S-Nitrosated human serum albumin dimer is not only a novel anti-tumor drug but also a potentiator for anti-tumor drugs with augmented EPR effects

Macromolecules have been developed as carriers of low-molecular-weight drugs in drug delivery systems (DDS) to improve their pharmacokinetic profile or to promote their uptake in tumor tissue via enhanced permeability and retention (EPR) effects. In the present study, recombinant human serum albumin dimer (AL-Dimer), which was designed by linking two human serum albumin (HSA) molecules with the amino acid linker (GGGGS)(2), significantly accumulated in tumor tissue even more than HSA Monomer (AL-Monomer) and appearing to have good retention in circulating blood in murine colon 26 (C26) tumor-bearing mice. Moreover, we developed S-nitrosated AL-Dimer (SNO-AL-Dimer) as a novel DDS compound containing AL-Dimer as a carrier, and nitric oxide (NO) as (i) an anticancer therapeutic drug/cell death inducer and (ii) an enhancer of the EPR effect. We observed that SNO-AL-Dimer treatment induced apoptosis of C26 tumor cells in vitro, depending on the concentration of NO. In in vivo experiments, SNO-AL-Dimer was found to specifically deliver large amounts of cytotoxic NO into tumor tissue but not into normal organs in C26 tumor-bearing mice as compared with control (untreated tumor-bearing mice) and SNO-AL-Monomer-treated mice. Intriguingly, S-nitrosation improved the uptake of AL-Dimer in tumor tissue through augmenting the EPR effect. These data suggest that SNO-AL-Dimer behaves not only as an anticancer therapeutic drug, but also as a potentiator of the EPR effect. Therefore, SNO-AL-Dimer would be a very appealing carrier for utilization of the EPR effect in future development of cancer therapeutics.     

Effect of berberine hydrochloride on grass carp Ctenopharyngodon idella serum bactericidal activity against Edwardsiella ictaluri

Bactericidal activity of grass carp (Ctenopharyngodon idella) serum was significantly enhanced when pre-treated with 15 mg l?1 or 3 mg l?1 of berberine hydrochloride, an effective component of several commonly used herbal medicines in aquaculture. The complement consumption experiment demonstrated that berberine hydrochloride can certainly activate fish complement system. The results of both experiments suggested that berberine hydrochloride could enhance the serum bactericidal activity in grass carp by activating the complement system and indicating the potential in the prevention or treatment of fish diseases.     

Detecting nonculturable bacteria in the active mycorrhizal zone of the pine mushroom <Emphasis Type="Italic">Tricholoma matsutake</Emphasis>

The fungus Tricholoma matsutake forms an ectomycorrhizal relationship with pine trees. Its sporocarps often develop in a circle, which is commonly known as a fairy ring. The fungus produces a solid, compact, white aggregate of mycelia and mycorrhizae beneath the fairy ring, which in Japanese is called a ’shiro’. In the present study, we used soil dilution plating and molecular techniques to analyze the bacterial communities within, beneath, and outside the T. matsutake fairy ring. Soil dilution plating confirmed previous reports that bacteria and actinomycetes are seldom present in the soil of the active mycorrhizal zone of the T. matsutake shiro. In addition, the results showed that the absence of bacteria was strongly correlated with the presence of T. matsutake mycorrhizae. The results demonstrate that bacteria, especially aerobic and heterotrophic forms, and actinomycetes, are strongly inhibited by T. matsutake. Indeed, neither bacteria nor actinomycetes were detected in 11.3% of 213 soil samples from the entire shiro area by culture-dependent methods. However, molecular techniques demonstrated that some bacteria, such as individual genera of Sphingomonas and Acidobacterium, were present in the active mycorrhizal zone, even though they were not detected in soil assays using the dilution plating technique.     

The genome characteristics and predicted function of methyl-group oxidation pathway in the obligate aceticlastic methanogens, Methanosaeta spp

