Origin of mannose-binding lectin-associated serine protease (MASP)-1 and MASP-3 involved in the lectin complement pathway traced back to the invertebrate,amphioxus

Mannose-binding lectin-associated serine proteases (MASPs) are involved in complement activation through the lectin pathway. To elucidate the phylogenetic origin of MASP and a primordial complement system, we cloned two MASP cDNAs from amphioxus (Branchiostoma belcheri) of the cephalochordates, considered to be the closest relative of vertebrates. The two sequences, orthologues of mammalian MASP-1 and MASP-3, were produced by alternative processing of RNA from a single gene consisting of a common H chain-encoding region and two L chain-encoding regions, a structure which is similar to that of the human MASP1/3 gene. We also isolated two MASP genes from the ascidian Halocynthia roretzi (urochordates) and found that each of them consists simply of an H chain-encoding region and a single L chain-encoding region. The difference in structure between the ascidian MASP genes and the amphioxus/mammalian MASP genes suggests that a prototype gene was converted to the MASP1/3-type gene possessing two L chain-encoding regions at an early stage of evolution before the divergence of amphioxus. This conclusion is supported by the presence of MASP-1 and MASP-3 homologues in almost all vertebrates, as demonstrated by the cloning of novel cDNA sequences representing lamprey (cyclostomes) MASP-1 and Xenopus MASP-3. The ancient origin of MASP-1 and MASP-3 suggests that they have crucial functions common to all species which emerged after cephalochordates.     

Both Notch1 and Notch2 contribute to the regulation of melanocyte homeostasis

Notch signaling affects a variety of mammalian stem cells, but there has been limited evidence that a specific Notch molecule regulates adult stem cells. Recently, it was reported that the reduced Notch signaling initiated at the embryonic stage results in a gradual hair graying phenotype after birth. Here we demonstrate that the oral administration of a gamma-secretase inhibitor (GSI) to wild-type adult C57/Bl6 mice led to a gradual increase in gray spots, which remained unchanged for at least 20 weeks after discontinuing the GSI. In GSI-treated mice, there was a severe decrease in unpigmented melanocytes in the bulge/subbulge region where melanocyte stem cells are located. While we confirmed that Notch1+/-Notch2+/- double heterozygous mice with a C57/Bl6 background were born with a normal hair color phenotype and gradually turned gray after the second hair cycle, in the c-kit mutant Wv background, Notch1+/- and Notch2+/- mice had larger white spots on the first appearance of hair than did the Wv/+ mice, which did not change throughout life. Notch1+/-Notch2+/-Wv/+ mice had white hair virtually all over the body at the first appearance of hair and the depigmentation continued to progress thereafter. Using a neural crest organ culture system, GSI blocked the generation of pigmented melanocytes when added to the culture during the period of melanoblast proliferation, but not during the period of differentiation. These observations imply roles of Notch signaling in both development of melanocyte during embryogenesis and maintenance of melanocyte stem cells in adulthood, while the degree of requirement is distinct in these settings: the latter is more sensitive than the former to the reduced Notch signaling. Furthermore, Notch1 and Notch2 cooperates with c-kit signaling during embryogenesis, and they cooperate with each other to regulate melanocyte homeostasis after birth.     

Ubiquitylation of terminal deoxynucleotidyltransferase inhibits its activity

Terminal deoxynucleotidyltransferase (TdT), which template-independently synthesizes DNA during V(D)J recombination in lymphoid cells, is ubiquitylated by a BPOZ-2/Cul3 complex, as the ubiquitin ligase, and then degraded by the 26 S proteasome. We show here that TdT is ubiquitylated by the Cul3-based ubiquitylation system in vitro. Because TdT could also be ubiquitylated in the absence of Cul/BPOZ-2, we determined that it could also be directly ubiquitylated by the E2 proteins UbcH5a/b/c and UbcH6, E3-independently. Furthermore, the ubiquitylated TdT inhibited its nucleotidyltransferase activity.     

A Codon-Optimized Bacterial Antibiotic Gene Used as Selection Marker for Stable Nuclear Transformation in the Marine Red Alga Pyropia yezoensis

Marine macroalgae play an important role in marine coastal ecosystems and are widely used as sea vegetation foodstuffs and for industrial purposes. Therefore, there have been increased demands for useful species and varieties of these macroalgae. However, genetic transformation in macroalgae has not yet been established. We have developed a dominant selection marker for stable nuclear transformation in the red macroalga Pyropia yezoensis. We engineered the coding region of the aminoglycoside phosphotransferase gene aph7″ from Streptomyces hygroscopicus to adapt codon usage of the nuclear genes of P. yezoensis. We designated this codon-optimized aph7″ gene as PyAph7. After bombarding P. yezoensis cells with plasmids containing PyAph7 under the control of their endogenous promoter, 1.9 thalli (or individuals) of hygromycin-resistant strains were isolated from a 10-mm square piece of the bombarded thallus. These transformants were stably maintained throughout the asexual life cycle. Stable expression of PyAph7was verified using Southern blot analysis and genomic PCR and RT-PCR analyses. PyAph7 proved to be a new versatile tool for stable nuclear transformation in P. yezoensis.     

