Lysine proximity significantly affects glycation of lysine-containing collagen model peptides

Advanced glycation end products (AGE) are known to cause diabetes complications in hyperglycemia patients. In this study we prepared hetero-trimers of collagen model peptides comprising Ac-(Pro-Hyp-Gly)(5)-Pro-Lys-Gly-(Pro-Hyp-Gly)(5)-Ala-NH(2) (4) and Ac-(Pro-Hyp-Gly)(11)-Ala-NH(2) (5) to investigate the clustering effect of lysine on AGE formation. The formation rate of carboxymethyllysine over several months was determined for the mixtures of peptides 4 and 5 at (3:0), (2:1) and (1:2) in the presence of glucose. The contents of carboxymethyllysine were significantly enhanced for (3:0) and (2:1) as compared with (1:2), suggesting that the proximity of lysine residues in the trimers accelerated formation of the AGE. Furthermore, a lysine dimerization moiety (GOLD) was identified for the first time from AGEs of glucose origin, which implied the significance of GOLD in oligomerization of collagens and other long-life proteins.     

Molecular analysis of X-linked inborn errors of purine metabolism: HPRT1 and PRPS1 mutations

Mutations of two enzyme genes, HPRT1 encoding hypoxanthine guanine phosphoribosyltransferase (HPRT) and PRPS1 encoding a catalytic subunit (PRS-I) of phosphoribosylpyrophosphate synthetase, cause X-linked inborn errors of purine metabolism. Analyzing these two genes, we have identified three HPRT1 mutations in Lesch-Nyhan families following our last report. One of them, a new mutation involving the deletion of 4224 bp from intron 4 to intron 5 and the insertion of an unknown 28 bp, has been identified. This mutation resulted in an enzyme polypeptide with six amino acids deleted due to abnormal mRNA skipping exon 5. The other HPRT1 mutations, a single base deletion (548delT, 183fs189X), and a point mutation causing a splicing error (532+1G>A, 163fs165X) were detected first in Japanese patients but have been reported in European families. On the other hand, in the analysis of PRPS1, no mutation was identified in any patient.     

Inhibition of hematopoietic prostaglandin D synthase improves allergic nasal blockage in guinea pigs

Although it has been suggested that prostaglandin (PG) D(2) is involved in the pathogenesis of allergic rhinitis, whether the inhibition of hematopoietic PGD(2) synthase (H-PGDS) shows beneficial effects on allergic rhinitis has been unclear. We evaluated the effects of a selective H-PGDS inhibitor, TFC-007, on nasal symptoms on Japanese cedar pollen-induced allergic rhinitis of guinea pigs. Sensitized animals were challenged with the pollen once a week. TFC-007 (30mg/kg, p.o.) given once before a challenge almost completely suppressed PGD(2) production in the nasal tissue early and late after the challenge. Although pre-treatment did not affect the incidences of sneezing and early phase nasal blockage, late phase nasal blockage was partially but significantly attenuated; however, nasal eosinophilia was not suppressed. In contrast, when TFC-007 was given once 1.5h after the challenge, the late phase response was not affected. Collectively, PGD(2) produced by H-PGDS early after an antigen challenge can participate in the induction of late phase nasal blockage, although the mechanism may be independent of eosinophil infilatration. The strategy for H-PGDS inhibition may be beneficial for allergic rhinitis therapy.     

Genetic isolation between the Western and Eastern Pacific populations of pronghorn spiny lobster Panulirus penicillatus

