CXCR4-derived synthetic peptides inducing anti-HIV-1 antibodies

Despite almost 30 years since the identification of the human immunodeficiency virus type I (HIV-1), development of effective AIDS vaccines has been hindered by the high mutability of HIV-1. The HIV-1 co-receptors CCR5 and CXCR4 are genetically stable, but viral proteins may mutate rapidly during the course of infection. CXCR4 is a seven transmembrane G protein-coupled receptor, possessing an N-terminal region (NT) and three extracellular loops (ECL1-3). Previous studies have shown that the CXCR4-ED-derived peptides inhibit the entry of HIV-1 by interacting with gp120, an HIV-1 envelope glycoprotein. In the present study, antigenicity of CXCR4-derived peptides has been investigated and the anti-HIV-1 effects of induced antisera have been assessed. It was found that CXCR4-ED-derived antigen molecules immunize mice, showing that the linear peptides have higher antigenicity than the cyclic peptides. The L1- and L2-induced antisera inhibited the HIV-1 entry significantly, while anti-N1 antibodies have no inhibitory activity. This study produced promising examples for the design of AIDS vaccines which target the human protein and can overcome mutability of HIV-1.     

Isolation and Sequencing of a Genomic Clone for Mouse Brain Specific Small RNA

We isolated a mouse genomic clone that hybridized with small RNA present in the cytoplasm of the brain. The RNA was about 150 nucleotides long. This RNA seemed to be specific to the brain, since it was not found in the liver or kidney. The clone DNA contained a sequence homologous to 82-nucleotide "identifier" core sequence of cDNA clones of rat. The sequence contained a split promoter for RNA polymerase III and was flanked by a 12-nucleotide direct repeat (ATAAATAATTTA).     

Anchorage-independent growth of mouse male germline stem cells in vitro

Spermatogenesis originates from a small number of spermatogonial stem cells that reside on the basement membrane and undergo self-renewal division to support spermatogenesis throughout the life of adult animals. Although the recent development of a technique to culture spermatogonial stem cells allowed reproduction of self-renewal division in vitro, much remains unknown about how spermatogonial stem cells are regulated. In this study, we found that spermatogonial stem cells could be cultured in an anchorage-independent manner, which is characteristic of stem cells from other types of self-renewing tissues. Although the cultured cells grew slowly (doubling time, approximately 4.7 days), they expressed markers of spermatogonia, and grew exponentially for at least 5 months to achieve 1.5 x 10(10) -fold expansion. The cultured cells underwent spermatogenesis following transplantation into the seminiferous tubules of infertile animals and fertile offspring were obtained by microinsemination of germ cells that had developed within the testes of recipients of the cultured cells. These results indicate that spermatogonial stem cells can undergo anchorage-independent, self-renewal division, and suggest that stem cells have the common property to survive and proliferate in the absence of exogenous substrata.     

Fluorescent-responsive synthetic C1b domains of protein kinase Cδ as reporters of specific high-affinity ligand binding

Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.     

Over-expression of the apple <Emphasis Type="Italic">spermidine synthase</Emphasis> gene in pear confers multiple abiotic stress tolerance by altering polyamine titers

An apple spermidine synthase (SPDS) gene (MdSPDS1) was verified to encode a functional protein by the complementation of the spe3 yeast mutant, which lacks the SPDS gene. To justify our hypothesis that apple SPDS is involved in abiotic stress responses and to obtain transgenic fruit trees tolerant to abiotic stresses as well, MdSPDS1-over-expressing transgenic European pear (Pyrus communis L. ‘Ballad’) plants were created by Agrobacterium-mediated transformation. A total of 21 transgenic lines showing various spermidine (Spd) titers and MdSPDS1 expression levels were obtained. Selected lines were exposed to salt (150 mM NaCl), osmosis (300 mM mannitol), and heavy metal (500 μM CuSO₄) stresses for evaluating their stress tolerances. Transgenic line no. 32, which was revealed to have the highest Spd accumulation and expression level of MdSPDS1, showed the strongest tolerance to these stresses. When growth increments, electrolyte leakage (EL), and values of thiobarbituric acid reactive substances (TBARS) were monitored, line no. 32 showed the lowest growth inhibition and the least increase in EL or TBARS under stress conditions. Spd titers in wild-type and transgenic lines showed diverse changes upon stresses, and these changes were not consistent with the changes in MdSPDS1 expressions. Moreover, there were no differences in the sodium concentration in the shoots between the wild type and line no. 32, whereas the copper concentration was higher in the wild type than in line no. 32. Although the mechanism(s) underlying the involvement of polyamines in stress responses is not known, these results suggest that the over-expression of the SPDS gene substantially increased the tolerance to multiple stresses by altering the polyamine titers in pear. Thus, MdSPDS1-over-expressing transgenic pear plants could be used to improve desert land and/or to repair polluted environments. Xiao-Peng Wen and Xiao-Ming Pang contributed equally to this work.     