In this work, we report the complete genome sequence of an obligate aceticlastic methanogen, Methanosaeta harundinacea 6Ac. Genome comparison indicated that the three cultured Methanosaeta spp., M. thermophila, M. concilii and M. harundinacea 6Ac, each carry an entire suite of genes encoding the proteins involved in the methyl-group oxidation pathway, a pathway whose function is not well documented in the obligately aceticlastic methanogens. Phylogenetic analysis showed that the methyl-group oxidation-involving proteins, Fwd, Mtd, Mch, and Mer from Methanosaeta strains cluster with the methylotrophic methanogens, and were not closely related to those from the hydrogenotrophic methanogens. Quantitative PCR detected the expression of all genes for this pathway, albeit ten times lower than the genes for aceticlastic methanogenesis in strain 6Ac. Western blots also revealed the expression of fwd and mch, genes involved in methyl-group oxidation. Moreover, (13)C-labeling experiments suggested that the Methanosaeta strains might use the pathway as a methyl oxidation shunt during the aceticlastic metabolism. Because the mch mutants of Methanosarcina barkeri or M. acetivorans failed to grow on acetate, we suggest that Methanosaeta may use methyl-group oxidation pathway to generate reducing equivalents, possibly for biomass synthesis. An fpo operon, which encodes an electron transport complex for the reduction of CoM-CoB heterodisulfide, was found in the three genomes of the Methanosaeta strains. However, an incomplete protein complex lacking the FpoF subunit was predicted, as the gene for this protein was absent. Thus, F(420)H(2) was predicted not to serve as the electron donor. In addition, two gene clusters encoding the two types of heterodisulfide reductase (Hdr), hdrABC, and hdrED, respectively, were found in the three Methanosaeta genomes. Quantitative PCR determined that the expression of hdrED was about ten times higher than hdrABC, suggesting that hdrED plays a major role in aceticlastic methanogenesis.     

Decreased Reactive Oxygen Species Production in Cells with Mitochondrial Haplogroups Associated with Longevity

Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function. Zhang and colleagues found a strikingly higher frequency of a C150T transition in the D-loop of mtDNA from centenarians and twins of an Italian population, and also demonstrated that this base substitution causes a remodeling of the mtDNA 151 replication origin in human leukocytes and fibroblasts [1]. The C150T transition is a polymorphism associated with several haplogroups. To determine whether haplogroups that carry the C150T transition display any phenotype that may be advantageous for longevity, we analyzed cybrids carrying or not the C150T transition. These cybrids were obtained by fusing cytoplasts derived from human fibroblasts with human mtDNA-less cells (ρ⁰ cells). We chose for cybrid construction and analysis haplogroup-matched pairs of fibroblast strains containing or not the C150T transition. In particular, we used, as one pair of mtDNA donors, a fibroblast strain of the U3a haplogroup, carrying the C150T transition and a strain of the U-K2 haplogroup, without the C150T transition, and as another pair, fibroblasts of the J2b haplogroup, carrying the C150T transition and of the J1c haplogroup, without the C150T transition. We have found no association of respiratory capacity, mtDNA level, mitochondrial gene expression level, or growth rate with the presence of the C150T transition. However, we have found that the cybrids with haplogroups that include the C150T transition have in common a lower reactive oxygen species (ROS) production rate than the haplogroup-matched cybrids without that transition. Thus, the lower ROS production rate may be a factor in the increased longevity associated with the U and the J2 haplogroups. Of further interest, we found that cybrids with the U3a haplogroup exhibited a higher respiration rate than the other cybrids examined.     

两个育性相关基因在几类小麦雄性不育材料中的表达研究

以1B/1R类K型不育系K3314A及其保持系3314B,非1B/1R类K型不育系732A及其保持系732B,YS温敏雄性不育小麦A3017为材料,提取不同温度处理下各时期小穗的总RNA进行反转录,对AY914051和AY660990两个目标基因进行半定量PCR和电泳分析,测定其在小麦中的表达变化趋势,以分析其与这几种不育系的育性相关关系,探讨这几种不育小麦败育的关键发育时期。结果表明,除AY914051基因在2种保持系3314B和732B中表达量变化不大之外,其它均有明显差异;2个目标基因与这几种雄性不育系的败育有关,但与育性相关程度不同,不育系两个目的基因的表达受温度变化影响程度明显大于保持系。由实验结果推测,1B/1R类K型不育系、非1B/1R类K型不育系和YS温敏雄性不育系的败育关键时期为单核期或单核期至二核期之间;温度差异可能会导致YS温敏雄性不育系的育性相关基因表达时期错位,错位后的表达差异积累可能最终导致其败育。     