The pronghorn spiny lobster, Panulirus penicillatus, is a circumtropical species which has the widest global distribution among all the species of spiny lobster, ranging throughout the entire Indo-Pacific region. Partial nucleotide sequences of mitochondrial DNA COI (1,142-1,207 bp) and 16S rDNA (535-546 bp) regions were determined for adult and phyllosoma larval samples collected from the Eastern Pacific (EP)(Galápagos Islands and its adjacent water), Central Pacific (CP)(Hawaii and Tuamotu) and the Western Pacific (WP)(Japan, Indonesia, Fiji, New Caledonia and Australia). Phylogenetic analyses revealed two distinct large clades corresponding to the geographic origin of samples (EP and CP+WP). No haplotype was shared between the two regional samples, and average nucleotide sequence divergence (Kimura's two parameter distance) between EP and CP+WP samples was 3.8±0.5% for COI and 1.0±0.4% for 16S rDNA, both of which were much larger than those within samples. The present results indicate that the Pacific population of the pronghorn spiny lobster is subdivided into two distinct populations (Eastern Pacific and Central to Western Pacific), with no gene flow between them. Although the pronghorn spiny lobster have long-lived teleplanic larvae, the vast expanse of Pacific Ocean with no islands and no shallow substrate which is known as the East Pacific Barrier appears to have isolated these two populations for a long time (c.a. 1MY).     

Disruption of Sept6, a fusion partner gene of MLL, does not affect ontogeny, leukemogenesis induced by MLL-SEPT6, or phenotype induced by the loss of Sept4

Septins are evolutionarily conserved GTP-binding proteins that can heteropolymerize into filaments. Recent studies have revealed that septins are involved in not only diverse normal cellular processes but also the pathogenesis of various diseases, including cancer. SEPT6 is ubiquitously expressed in tissues and one of the fusion partner genes of MLL in the 11q23 translocations implicated in acute leukemia. However, the roles of this septin in vivo remain elusive. We have developed Sept6-deficient mice that exhibited neither gross abnormalities, changes in cytokinesis, nor spontaneous malignancy. Sept6 deficiency did not cause any quantitative changes in any of the septins evaluated in this study, nor did it cause any additional changes in the Sept4-deficient mice. Even the depletion of Sept11, a close homolog of Sept6, did not affect the Sept6-null cells in vitro, thus implying a high degree of redundancy in the septin system. Furthermore, a loss of Sept6 did not alter the phenotype of myeloproliferative disease induced by MLL-SEPT6, thus suggesting that Sept6 does not function as a tumor suppressor. To our knowledge, this is the first report demonstrating that a disruption of the translocation partner gene of MLL in 11q23 translocation does not contribute to leukemogenesis by the MLL fusion gene.     

Phospholipase C-delta1 and -delta3 are essential in the trophoblast for placental development

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in phosphoinositide turnover and is involved in a variety of physiological functions. We analyzed PLCdelta1 knockout mice and found that PLCdelta1 is required for the maintenance of skin homeostasis. However, there were no remarkable abnormalities except hair loss and runting in PLCdelta1 knockout mice, even though PLCdelta1 is broadly distributed. Here, we report that mice lacking both PLCdelta1 and PLCdelta3 died at embryonic day 11.5 (E11.5) to E13.5. PLCdelta1/PLCdelta3 double-knockout mice exhibited severe disruption of the normal labyrinth architecture in the placenta and decreased placental vascularization, as well as abnormal proliferation and apoptosis of trophoblasts in the labyrinth area. Furthermore, PLCdelta1/PLCdelta3 double-knockout embryos supplied with a normal placenta by the tetraploid aggregation method survived beyond E14.5, clearly indicating that the embryonic lethality is caused by a defect in trophoblasts. On the basis of these results, we conclude that PLCdelta1 and PLCdelta3 are essential in trophoblasts for placental development.     

