IL 2 restores memory B cell activation by antigen-specific T cell clone variants

It has recently been demonstrated that there are at least two separate pathways by which a single keyhole limpet hemocyanin (KLH) reactive T cell clone can induce B cell differentiation. With the use of the high-dose antigen-driven system (10 micrograms/ml trinitrophenyl (TNP)-KLH), a KLH-specific T cell clone was able to induce a primary anti-TNP response in unprimed B cells. In the presence of aliquots of the same T cell clone, a low-dose of antigen (5 X 10(-2) micrograms/ml TNP-KLH) induced an immunoglobulin (Ig)G response in primed B cells. It has also been demonstrated that there are variant subclones of such KLH-specific helper T cell clones that are unable to provide antigen-specific help in the presence of low-dose antigen but maintain the high-dose antigen-driven helper response. This study was undertaken to investigate whether interleukin 2 (IL 2) had some activity in the low-dose, antigen-driven response induced by the T cell clone. With the use of a variant T cell clone (which lost low-dose, antigen-driven helper activity), it was demonstrated that IL 2 was capable of reconstituting the low-dose, antigen-driven helper activity. To investigate whether accessory cells were required in this system, we removed the adherent cell population from the primed spleen cells added to culture. Interestingly, removal of the G10-adherent cells eliminated the low-dose, antigen-driven response induced by IL 2. Additionally by add-back experiments, we were able to demonstrate that the necessary adherent cell population did not require major histocompatibility complex (MHC) restriction for reconstitution of the IL 2-dependent, low-dose, antigen-driven response. Furthermore, 1% concanavalin A (Con A) supernatant (Sn), but not interleukin 1 (IL 1), could replace this adherent cell function. These data suggest that in this system, IL 2 bypasses the MHC-restricted interaction between T cells and antigen-charged adherent cells; B cells can present antigen to cloned helper T cells efficiently for primary responses but need an added factor(s) to induce IgG production; and adherent cells are essential for IgG production in primed B cells, possibly through the release of soluble factor(s) included in Con A Sn.     

CRF-immunoreactive nerve fibers in the circumventricular organs of the monkey,Macaca fuscata

Summary The occurrence of CRF (corticotropin-releasing factor)-immunoreactive nerve fibers in the circumventricular organs of adult male monkeys, Macaca fuscata, was studied on serially sectioned brains, by means of the peroxidase-antiperoxidase technique in combination with a highly specific and sensitive CRF antiserum. CRF-containing nerve fibers were found in high concentrations in the infundibulum and, in addition, in small numbers in the posterior lobe, organum vasculosum laminae terminalis, subfornical organ, and area postrema; they were missing in the pineal body and the subcommissural organ. The CRF immunoreactive nerve fibers distributed in these organs were located in the proximity of the blood vessels.Supported by a grant (No. 56440022) from the Ministry of Education, Science, and Culture, Japan     

Endogenous retroviral LTR DNA sequences as markers for individual human chromosomes.

The human genome carries multiple copies of sequences related to endogenous retroviral genomes that include long terminal repeat (LTR) sequences. We used the LTR of one such viral genome, called HERV-A, as a probe in Southern analysis to examine the distribution profiles of the hybridizing DNA in the genomes of twelve human x rodent hybrid cell lines carrying one or a few human chromosomes, and in the DNA samples prepared from six sorted, individual chromosomes. The HERV-A sequence was found to be widely distributed among different chromosomes and the Southern patterns for chromosomes 5, the X, and the Y, each obtained in duplicate from independently prepared cell lines or sorted chromosomes, were matched. Chromosome-specific Southern profiles can be used to monitor chromosomes in hybrid cells or to characterize chromosome aberrations, such as deletions.     

Anatomic and chromosomal anomalies in 639 spontaneous abortuses

Summary A total of 639 spontaneous abortuses collected in a maternity hospital were set up in culture. This sample included 565 unselected consecutive abortuses and 74 selected abortuses ascertained by morphology and/or clinical history. Among these, 339 were incomplete specimens with no recovered embryo or fetus, 110 were anatomically apparently normal and 190 were grossly abnormal. In the unselected series, 565 specimens were cultured and 402 were karyotyped; 215 (53.5%) were chromosomally abnormal. In the selected series, 74 specimens were set up in culture and 45 were karyotyped; 26 (57.7%) had an abnormal karyotype. In all, successful karyotyping was done on 447 abortuses (70%), of which 339 were studied with banding. One or more major chromosome abnormalities were detected in 241 (54%) of the karyotyped cases, 230 of which were numerical anomalies and 11 structural anomalies. Numerical anomalies included primary autosomal trisomies (31% of the total karyotyped), 45,X (10%), triploidy (6.5%), and tetraploidy (1.8%). Of the 22 types of autosomal trisomies possible, all except those for 1, 5, 17, and 19 were identified. An abortus with a 49,XX,+2,+5,+8 karyotype was detected. The excess autosomal material present in the triple trisomic abortus corresponded to 10% of the haploid autosomal complement. Of the 11 abortuses with structural abnormalities, seven were inherited while the other four were sporadic. The survival rate of 45,X conceptuses was estimated to be one in 300.     

