Murine p53 inhibits the function but not the formation of SV40 T antigen hexamers and stimulates T antigen RNA helicase activity

We have characterized the effects of p53 on several biochemical activities of simian virus 40 (SV40) large tumor (T) antigen. While p53 induced a strong inhibition of the T antigen DNA helicase activity, surprisingly, its RNA helicase activity was stimulated. This supports the liklihood that the DNA and RNA helicase activities of T antigen reflect discrete functions. p53 did not significantly affect the ATP-dependent conversion of T antigen monomers to hexamers. However, the ability of these hexamers to assemble on a DNA fragment containing the viral origin was impaired by p53. Thus, these results suggest that p53 inhibits the function but not the formation of T antigen multimers. This conclusion was further supported by the observation that the addition of a purified p53:T antigen complex was as inhihitory as free p53 to the DNA helicase activity of free T antigen. Thus our data indicates that the targets of p53 inhibition are the functional units of T antigen, namely the hexamers.     

低温胁迫下凤眼莲叶片的适应——脱落酸和可溶性蛋白质含量升高

本文报道低温胁迫下风眼莲叶片脱落酸(ABA)、可溶性蛋白质和水势的测定结果。低温胁迫时脱落酸和可溶性蛋白质含量远高于对照,(前者含量最高可达对照的4倍,后者可达到对照的2.75倍),而且脱落酸和蛋白质含量随温度降低而升高。蛋白质的生物合成抑制剂亚胺环己酮证明,可溶性蛋白质含量升高,原因是有部分是新合成的。在各种低温处理下获得了几乎相同于对照的叶片水势。我们推测:低温胁迫下,脱落酸水平的相应变化不是由于低温诱导水分胁迫所致,而是低温胁迫本身诱导。     

Defining the minimum size of a hydrophobic cluster in two-stranded alpha-helical coiled-coils: effects on protein stability

The alpha-helical coiled-coil motif is characterized by a heptad repeat pattern (abcdefg)(n) in which residues a and d form the hydrophobic core. Long coiled-coils (e.g., tropomyosin, 284 residues per polypeptide chain) typically do not have a continuous hydrophobic core of stabilizing residues, but rather one that consists of alternating clusters of stabilizing and destabilizing residues. We have arbitrarily defined a cluster as a minimum of three consecutive stabilizing or destabilizing residues in the hydrophobic core. We report here on a series of two-stranded, disulfide-bridged parallel alpha-helical coiled-coils that contain a central cassette of three consecutive hydrophobic core positions (d, a, and d) with a destabilizing cluster of three consecutive Ala residues in the hydrophobic core on each side of the cassette. The effect of adding one to three stabilizing hydrophobes in these positions (Leu or Ile; denoted as [see text]) was investigated. Alanine residues (denoted as [see text]) are used to represent destabilizing residues. The peptide with three Ala residues in the d a d cassette positions ([see text]) was among the least stable coiled-coil (T(m) = 39.3 degrees C and Urea(1/2) = 1.9 M). Surprisingly, the addition of one stabilizing hydrophobe (Leu) to the cassette or two stabilizing hydrophobes (Leu), still interspersed by an Ala in the cassette ([see text]), also did not lead to any gain in stability. However, peptides with two adjacent hydrophobes in the cassette ([see text])([see text]) did show a gain in stability of 0.9 kcal/mole over the peptide with two interspersed hydrophobes ([see text]). Because the latter three peptides have the same inherent hydrophobicity, the juxtaposition of stabilizing hydrophobes leads to a synergistic effect, and thus a clustering effect. The addition of a third stabilizing hydrophobe to the cassette ([see text]) resulted in a further synergistic gain in stability of 1.7 kcal/mole (T(m) = 54.1 degrees C and Urea(1/2) = 3.3M). Therefore, the role of hydrophobicity in the hydrophobic core of coiled-coils is extremely context dependent and clustering is an important aspect of protein folding and stability.     

