Hematopoietic stem cells (HSCs) give rise to variety of hematopoietic cells via pluripotential progenitors and lineage-committed progenitors and are responsible for blood production throughout adult life. Amplification of HSCs or progenitors represents a potentially powerful approach to the treatment of various blood disorders and to applying gene therapy by bone marrow transplantation. Lnk is an adaptor protein regulating the production of B cells. Here we show that Lnk is also expressed in hematopoietic progenitors in bone marrow, and that in the absence of Lnk, the number and the hematopoietic ability of progenitors are significantly increased. Augmented growth signals through c-Kit partly contributed to the enhanced hematopoiesis by lnk − / − cells. Lnk was phosphorylated by and associated with c-Kit, and selectively inhibited c-Kit–mediated proliferation by attenuating phosphorylation of Gab2 and activation of mitogen-activated protein kinase cascade. These observations indicate that Lnk plays critical roles in the expansion and function of early hematopoietic progenitors, and provide useful clues for the amplification of hematopoietic progenitor cells.

The crystal structure of the L-asparaginase from the hyperthermophilic archaeon Pyrococcus horikoshii (PhA) was determined by the multiwavelength anomalous diffraction (MAD) method and was refined to a resolution of 2.16 angstroms with a crystallographic R factor and free R factor of 21.1 and 25.3%, respectively. This is the first report of the three-dimensional structure of a type I L-asparaginase. These enyzmes are known as cytosolic L-asparaginases with lower affinities for substrate than the type II L-asparaginases. Although the overall fold of PhA was closely related to the structure of the well characterized type II L-asparaginase, structural differences were also detected. PhA forms a homodimer that corresponds to half the homotetramer of type II L-asparaginases. Structure comparison at the active site reveals that most catalytic residues are conserved except for two residues that recognize the amino group of the substrate. Additionally, a remarkable structural difference is found in the so-called 'active-site flexible loop'. In PhA this loop is stabilized by beta-hairpin formation and by elaborate interactions with the type-I-specific alpha-helical region derived from the other subunit forming the PhA dimer. The flexible loop of the type II enzyme is considered to serve as a mobile gate to the active site. Therefore, the loop stabilization observed in the PhA structure may cause limitation of the access of the substrate to the active site.

SynopsisThe first X-ray structure of the catalytic nucleotide-binding subunit A of the A 1 -ATPase has been determined at 2.55 Å resolution.Abstract H + -transporting ATP synthase is a multi-subunit enzyme involved in the production of ATP, which is essential molecule for living organisms as a source of energy. Archaeal A-type ATPase (A-ATPase) is thought to act as a functional ATP synthase in Archaea and is thought to have chimeric properties of F-ATPase and V-ATPase.From the previous structural studies of F-ATPase, it is indicated that the major nucleotide-binding subunits α and β consist of three domains. The catalytic nucleotide-binding subunit A of V/A-ATPase contains an insertion of about 90 residues, which is absent from the F 1 -β subunit. Here we describe the first X-ray structure of the catalytic nucleotide-binding subunit A of the A 1 -ATPase determined at 2.55 Å resolution. A 1 -ATPase subunit A from Pyrococcus horikoshii consists of four domains. A novel domain, including a part of this insertion, corresponds to the "knob-like structure" observed in electron microscopy of A 1 -ATPase. Based on the structure, it is highly likely that this inserted domain is related to the peripheral stalk common to the A-and V-ATPases. The arrangement of this inserted domain suggests that this region plays an important role in A-ATPase as well as in V-ATPase.

