The Blood Island is a Site of Formation of the Primary Embryo Thrombocyte in the Chick Blastoderm

Using the immunohistological technique we inquired at what developmental stage and in which site of chick blastoderm does the embryo thrombocyte (ET) begin to differentiate. An anti-ET antibody was raised against rabbits by injecting ETs isolated from blood of 10 day chick embryos. By applying the indirect staining method to smear preparations of blood collected from developing embryos it was confirmed that cytoplasm of the ET showed more intense staining than that of the erythroid cell and that the ET population could be distinguished from the erythrocyte population by this antibody. Cells showing the intense staining could be detected first in blood islands of the area opaca vasculosa of stage 9+ blastoderms. These embryo thromboblasts were found singly or in groups of a small number at dorsal periphery of cell clusters in the blood island. The electron microscopy revealed that embryo thromboblasts appeared in the same position in the stage 9+ blastoderm. At stage 10+ or later embryo thromboblasts were also present adhering to the vascular endothelium or free in the vessel lumen. We conclude that ETs start differentiating from primitive mesenchymal cells localized in the blood island of the area opaca vasculosa at stage 9 or earlier, migrate thereafter to vessel lumen, and enter the blood stream.     

HLA antibody responses in HLA class I transgenic mice

In a previous report we described how cross-immunizations of pairs of transgenic mice expressing different HLA class I antigens led to the production of antibodies directed exclusively at polymorphic epitopes. This was ascribed to self-tolerance of HLA that prevents immune responses to monomorphic epitopes and focuses responses on polymorphic ones. In the present report we extend our findings and demonstrate that immunizations of class I transgenic mice with HLA transfected mouse fibrosarcoma as well as with human lymphoblastoid cells also preferentially yield antibodies to polymorphic epitopes. This was the case whether or not immunizations were carried out across locus barriers [e.g., Tg (HLA-A *0201) or Tg (HLA-Cw*0301) transgenic mice immunized with HLA-B27 transfectants] or within the same locus [e.g., Tg (HLA-B*1302) transgenic mice immunized with HLA-B27 transfectants or B27-expressing lympho-blastoid cell]. Use of an extended immunization protocol with four or more booster injections favored antibodies of IgG isotype with affinities high enough to lyse normal peripheral blood lymphocytes (PBLs) in complement-dependent cytotoxicity assays and to immunoprecipitate HLA antigens. The specificities covered by the monoclonal antibodies (mAbs) could be either broad or narrow, depending on the genetic distance of the HLA antigens or alleles involved. For instance, a Tg(HLA-B*1302) transgenic mouse immunized with B27 produced both broad B7/B27-specific antibodies, Bw4-specific antibodies, and one antibody reacting with all B alleles except B13 and with some C alleles. On the other hand, a Tg(HLA-B*1302) transgenic mouse immunized with Bw47 transfectants responded narrowly with an antibody to Bw60 and Bw47. Thus it appears that by choosing appropriate recipient mice and closely related or more distant HLA antigens, antibodies of a programmed specificity can be generated. Address correspondence and offprint requests to: U. Hämmerling.     

Telomere elongation by a mutant tankyrase 1 without TRF1 poly(ADP-ribosyl)ation

Telomeres are the capping structures of the eukaryotic chromosome ends. Tankyrase 1 is a poly(ADP-ribose) polymerase that elongates telomeres in a telomerase-dependent manner. This function of tankyrase 1 is mediated by down-regulation of TRF1, a negative regulator of telomere access to telomerase. Namely, tankyrase 1 poly(ADP-ribosyl)ates (PARsylates) TRF1, which in turn dissociates TRF1 from telomeres. The resulting telomeres become better substrates for telomerase-mediated DNA extension. Tankyrase 1 has five independent TRF1 binding sites, ARC (ANK repeat cluster) I to V. Among them, the most C-terminal ARC V is required for TRF1 PARsylation and its release from telomeres. By contrast, functional significance of other four ARCs remains elusive. In this study, we generated a mutant tankyrase 1 that had inactive ARC IV and lacked ARC V but elongated telomeres without TRF1 PARsylation. Consistent with the failure in PARsylation, this mutant only marginally released TRF1 from telomeres. Still, it decreased telomere binding of POT1, a downstream effector of TRF1-mediated telomere length control, and elongated the telomeric 3'-overhang as the wild-type tankyrase 1 did. Thus even without TRF1 PARsylation, this mutant tankyrase 1 seemed to loosen the closed structure of the telomeric heterochromatin. These findings suggest a new role for multiple ARCs in telomere extension by tankyrase 1.     

Capability of wild Rosa rugosa and its varieties and hybrids to produce sesquiterpene components in leaf glandular trichomes.

The sesquiterpene contents in leaves of wild Rosa rugosa and of sixty-one hybrid rugosas were quantitatively measured by a GC analysis. In this group of samples, the greater the number of glandular trichomes the hybrid rugosas possessed on their leaves, the larger the amount of sesquiterpenes they accumulated. In contrast, those having no leaf glandular hairs contained only a trace amount of sesquiterpene components. The concentrations of bisaborosaol A (1) and carota-1,4-dienaldehyde (2) as representative sesquiterpenes of R. rugosa were positively correlated with the density of the glandular trichomes. Furthermore, an approximately regular correlation was observed between the concentrations of 1 and 2 in most of the sesquiterpene-producing hybrid rugosas, regardless of their productivity. This suggests that a major part of these hybrid rugosas have inherited from R. rugosa the ability to produce two skeletally different sesquiterpenes in parallel with a phenotype to develop leaf glandular trichomes. This investigation also led to discovering 1-dominant (e.g., Amelie Gravereaux and Purple Pavement), 2-dominant (e.g., David Thompson), and other-dominant (e.g., Martin Frobisher) types of sesquiterpene-producing hybrid rugosas.     