Morphological Response of the Halophilic Fungal Genus Wallemia to High Salinity

The basidiomycetous genus Wallemia is an active inhabitant of hypersaline environments, and it has recently been described as comprising three halophilic and xerophilic species: Wallemia ichthyophaga, Wallemia muriae, and Wallemia sebi. Considering the important protective role the fungal cell wall has under fluctuating physicochemical environments, this study was focused on cell morphology changes, with particular emphasis on the structure of the cell wall, when these fungi were grown in media with low and high salinities. We compared the influence of salinity on the morphological characteristics of Wallemia spp. by light, transmission, and focused-ion-beam/scanning electron microscopy. W. ichthyophaga was the only species of this genus that was metabolically active at saturated NaCl concentrations. W. ichthyophaga grew in multicellular clumps and adapted to the high salinity with a significant increase in cell wall thickness. The other two species, W. muriae and W. sebi, also demonstrated adaptive responses to the high NaCl concentration, showing in particular an increased size of mycelial pellets at the high salinities, with an increase in cell wall thickness that was less pronounced. The comparison of all three of the Wallemia spp. supports previous findings relating to the extremely halophilic character of the phylogenetically distant W. ichthyophaga and demonstrates that, through morphological adaptations, the eukaryotic Wallemia spp. are representative of eukaryotic organisms that have successfully adapted to life in extremely saline environments.Hypersaline habitats had long been considered to be populated almost exclusively by prokaryotic organisms and the research on hypersaline environments had consequently been monopolized by bacteriologists. In 2000, the first reports appeared showing that fungi are active inhabitants of solar salterns (20). Until then, fungi able to survive in environments with a low amount of biologically available water (low water activity [a_w]) were only known as contaminants of foods preserved with high concentrations of salt or sugar. Since their first discovery in salterns, many new species have been discovered in natural hypersaline environments around the world, including some species that were previously known only as food-borne contaminants. Due to these discoveries, fungi are now recognized as an integral part of indigenous halophilic microbial communities since they can grow and adjust across the whole salinity range, from freshwater to almost saturated NaCl solutions (49). Most fungi differ from the majority of halophilic prokaryotes (16): they tend to be extremely halotolerant rather than halophilic and do not require salt to remain viable, with the exception of Wallemia spp.The order Wallemiales (Wallemiomycetes, Basidiomycota) was only recently introduced to define the single genus Wallemia, a phylogenetic maverick in the Basidiomycota (49). Until 2005, this genus contained only the species W. sebi. However, taxonomic analyses of isolates from sweet, salty, and dried foods (41) and from hypersaline evaporation ponds in the Mediterranean Sea, the Caribbean, and the Dead Sea (45, 49) have resolved this genus into three species: W. ichthyophaga, W. muriae, and W. sebi. The first two of these three Wallemia spp. require additional solutes in the growth media, and W. ichthyophaga is the most halophilic eukaryote described to date, since it cannot grow without the addition of 9% NaCl (wt/vol), and it still shows growth at a_w of 0.77, equivalent to 30% NaCl (wt/vol) (49).The survival, and especially the growth, of microorganisms in highly saline environments requires numerous adaptations (6, 18, 21, 34). The dominant representatives and the most thoroughly investigated halophilic fungi in hypersaline waters of the salterns are the black yeasts, and particularly the model organism Hortaea werneckii (20). An important level of adaptation of the black yeasts to high salinity is seen in their extremophilic ecotype, which is characterized by a special meristematic morphology and changes in cell wall structure and pigmentation (27). Other fungal osmoadaptations include the accumulation of osmolytes (27, 28, 40), the extrusion of sodium (5), modification of the plasma membrane (44) and the cell wall, and even changes in fungal colony morphology (27).The fungal cell wall is the first line of defense against environmental stress; therefore, adaptation at the cell wall level is expected to have one of the most important roles for successful growth at a low a_w (24, 32). The cell wall is essential for maintaining the osmotic homeostasis of cells, since it protects them against mechanical damage as well as high concentrations of salts (7). The central fibrillar glycan network of the cell wall is embedded in highly flexible amorphous cement, which allows considerable stretching with changing osmotic pressure (14, 29). Its balance between a rigid and a dynamic structure influences the shape of cells (14) and enables growth and hyphal branching (11).Since the species within the genus Wallemia have been recognized only recently (49), little is known about their mechanisms of adaptation to high salinity. To investigate the effects of low and high NaCl concentrations on cell morphology, with particular emphasis on cell wall ultrastructure, we compared W. ichthyophaga, the most halophilic fungal species known thus far, with the related xerophilic W. muriae and W. sebi. Micrographs were prepared by using light, transmission, and scanning electron microscopy. The results reveal how this eukaryotic genus uses adaptations at the cell wall level for thriving in extremely saline environments.     