ent-Eudesmane sesquiterpenoids, galloyl esters of the oak lactone precursor, and a 3-O-methylellagic acid glycoside from the wood of Platycarya strobilacea

ent-Eudesmane sesquiterpenoids, 8,11-dihydroxy-2,4-cycloeudesmane, 11-hydroxy-2,4-cycloeudesman-8-one and 2,4-cyclo-7(11)-eudesmen-8-one, were isolated from the wood of Platycarya strobilacea, which has been used as an aromatic tree since at least the 18th century. On charring the wood, 2,4-cyclo-7(11)-eudesmen-8-one was detected in the smoke. In the charred wood, the concentrations of ellagitannins, such as galloyl pedunculagin, dramatically decreased, whereas concentrations of pentagalloyl glucose, and other gallotannins were relatively stable. In addition, two other compounds, the 6′-O-m- and p-digalloyl oak lactone precursor and the 3-O-methylellagic acid 4′-O-(4″-O-galloyl)-xylopyranoside, were isolated from the charred wood along with m- and p-digallic acid.     

Role of AtPolζ, AtRev1, and AtPolη in UV light-induced mutagenesis in Arabidopsis

Translesion synthesis (TLS) is a DNA damage tolerance mechanism in which DNA lesions are bypassed by specific polymerases. To investigate the role of TLS activities in ultraviolet light-induced somatic mutations, we analyzed Arabidopsis (Arabidopsis thaliana) disruptants of AtREV3, AtREV1, and/or AtPOLH genes that encode TLS-type polymerases. The mutation frequency in rev3-1 or rev1-1 mutants decreased compared with that in the wild type, suggesting that AtPolζ and AtRev1 perform mutagenic bypass events, whereas the mutation frequency in the polh-1 mutant increased, suggesting that AtPolη performs nonmutagenic bypass events with respect to ultraviolet light-induced lesions. The rev3-1 rev1-1 double mutant showed almost the same mutation frequency as the rev1-1 single mutant. The increased mutation frequency found in polh-1 was completely suppressed in the rev3-1 polh-1 double mutant, indicating that AtPolζ is responsible for the increased mutations found in polh-1. In summary, these results suggest that AtPolζ and AtRev1 are involved in the same (error-prone) TLS pathway that is independent from the other (error-free) TLS pathway mediated by AtPolη.     

Physical Interaction between Bacterial Heat Shock Protein (Hsp) 90 and Hsp70 Chaperones Mediates Their Cooperative Action to Refold Denatured Proteins

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.     

Cytotaxonomic study of ferns of Yunnan,southwestern China

A cytotaxonomic study was made of 90 fern species of Yunnan, southwestern China, based on collections from northwestern, central, and southwestern Yunnan and a few Cheng's collections. The results verified most of the formerly reported basic chromosome numbers of Chinese genera, and recorded for the first time the basic numbers ofGymnogrammitis andSorolepidium and Chinese members of several other genera. Cytotaxonomy of some problematical genera was discussed. Biogeographical relationships between Japanese, Chinese, and Himalayan ferns were compared with special reference to local cyto-reproductive variations inConiogramme, Deparia, Onychium, andPteris cretica. The present evidence indicates that most triploid species examined are agamosporous, as general in filicalean ferns, and also suggests the sexual 32-spored sporogenesis inHypodematium crenatum andSorolepidium graciale as in Lindsaeaceae.     

A newly isolatedBacillus stearotheromophilus K1041 and its transformation by electroporation

Summary Optimal conditions for the plasmid transformation of a newly isolatedBacillus stearothermophilus K1041 by electroporation were investigated. The optimal conditions allowed a transformation efficiency of 5.8×10⁵ transformants per μg plasmid pUB110.