Identification of the 4-hydroxycinnamate decarboxylase (PAD) gene of Klebsiella oxytoca

A 7.1-kbp DNA fragment isolated from a wild strain of Klebsiella oxytoca was sequenced, leading to the identification of 10 open-reading frames (ORFs), including a 504-bp Pad gene. The Pad gene of the Gram-negative bacterium was subsequently expressed in Escherichia coli as a chimeric Pad. The deduced amino acid (AA) sequence of the Pad gene from wild-type K. oxytoca showed approximately 50% homology to those of other bacterial PADs from Gram-positive bacilli plus a coccus. These data and a genomic library search of some gamma-proteobacteria, including E. coli and Vibrio sp., indicated that PAD of K. oxytoca is a member of the bacterial PAD family characteristic of Gram-negative bacteria. Using Pad-specific PCR primers designed from the Gram-negative bacterial Pad of K. oxytoca, Pad genes of two further strains of K. oxytoca, another wild isolate and JCM 1665 and two PAD-positive Enterobacter spp. were successfully amplified for specific Pad detection.     

Importin-beta and the small guanosine triphosphatase Ran mediate chromosome loading of the human chromokinesin Kid

Nucleocytoplasmic transport factors mediate various cellular processes, including nuclear transport, spindle assembly, and nuclear envelope/pore formation. In this paper, we identify the chromokinesin human kinesin-like DNA binding protein (hKid) as an import cargo of the importin-alpha/beta transport pathway and determine its nuclear localization signals (NLSs). Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells. In digitonin-permeabilized mitotic cells, hKid was bound only to the spindle and not to the chromosomes themselves. Surprisingly, hKid bound to importin-alpha/beta was efficiently targeted to mitotic chromosomes. The addition of Ran-guanosine diphosphate and an energy source, which generates Ran-guanosine triphosphate (GTP) locally at mitotic chromosomes, enhanced the importin-beta-mediated chromosome loading of hKid. Our results indicate that the association of importin-beta and -alpha with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP-mediated cargo release promotes the accumulation of hKid on chromosomes. Thus, this study demonstrates a novel nucleocytoplasmic transport factor-mediated mechanism for targeting proteins to mitotic chromosomes.     

Phylogeny of gamma-polyglutamic acid-producing Bacillus strains isolated from a fermented locust bean product manufactured in West Africa

Twenty-five Bacillus strains capable of producing gamma-polyglutamic acid (PGA) were isolated from fermented locust bean products manufactured in the savanna area of Ghana. To clarify the phylogeny of these PGA-producing strains, phylogenetic analyses based on sequences of 16S rDNA, rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) genes were performed. A phylogenetic tree based on 16S rDNA indicated that ten isolates were clustered in the same group of Bacillus subtilis. Another ten isolates were located in the cluster of B. amyloliquefaciens, and the remaining isolates were identified as B. pumilus (three isolates) and B. licheniformis (two isolates), respectively. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. Thirty-four strains in 27 species belonging to the genus Bacillus and its neighbors were also investigated for PGA production. It was found that PGA was produced by B. amyloliquefaciens NBRC 14141 and NBRC 15535(T), B. atrophaeus NBRC 15539(T), B. licheniformis NBRC 12107, B. mojavensis NBRC 15718(T), B. pumilus NBRC 12094, B. subtilis NBRC 16449, and Lysinibacillus sphaericus NBRC 3525. Except for L. sphaericus, the above Bacillus species are very closely related in phylogeny, indicating that PGA-producing Bacillus strains constitute a cluster.     

Design,synthesis, and taste evaluation of a high-intensity umami-imparting oxazole-based compound

Umami taste is imparted predominantly by monosodium glutamate (MSG) and 5′-ribonucleotides. Recently, several different classes of hydrophobic umami-imparting compounds, the structures of which are quite different from MSG, have been reported. To obtain a novel umami-imparting compound, N-cinnamoyl phenethylamine was chosen as the lead compound, and a rational structure-optimization study was conducted on the basis of the pharmacophore model of previously reported compounds. The extremely potent umami-imparting compound 2-[[[2-[(1E)-2-(1,3-benzodioxol-5-yl)ethenyl]-4-oxazolyle]methoxy]methyl]pyridine, which exhibits 27,000 times the umami taste of MSG, was found. Its terminal pyridine residue and linear structure are suggested to be responsible for its strong activity. The time taken to reach maximum taste intensity exhibited by it, as determined by the time-intensity method, is 22.0 s, whereas the maximum taste intensity of MSG occurs immediately. This distinct difference in the time-course taste profile may be due to the hydrophobicity and strong receptor affinity of the new compound.     

Protective effect of epigallocatechin gallate and esculetin on oxidative DNA damage induced by psoralen plus ultraviolet-A therapy

We examined antioxidants exhibiting no effects on DNA cross-linking, which is the basis of psoralen and ultraviolet-A therapy for skin diseases, and suppressing oxidative DNA damage incidental to the therapy. Epigallocatechin gallate and esculetin effectively suppressed oxidative DNA damage with little effect on the formation of DNA cross-linking. These antioxidants might be useful in suppressing the adverse reaction induced by this therapy.