Pyrrolinone derivatives as a new class of P2X3 receptor antagonists Part 2: Discovery of orally bioavailable compounds

Some P2X3 receptor antagonists have been developed as new therapeutic drugs for pain. We discovered a novel chemotype of P2X3 receptor antagonists with a pyrrolinone skeleton. Because of SAR studies to improve bioavailability of lead compound 2, compound (R)-24 was identified, which showed an analgesic effect against neuropathic pain by oral administration. We constructed a human P2X3 homology model as a template for the zebrafish P2X4 receptor, which agreed with SAR studies of pyrrolinone derivatives.     

Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses

2-Hydroxy fatty acids (2-HFAs) are predominantly present in sphingolipids and have important physicochemical and physiological functions in eukaryotic cells. Recent studies from our group demonstrated that sphingolipid fatty acid 2-hydroxylase (FAH) is required for the function of Arabidopsis (Arabidopsis thaliana) Bax inhibitor-1 (AtBI-1), which is an endoplasmic reticulum membrane-localized cell death suppressor. However, little is known about the function of two Arabidopsis FAH homologs (AtFAH1 and AtFAH2), and it remains unclear whether 2-HFAs participate in cell death regulation. In this study, we found that both AtFAH1 and AtFAH2 had FAH activity, and the interaction with Arabidopsis cytochrome b₅ was needed for the sufficient activity. 2-HFA analysis of AtFAH1 knockdown lines and atfah2 mutant showed that AtFAH1 mainly 2-hydroxylated very-long-chain fatty acid (VLCFA), whereas AtFAH2 selectively 2-hydroxylated palmitic acid in Arabidopsis. In addition, 2-HFAs were related to resistance to oxidative stress, and AtFAH1 or 2-hydroxy VLCFA showed particularly strong responses to oxidative stress. Furthermore, AtFAH1 interacted with AtBI-1 via cytochrome b₅ more preferentially than AtFAH2. Our results suggest that AtFAH1 and AtFAH2 are functionally different FAHs, and that AtFAH1 or 2-hydroxy VLCFA is a key factor in AtBI-1-mediated cell death suppression.Sphingolipids are a large class of lipids present ubiquitously in eukaryotic cells and involved in various cellular processes such as signal transduction, protein transport, and programmed cell death as structural components of the plasma membrane and tonoplast and as signaling molecules (Dunn et al., 2004; Pata et al., 2010). These numerous functions are based on the structural diversity of sphingolipids. Complex sphingolipids are largely formed by a variety of head groups and a ceramide, which is comprised of a long-chain base amide linked to a fatty acid. One of the main characteristics of sphingolipid fatty acids is that large number of sphingolipids contained very-long-chain fatty acids (VLCFAs; fatty acids with ≥ C20; Raffaele et al., 2009). Another feature is the 2-hydroxylation of fatty acids (Alderson et al., 2005). The 2-hydroxy fatty acid (2-HFA)-containing sphingolipids are present in most organisms, including yeast (Saccharomyces cerevisiae), vertebrates, and plants. Especially in higher plants, 2-HFAs are contained in more than 90% complex sphingolipids such as glucosylceramides and glycosylinositolphosphorylceramides (Imai et al., 1995; Pata et al., 2010).The 2-hydroxylation of sphingolipid N-acyl chains is catalyzed in yeast and mammals by the enzyme sphingolipid fatty acid 2-hydroxylase (ScFAH1 and FA2H, respectively, and we refer to them as FAH in this report; Mitchell and Martin, 1997; Alderson et al., 2004; Hama, 2010). FAH is an endoplasmic reticulum (ER)-localized membrane protein composed of two domains: an N-terminal cytochrome b₅ (Cb5)-like domain and a putative catalytic site with the His motif (HXXHH), which coordinates the nonheme diiron cluster at the active site of the enzyme and is highly conserved among membrane-bound desaturases and hydroxylases. Mammalian FA2H is reported to catalyze the 2-hydroxylation of free fatty acid in vitro, which was dependent on a reconstituted electron transport system (Alderson et al., 2005). In mammals, FA2H-synthesized 2-HFAs abundantly accumulate in epidermal keratinocytes and the myelin-forming cells of the nervous system (Alderson et al., 2006; Uchida et al., 2007; Maldonado et al., 2008). Loss of FA2H in mouse altered the composition and physicochemical properties of sebum, causing alopecia (Maier et al., 2011). In addition, FA2H deficiency also causes a wide range of neurodegeneration phenotypes such as spastic paraplegia, leukodystrophy, and brain ion deposition (Schneider and Bhatia, 2010). Moreover, FA2H is a negative regulator of the cell cycle and facilitates dibutyryl-cyclic AMP-induced cell cycle exit in D6P2T schwannoma cells (Alderson and Hama, 2009). These studies suggest that 2-hydroxylation of fatty acids is important in various cellular functions in mammals. However, there had been few reports about plant FAHs in the past.Recently, we suggested that FAH could mediate the function of Bax inhibitor-1 (BI-1), which is an ER membrane-localized cell death suppressor widely conserved in animals and plants (Xu and Reed, 1998; Kawai-Yamada et al., 2001; Nagano et al., 2009). Arabidopsis (Arabidopsis thaliana) BI-1 (AtBI-1) plays important roles in the resistance to various biotic and abiotic stresses including pathogens, ER stress, ion stress, and oxidative stress generating reactive oxygen species (ROS; Kawai-Yamada et al., 2001, 2004, 2009; Watanabe and Lam, 2006, 2008; Ihara-Ohori et al., 2007; Ishikawa et al., 2010). Our recent work indicated that AtBI-1 interacted with ScFAH1, and that deletion of ScFAH1 prevented AtBI-1 from suppression of cell death induced by mammalian proapoptotic protein, Bax, in yeast cells (Nagano et al., 2009). In Arabidopsis, there are two highly identical FAH homologs, AtFAH1 and AtFAH2, which possess several His motifs like their yeast and mammalian counterparts. However, AtFAH1 and AtFAH2 lack a Cb5-like domain, which provides electrons to ScFAH1 and FA2H (Mitchell and Martin, 1997; Alderson et al., 2004; Nagano et al., 2009). Instead, we demonstrated the interaction between both AtFAHs and Arabidopsis Cb5s (AtCb5s), which are localized in the ER membrane or mitochondrial outer membrane, by using a split-ubiquitin yeast two-hybrid system. In addition, AtBI-1 also interacted with AtCb5s. Moreover, overexpression of AtBI-1 increased the amounts of 2-hydroxy VLCFAs. These results allowed us to speculate that AtBI-1 interacts with AtFAHs via AtCb5 to activate the synthesis of 2-hydroxy VLCFA in Arabidopsis, which leads to the suppression of cell death. However, it remains unclear whether 2-hydroxy VLCFA is related to the regulation of cell death or stress responses. Furthermore, little is known about the function of AtFAH1 and AtFAH2 in 2-HFA synthesis of plant cells.The results of this study confirmed that both AtFAHs are FAHs, and identified functional differences between AtFAH1 and AtFAH2 in fatty acid 2-hydroxylation in Arabidopsis. In addition, AtFAH1 modified the resistance to oxidative stress more strongly than AtFAH2, which was consistent with the preferential interaction with AtBI-1.     