The combined use of P(2-MeOC 6 H 4 ) 3 (L1) and TMEDA as ligands effectively prevents defect formation in palladiumcatalyzed direct arylation polymerization (DArP) to give donor− acceptor type alternating copolymers (DA polymers) with unsubstituted 2,2′-bithiophene units. When only L1 is used as a ligand, the reaction of 5,6-difluoro-4,7-bis) in toluene at 100 °C forms a notable amount of insoluble materials via branching and cross-linking. In contrast, in the presence of L1 and TMEDA, the formation of insoluble materials is completely suppressed, and PffBT4T [poly(1a-alt-2)] with a well-controlled structure and high molecular weight is obtained (M n = 88100). Similarly, the reaction of 5,10-bis[5-bromo-4-(2-decyltetradecyl)thiophene-2-yl]naphtho[1,2-c:5,6-c′]bis[1,2,5]thiadiazole (1b-Br) with 2-H in toluene at 110 °C forms PNTz4T[poly(1b-alt-2)] with M n = 43800. The resulting polymers function as good components of bulk-heterojunction type organic solar cells (OSCs) using PC 71 BM. In particular, PNTz4T performs the best of all the DArP polymers reported so far (PCE = 9.0(1)%).

No abstract

Cytosolic phospholipase A2 (PLase A2) is activated by low Ca2+ concentrations and translocates from the cytosol to the cell membrane, releasing arachidonic acid; the arachidonic acid cascade then leads to the production of many inflammatory mediators. The aim of this study, accordingly, was to investigate the role of phospholipid metabolism in the intestinal mucosa in inflammatory bowel disease (IBD). Surgically resected specimens from patients with Crohn's disease (CD), ulcerative colitis (UC), and colrectal cancer (non-cancerous tissue; as a control) were submitted to phospholipid analysis and a PLase A2 assay, which measures the degradation of endogenous mucosal phospholipids. A high percentage of plasmenylethanolamine (plas.E) was detected in the glycerophospholipid fraction of CD mucosa. The arachidonic acid content of the phosphatidylethanolamine plus plas.E subfraction was higher in inflamed than in intact mucosa in CD. PLaseA2 activity, resulting in lysophosphatidyl ethanolamine production, was detected only in inflamed mucosa from CD and UC patients, but not in normal mucosa from controls. PLaseA2 activity was highest in moderately inflamed mucosa adjacent to a severely ulcerated area. The PLaseA2 that reacts with endogenous phosphatidylcholine (PC) to form lysoPC was found irrespective of the presence of inflammation. The PLaseA2 that reacts with ethanolamine-containing phospholipids is more closely related to inflammation than other PLaseA2 isoenzymes in IBD mucosa.

We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluate the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, is suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.

Background. Sialyl Lea antigen and sialyl Lex antigen are cancer‐associated carbohydrate antigens. Previous immunohistologic and immunochemical studies have shown that these antigens are preferentially expressed in gastric cancer and colonic cancer and that they possibly are related to the metastatic potential of the cancer cells. The biosynthesis of these antigens is completed by fucosyltransferases, but it has not been reported how fucosyltransferases control the expression of these carbohydrate antigens concerning the invasive potential of the cancer. Methods. The authors established an assay system for measuring the activity of α1 → 4 fucosyltransferase (sialyl Lea synthase) and α1 → 3 fucosyltransferase (sialyl Lex synthase) with a high‐pressure liquid chromatography system (HPLC). The activity was measured in various parts of normal and cancerous gastric and colonic tissue and compared with the expression of sialyl Lea, sialyl Lex, Lea, and Lex antigens determined in a solid‐phase enzyme‐linked immunosolvent assay (EIA). Results. Sialyl Lea synthase was detected in most normal or malignant mucosa of gastric and colonic tissues, regardless of anatomic locations. Sialyl Lex synthase activity generally was low in the normal gastric mucosa, whereas the activity was higher in 77% (7 of 9) of gastric cancer tissues than in corresponding normal tissues with enhanced expression of sialyl Lex antigen in most patients (5 of 7). In the large intestine, the activity of sialyl Lea synthase and sialyl Lex synthase was correlated. Although enhanced expression of sialyl Lex in colonic cancer was observed in 86% (12 of 14) of all patients, concomitant higher sialyl Lex synthase activity than that in normal tissue was observed in only 58% (7 of 12) of patients. Conclusions. The expression of sialyl Lea and sialyl Lex antigens in the stomach and the colon was not controlled solely by fucosyltransferases but by a more complicated system involving other glycosyltransferases. Cancer 1994; 73:1552–61.