Structural and functional evolution of three cardiac natriuretic peptides

Natriuretic peptides (NPs) are a group of hormones playing important roles in cardiovascular and osmoregulatory systems in vertebrates. Among the NP subtypes, atrial NP (ANP), B-type NP (BNP), and ventricular NP (VNP) are circulating hormones expressed exclusively in the heart (cardiac NPs). The constitution of cardiac NPs is variable among species of vertebrates. In order to understand the evolutionary and functional significance of such variation, we performed a systematic survey of cardiac NP cDNAs in nine taxonomically diverse teleosts inhabiting environments of varying salinity. The discovery of the coexistence of the ANP, BNP, and VNP genes in the eel and rainbow trout suggested that the ancestral teleost had all three cardiac NPs. As the VNP cDNA was undetectable in ayu and six species of Neoteleostei, it is possible that VNP was lost before the divergence of Osmeroidei. The ANP gene was also undetectable in the medaka. Thus, only the BNP gene is universal in species examined in the present study. Synthetic medaka BNP preferentially activated two medaka GC-A-type receptors, suggesting that the three cardiac NPs share the same receptor. However, the regulation of BNP expression may be the most strict because ATTTA repeats in the 3'-untranslated region and the dibasic motif in the ring are conserved among teleosts and tetrapods. Linkage analyses in the rainbow trout located ANP, BNP, and VNP genes on the same chromosome, which suggested the generation of the VNP gene by tandem duplication as observed with ANP and BNP genes. If the duplication occurred before the divergence of tetrapods and teleosts, VNP may exist in the tetrapod lineage.     

Differential spontaneous killing of human and murine tumour cell lines by leucocyte subpopulations from carp peripheral blood leucocytes

Cell populations from carp (Cyprinus carpio L.) peripheral blood leucocytes (PBLs) were examined for nonspecific cytotoxicities. By using monoclonal antibodies (MAbs) against carp thrombocytes (TCL-HB8) and both neutrophils and monocytes (TCL-BE8), PBLs with a density of 1.08 g ml-1 were separated into three fractions: thrombocytes, a mixture of neutrophils and monocytes, and other cells (mainly lymphocytes), and the separated cells were tested for cytotoxic activities against mammalian tumour cell lines (K562, HeLa, P815 and Yac-1 cell). Consequently, the mixture of neutrophils and monocytes exhibited cytolysis against these target cells, whereas the lymphocyte-rich and thrombocyte fractions did not show any cytolysis. To isolate only neutrophils, which do not contain monocytes, the MAb (TCL-BE8) positive cells from PBLs with a density of 1.08-1.09 g ml-1 were separated. Pure isolated neutrophils showed cytotoxic activities against K562 cells, but not P815 cells. Furthermore, analysis of the cytolytic mechanisms indicated that killing of these cells depended on H2O2 or HOCl. These results suggest that both neutrophils and monocytes are effectors for nonspecific cytotoxicity in carp PBLs, and neutrophils may be distinct from monocytes in their reactivity in cytolysis, including target cell selectivity and/or target cell sensitivity, and the cytolytic pathway. In carp, cytotoxicity of target cells can be mediated by several populations of their leucocytes which have cytotoxic capacities with various recognition and cytolytic mechanisms.     

Developmentally programmed remodeling of the Drosophila olfactory circuit

Neural circuits are often remodeled after initial connections are established. The mechanisms by which remodeling occurs, in particular whether and how synaptically connected neurons coordinate their reorganization, are poorly understood. In Drosophila, olfactory projection neurons (PNs) receive input by synapsing with olfactory receptor neurons in the antennal lobe and relay information to the mushroom body (MB) calyx and lateral horn. Here we show that embryonic-born PNs participate in both the larval and adult olfactory circuits. In the larva, these neurons generally innervate a single glomerulus in the antennal lobe and one or two glomerulus-like substructures in the MB calyx. They persist in the adult olfactory circuit and are prespecified by birth order to innervate a subset of glomeruli distinct from larval-born PNs. Developmental studies indicate that these neurons undergo stereotyped pruning of their dendrites and axon terminal branches locally during early metamorphosis. Electron microscopy analysis reveals that these PNs synapse with MB gamma neurons in the larval calyx and that these synaptic profiles are engulfed by glia during early metamorphosis. As with MB gamma neurons, PN pruning requires cell-autonomous reception of the nuclear hormone ecdysone. Thus, these synaptic partners are independently programmed to prune their dendrites and axons.     

Proteomic analysis of slow- and fast-twitch skeletal muscles

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.