Adiponectin and visceral fat associate with cardiovascular risk factors

Objective: To examine the combined effect of CT‐measured visceral fat area (VFA) and adiponectin level against the clustering of metabolic risk factors. Design and Methods: The subjects were 6,996 Japanese. The subjects were divided according to the combinations of VFA and adiponectin level quartiles and the odds ratio for multiple risk factors of metabolic syndrome were calculated (adjusted for age and lifestyle factors using logistic regression analyses). Group with the lowest VFA and the highest adiponectin level was used as a reference. The correlation between adiponectin level and each metabolic risk factor was evaluated. Results: The strongest correlation was observed between adiponectin level and high‐density lipoprotein cholesterol levels (r = 0.369 and 0.439 for men and women). Both VFA and adiponectin level were independently associated with the clustering of metabolic risk factors (interaction P = 0.58 and 0.11 for men and women). The odds ratio for the clustering of metabolic risk factors in the group with the highest VFA and the lowest adiponectin level, compared with the group with the lowest VFA and the highest adiponectin level, was 12.7 (9.7‐16.6) for men and 13.5 (6.0‐30.2) for women. Conclusion: The ability to detect metabolic syndrome could be improved by examining adiponectin level in conjunction with VFA.     

Vinculin and Rab5 Complex Is Requited for Uptake of Staphyrococcus aureus and Interleukin-6 Expression

Vinculin, a 116-kDa membrane cytoskeletal protein, is an important molecule for cell adhesion; however, little is known about its other cellular functions. Here, we demonstrated that vinculin binds to Rab5 and is required for Staphylococcus aureus (S. aureus) uptake in cells. Viunculin directly bound to Rab5 and enhanced the activation of S. aureus uptake. Over-expression of active vinculin mutants enhanced S. aureus uptake, whereas over-expression of an inactive vinculin mutant decreased S. aureus uptake. Vinculin bound to Rab5 at the N-terminal region (1-258) of vinculin. Vinculin and Rab5 were involved in the S. aureus-induced phosphorylation of MAP kinases (p38, Erk, and JNK) and IL-6 expression. Finally, vinculin and Rab5 knockdown reduced infection of S. aureus, phosphorylation of MAPKs and IL-6 expression in murine lungs. Our results suggest that vinculin binds to Rab5 and that these two molecules cooperatively enhance bacterial infection and the inflammatory response.     

Use of a Gluconobacter frateurii mutant to prevent dihydroxyacetone accumulation during glyceric acid production from glycerol

To prevent dihydroxyacetone (DHA) by-production during glyceric acid (GA) production from glycerol using Gluconobacter frateurii, we used a G. frateurii THD32 mutant, ΔsldA, in which the glycerol dehydrogenase subunit-encoding gene (sldA) was disrupted, but ΔsldA grew much more slowly than the wild type, growth starting after a lag of 3 d under the same culture conditions. The addition of 1% w/v D-sorbitol to the medium improved both the growth and the GA productivity of the mutant, and ΔsldA produced 89.1 g/l GA during 4 d of incubation without DHA accumulation.     

Separation of active and inactive (nonphosphorylating) Ca(2+)-ATPase in sarcoplasmic reticulum subfractions from low-frequency-stimulated rabbit muscle.

Chronic low-frequency stimulation elicits in rabbit fast-twitch muscle a partial inactivation of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase and Ca(2+)-uptake activities. Inactive Ca(2+)-ATPase was enriched in a light microsomal fraction by sucrose density gradient centrifugation after calcium oxalate loading in the presence of ATP. This fraction showed a reduced specific activity and phosphoprotein formation of the Ca(2+)-transport ATPase. These results suggest that the inactivation of the Ca(2+)-ATPase as induced by increased contractile activity, is confined to a specific SR vesicle population.     

Enhanced Exposure of Human Immunodeficiency Virus Type 1 Primary Isolate Neutralization Epitopes through Binding of CD4 Mimetic Compounds