Effect of the methylation of uracil and/or glycine on their mutual interaction

In order to simulate the hydrogen bonding and proton transfer (PT) in protein-DNA/RNA interactions, a series of simplified models were employed and investigated in the gas phase. These models included various neutral, anionic and cationic glycine-uracil dimers, and their methylated derivatives generated by the mono- or dimethylation of glycine and/or uracil moieties of the dimer. The results reveal that the only process that can occur in the neutral complexes is a double-PT process leading to proton exchange between the two moieties (i.e., point mutation). The first methyl substitute can reduce the activation energy of the PT process and thus promote the isomerization of the two moieties; further methylation can reduce the isomerization in only some of the cases. In the anionic complexes, only the one-way PT (i.e., amino acid → nucleic acid base) process is energetically favorable, and this PT process is an interesting barrier-free one (BFPT), with the attached electron locating itself at the base moiety. Methylation will disfavor BFPT, but it cannot alter the nature of BFPT. In the cationic complexes, three different PT processes can occur. These processes can transform mutually by adjusting either or both of the methylated sites and methyl number, indicating that the methylation can regulate the dynamics of these PT processes.     

辽宁省城市热岛强度特征及等级划分

根据气象站资料累积时间、资料连续性、站点位置、城郊站间距离、站点数量、迁站情况,对辽宁省61个气象站进行筛选,并利用选出的城郊代表站的月和年气温数据,分析了辽宁省热岛强度的变化范围及幅度,确定了热岛强度等级.结果表明:辽宁省铁岭站、大连站、鞍山站、朝阳站、丹东站、锦州站6个气象站可作为城市气温的代表站,台安站等18个气象站可作为郊区气温的代表站;1980-2009年,辽宁省6个代表城市的热岛强度年变化不同,除铁岭为微弱下降外,其他5市均表现为上升趋势,铁岭年热岛强度较强,大连年热岛强度较弱;6个代表城市的月热岛强度变化也不同,丹东、锦州、铁岭的月热岛强度呈明显的“U”型分布,6市最大热岛强度均出现在1月,最小热岛强度均出现在5-8月,具有冬季强、夏季弱的特点.6市热岛强度的年、月变化范围分别为0.57～2.15℃和-0.70～4.60℃,年和月热岛强度分别有97.8％和72.3％集中在0.5～2.0℃.辽宁省热岛强度可划分为无、弱、强和极强4个等级.     

Fibril formation of hsp10 homologue proteins and determination of fibril core regions: differences in fibril core regions dependent on subtle differences in amino acid sequence

Heat shock protein 10 (hsp10) is a member of the molecular chaperones and works with hsp60 in mediating various protein folding reactions. GroES is a representative protein of hsp10 from Escherichia coli. Recently, we found that GroES formed a typical amyloid fibril from a guanidine hydrochloride (Gdn-HCl) unfolded state at neutral pH. Here, we report that other hsp10 homologues, such as human hsp10 (Hhsp10), rat mitochondrial hsp10 (Rhsp10), Gp31 from T4 phage, and hsp10 from the hyperthermophilic bacteria Thermotoga maritima, also form amyloid fibrils from an unfolded state. Interestingly, whereas GroES formed fibrils from either the Gdn-HCl unfolded state (at neutral pH) or the acidic unfolded state (at pH 2.0-3.0), Hhsp10, Rhsp10, and Gp31 formed fibrils from only the acidic unfolded state. Core peptide regions of these protein fibrils were determined by proteolysis treatment followed by a combination of Edman degradation and mass spectroscopy analyses of the protease-resistant peptides. The core peptides of GroES fibrils were identical for fibrils formed from the Gdn-HCl unfolded state and those formed from the acidic unfolded state. However, a peptide with a different sequence was isolated from fibrils of Hhsp10 and Rhsp10. With the use of synthesized peptides of the determined core regions, it was also confirmed that the identified regions were capable of fibril formation. These findings suggested that GroES homologues formed typical amyloid fibrils under acidic unfolding conditions but that the fibril core structures were different, perhaps owing to differences in local amino acid sequences.     

Small intestine CD4+ T cells are profoundly depleted during acute simian-human immunodeficiency virus infection, regardless of viral pathogenicity

To analyze the relationship between acute virus-induced injury and the subsequent disease phenotype, we compared the virus replication and CD4(+) T-cell profiles for monkeys infected with isogenic highly pathogenic (KS661) and moderately pathogenic (#64) simian-human immunodeficiency viruses (SHIVs). Intrarectal infusion of SHIV-KS661 resulted in rapid, systemic, and massive virus replication, while SHIV-#64 replicated more slowly and reached lower titers. Whereas KS661 systemically depleted CD4(+) T cells, #64 caused significant CD4(+) T-cell depletion only in the small intestine. We conclude that SHIV, regardless of pathogenicity, can cause injury to the small intestine and leads to CD4(+) T-cell depletion in infected animals during acute infection.