, which were called the south type, the north type, and the third type. An analysis of the nucleotide sequences of chloroplast DNA also distinguished the same entities. Crossing tests showed that the south type was completely cross-sterile with the other two types, and that the other two were considerably cross-sterile with each other. These results suggest that the three entities should be regarded as different biological species. Although the south type and the other two meet in several regions, complete cross-sterility between them seems to sustain their genetic distinctiveness in spite of occasional crossing. The results from the present study suggest that widely distributed fern species are apt to comprise several cryptic species.     

Estimation of the highest chromosome number of eukaryotes based on the minimum interaction theory

According to the minimum interaction theory, the chromosome evolution of eukaryotes proceeds as a whole toward increasing the chromosome number. This raises the following two questions: what was the starting chromosome number of eukaryotes and does the chromosome number increase infinitely? We attempted to provide a theoretical framework to resolve these questions. We propose that the species with n=2 observed in Protozoa, Platyhelminthes, Annelid, Algae, Fungi and higher plants would be chromosomal relicts conserving the karyotypes of ancestral eukaryotes. We also propose that the ideal highest number of eukaryotes (n(max)) can be given by an inverse of the minimum terminal interference distance (It(min)) in crossing-over (n(max)=100/It(min)). AsIt(min) =0.6 in mammals, n(max) approximately 166. On the other hand, the value estimated by computer simulations is somewhat lower with n(max)=133-138. Our arguments can be applied to other eukaryotes, if they have a localized centromere and the ratio of total synaptonemal complex/nuclear volume is comparable to that of mammals. We revealed that the index of gene shuffling per karyotypes (G) by means of the total number of gamete types with different gene combinations can be formulated asG =2(n+Fxi), where Fxi means interstitial chiasma frequency per cell corresponding to crossing-over mediated by the recombination nodule. The Fxi value increases in proportion to the n value in areas where n<40, but decreases gradually when n>40 and becomes zero when n>83. Therefore, in the ultimate karyotype with n(max)=166, FXi=0 andG =2(n)=2(166), where gene shuffling is guaranteed by the random orientation of chromosomes at the equatorial plate of meiotic metaphase I.     

A Novel Theory on the Origin of the Genetic Code: A GNC-SNS Hypothesis

We have previously proposed an SNS hypothesis on the origin of the genetic code (Ikehara and Yoshida 1998). The hypothesis predicts that the universal genetic code originated from the SNS code composed of 16 codons and 10 amino acids (S and N mean G or C and either of four bases, respectively). But, it must have been very difficult to create the SNS code at one stroke in the beginning. Therefore, we searched for a simpler code than the SNS code, which could still encode water-soluble globular proteins with appropriate three-dimensional structures at a high probability using four conditions for globular protein formation (hydropathy, α-helix, β-sheet, and β-turn formations). Four amino acids (Gly [G], Ala [A], Asp [D], and Val [V]) encoded by the GNC code satisfied the four structural conditions well, but other codes in rows and columns in the universal genetic code table do not, except for the GNG code, a slightly modified form of the GNC code. Three three-amino acid systems ([D], Leu and Tyr; [D], Tyr and Met; Glu, Pro and Ile) also satisfied the above four conditions. But, some amino acids in the three systems are far more complex than those encoded by the GNC code. In addition, the amino acids in the three-amino acid systems are scattered in the universal genetic code table. Thus, we concluded that the universal genetic code originated not from a three-amino acid system but from a four-amino acid system, the GNC code encoding [GADV]-proteins, as the most primitive genetic code. Received: 11 June 2001 / Accepted: 11 October 2001