N-(4-Chlorophenyl)-N′-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamide (NBD-556) is a low-molecular-weight compound that reportedly blocks the interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and its receptor CD4. We investigated whether the enhancement of binding of anti-gp120 monoclonal antibodies (MAbs) toward envelope (Env) protein with NBD-556 are similar to those of soluble CD4 (sCD4) by comparing the binding profiles of the individual MAbs to Env-expressing cell surfaces. In flow cytometric analyses, the binding profiles of anti-CD4-induced epitope (CD4i) MAbs toward NBD-556-pretreated Env-expressing cell surfaces were similar to the binding profiles toward sCD4-pretreated cell surfaces. To investigate the binding position of NBD-556 on gp120, we induced HIV-1 variants that were resistant to NBD-556 and sCD4 in vitro. At passage 21 in the presence of 50 μM NBD-556, two amino acid substitutions (S375N in C3 and A433T in C4) were identified. On the other hand, in the selection with sCD4, seven mutations (E211G, P212L, V255E, N280K, S375N, G380R, and G431E) appeared during the passages. The profiles of the mutations after the selections with NBD-556 and sCD4 were very similar in their three-dimensional positions. Moreover, combinations of NBD-556 with anti-gp120 MAbs showed highly synergistic interactions against HIV-1. We further found that after enhancing the neutralizing activity by adding NBD-556, the contemporaneous virus became highly sensitive to antibodies in the patient''s plasma. These findings suggest that small compounds such as NBDs may enhance the neutralizing activities of CD4i and anti-V3 antibodies in vivo.Human immunodeficiency virus type 1 (HIV-1) replicates continuously in the face of a strong antibody (Ab) response, although Abs effectively control many viral infections (3). Neutralizing Abs (NAbs) are directed against the HIV-1 envelope (Env) protein, which is a heterodimer comprising an extensively glycosylated CD4-binding subunit (gp120) and an associated transmembrane protein (gp41). Env proteins are present on the virion surface as “spikes” composed of trimers of three gp120-gp41 complexes (20, 21, 29). These spikes resist neutralization through epitope occlusion within the oligomer, extensive glycosylation, extension of variable loops from the surface of the complex, and steric and conformational blocking of receptor binding sites (16, 18, 20).Ab access to conserved regions is further limited because viral entry is a stepwise process involving conformational changes that lead to only transient exposure of conserved domains such as the coreceptor binding site (4, 5). However, some early strains of HIV-1 appear to be highly susceptible to neutralization by Abs (1, 10). For instance, subtype A HIV-1 envelopes from the early stage of infection exhibit a broad range of neutralization sensitivities to both autologous and heterologous plasma (1), suggesting that at least a subset of the envelopes have some preserved and/or exposed neutralization epitopes. It is well known that the potential for neutralizing properties of particular Abs is enhanced after binding of soluble CD4 (sCD4), especially NAbs against CD4-induced epitopes (CD4i Abs) (27) and some anti-V3 Abs (22). CD4i Abs are detected in plasma samples from many patients at an early stage of HIV-1 infection (9). Consequently, we hypothesize that small compounds such as sCD4 can enhance the neutralizing activities of CD4i Abs and some anti-V3 Abs not only in vitro but also in vivo.In a previous report, two low-molecular-weight compounds that presumably interfere with viral entry of HIV-1 into cells were described (35). These two N-phenyl-N′-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamide analogs, NBD-556 and NBD-557, comprise a novel class of HIV-1 entry inhibitors that block the interaction between gp120 and CD4. These compounds were found to be equally potent inhibitors of both X4 and R5 viruses in CXCR4- and CCR5-expressing cell lines, respectively (35). Schön et al. (25) also reported that NBD-556 binds to gp120 in a process characterized by a large favorable change in enthalpy that is partially compensated for by a large unfavorable entropy change, representing a thermodynamic signature similar to that observed for binding of sCD4 to gp120. In a recent study, Madani et al. (23) reported the following findings: (i) NBD-556 binds within the Phe43 cavity, a highly conserved and functionally important pocket formed as gp120 assumes the CD4-bound conformation; (ii) the NBD-556 phenyl ring projects into the Phe43 cavity; (iii) the enhancement of CD4-independent infection by NBD-556 requires the induction of conformational changes in gp120; and (iv) increased affinities of NBD-556 analogs toward gp120 improve the antiviral potency during infection of CD4-expressing cells. The latter two studies demonstrated that low-molecular-weight compounds such as NBDs can induce conformational changes in the HIV-1 gp120 glycoprotein similar to those observed upon sCD4 binding (23, 25). The authors of these studies concluded that their data supported the importance of gp120 residues near the Phe43 cavity in binding to NBD-556 and lent credence to the docked binding mode.In the present study, we investigated the binding position of NBD-556 on gp120 by inducing HIV-1 variants that were resistant to NBD-556 by exposing HIV-1_IIIB to increasing concentrations of the compound in vitro. We also induced sCD4-resistant HIV-1_IIIB variants and compared the profile of the sCD4-resistant mutations to that of the NBD-556-resistant mutations. We subsequently examined the virological properties of pseudotyped HIV-1 clones carrying the NBD-556 and sCD4 resistance-associated env gene mutations. Our findings provide a foundation for understanding the interaction of NBD-556 with the CD4-binding site of HIV-1 gp120. We also evaluated the anti-HIV-1 interactions between plasma NAbs and NBD-556 in vitro and considered the possibility of using the data as a key to opening the shield covering the conserved epitopes targeted by NAbs.(This study was presented in part at the 15th Conference on Retroviruses and Opportunistic Infection, Boston, MA, 3 to 6 February 2008 [Abstract 736].)