N-acetylglucosaminyltransferase III antagonizes the effect of N-acetylglucosaminyltransferase V on alpha3beta1 integrin-mediated cell migration

N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of beta1,6-GlcNAc branching of N-glycans, which contributes to metastasis. N-acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulting in the suppression of metastasis. It has long been hypothesized that the suppression of GnT-V product formation by the action of GnT-III would also exist in vivo, which will consequently lead to the inhibition of biological functions of GnT-V. To test this, we draw a comparison among MKN45 cells, which were transfected with GnT-III, GnT-V, or both, respectively. We found that alpha3beta1 integrin-mediated cell migration on laminin 5 was greatly enhanced in the case of GnT-V transfectant. This enhanced cell migration was significantly blocked after the introduction of GnT-III. Consistently, an increase in bisected GlcNAc but a decrease in beta1,6-GlcNAc-branched N-glycans on integrin alpha3 subunit was observed in the double transfectants of GnT-III and GnT-V. Conversely, GnT-III knockdown resulted in increased migration on laminin 5, concomitant with an increase in beta1,6-GlcNAc-branched N-glycans on the alpha3 subunit in CHP134 cells, a human neuroblastoma cell line. Therefore, in this study, the priority of GnT-III for the modification of the alpha3 subunit may be an explanation for why GnT-III inhibits GnT-V-induced cell migration. Taken together, our results demonstrate for the first time that GnT-III and GnT-V can competitively modify the same target glycoprotein and furthermore positively or negatively regulate its biological functions.