薜荔（Ficus pumila L.）传粉的代价

种间关系中一方以自身生长过程中的一部分物质和能量为对方所利用的现象便是一个物种为对方付出的代价。在中性的种间关系中,两个物种彼此不受影响,无所谓代价;在偏性的种间关系中,受害者付出无谓的代价,受益者得到无偿的利益（如寄主与寄生物、被捕食者与捕食者之间...     

成人及新生儿颅骨铜和锌含量的初步研究

本文系用原子吸收光谱法测定了西安地区10例成人尸体和15例新生儿尸体的颅盖骨内Cu和zn的含量。结果求得成人颅骨中Cu正常含量(均数±标准差)为4.48±3.78mg/kg(干组织重);zn为597.05±472.54mg/kg。新生儿颅骨中Cu和zn的含量分别为1.96±0.76mg/kg;1160.38±859.71mg/kg。结果表明,成人颅骨内Cu含量高于新生儿,而成人颅骨内Zn含量显著低于新生儿。     

An ordered EST catalogue and gene expression profiles of cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis>) at key growth stages

A cDNA library was constructed from the root tissues of cassava variety Huanan 124 at the root bulking stage. A total of 9,600 cDNA clones from the library were sequenced with single-pass from the 5′-terminus to establish a catalogue of expressed sequence tags (ESTs). Assembly of the resulting EST sequences resulted in 2,878 putative unigenes. Blastn analysis showed that 62.6% of the unigenes matched with known cassava ESTs and the rest had no ‘hits’ against the cassava database in the integrative PlantGDB database. Blastx analysis showed that 1,715 (59.59%) of the unigenes matched with one or more GenBank protein entries and 1,163 (40.41%) had no ‘hits’. A cDNA microarray with 2,878 unigenes was developed and used to analyze gene expression profiling of Huanan 124 at key growth stages including seedling, formation of root system, root bulking, and starch maturity. Array data analysis revealed that (1) the higher ratio of up-regulated ribosome-related genes was accompanied by a high ratio of up-regulated ubiquitin, proteasome-related and protease genes in cassava roots; (2) starch formation and degradation simultaneously occur at the early stages of root development but starch degradation is declined partially due to decrease in UDP-glucose dehydrogenase activity with root maturity; (3) starch may also be synthesized in situ in roots; (4) starch synthesis, translocation, and accumulation are also associated probably with signaling pathways that parallel Wnt, LAM, TCS and ErbB signaling pathways in animals; (5) constitutive expression of stress-responsive genes may be due to the adaptation of cassava to harsh environments during long-term evolution.     

Neurons and Neuronal Stem Cells Survive in Glucose-Free Lactate and in High Glucose Cell Culture Medium During Normoxia and Anoxia

Several questions concerning the survival of isolated neurons and neuronal stem and progenitor cells (NPCs) have not been answered in the past: (1) If lactate is discussed as a major physiological substrate of neurons, do neurons and NPCs survive in a glucose-free lactate environment? (2) If elevated levels of glucose are detrimental to neuronal survival during ischemia, do high concentrations of glucose (up to 40 mmol/L) damage neurons and NPCs? (3) Which is the detrimental factor in oxygen glucose deprivation (OGD), lack of oxygen, lack of glucose, or the combination of both? Therefore, in the present study, we exposed rat cortical neurons and NPCs to different concentrations of d-glucose ranging from 0 to 40 mmol/L, or 10 and 20 mmol/L l-lactate under normoxic and anoxic conditions, as well as in OGD. After 24 h, we measured cellular viability by biochemical assays and automated cytochemical morphometry, pH values, bicarbonate, lactate and glucose concentrations in the cell culture media, and caspases activities. We found that (1) neurons and NPCs survived in a glucose-free lactate environment at least up to 24 h, (2) high glucose concentrations >5 mmol/L had no effect on cell viability, and (3) cell viability was reduced in normoxic glucose deprivation to 50% compared to 10 mmol/L glucose, whereas cell viability in OGD did not differ from that in anoxia with lactate which reduced cell viability to 30%. Total caspases activities were increased in the anoxic glucose groups only. Our data indicate that (1) neurons and NPCs can survive with lactate as exclusive metabolic substrate, (2) the viability of isolated neurons and NPCs is not impaired by high glucose concentrations during normoxia or anoxia, and (3) in OGD, low glucose concentrations, but not low oxygen levels are detrimental for neurons and NPCs.