Components of the human spindle checkpoint control mechanism localize specifically to the active centromere on dicentric chromosomes

The spindle checkpoint control mechanism functions to ensure faithful chromosome segregation by delaying cell division until all chromosomes are correctly oriented on the mitotic spindle. Initially identified in budding yeast, several mammalian spindle checkpoint-associated proteins have recently been identified and partially characterized. These proteins associate with all active human centromeres, including neocentromeres, in the early stages of mitosis prior to the commencement of anaphase. We have examined the status of proteins associated with the checkpoint protein complex (BUB1, BUBR1, BUB3, MAD2), the anaphase-promoting complex (Tsg24, p55CDC), and other proteins associated with mitotic checkpoint control (ERK1, 3F3/2 epitope, hZW10), on a human dicentric chromosome. Each of these proteins was found to specifically associate with only the active centromere, suggesting that only active centromeres participate in the spindle checkpoint. This finding complements previous studies on multicentric chromosomes demonstrating specific association of structural and motor-related centromere proteins with active centromeres, and suggests that centromere inactivation is accompanied by loss of all functionally important centromere proteins.     

High Temperature Stress Resistance of Escherichia coli Induced by a Tobacco Class I Low Molecular Weight Heat-Shock Protein

TLHS1 is a class I low molecular weight heat-shock protein (LMW HSP) of tobacco (Nicotiana tabacum). For a functional study of TLHS1, a recombinant DNA coding for TLHS1 with a hexahistidine tag at the aminoterminus was constructed and expressed in Escherichia coli. An expressed fusion protein, H₆TLHS1, was purified using a Ni²⁺ affinity column and a Sephacryl S400 HR column. A polyclonal antibody against H₆TLHS1 was produced to follow the fate of H₆TLHS1 in E. coli. The fusion protein in E. coli maintained its solubility at a temperature of up to 90°C and most of the proteins in the E. coli cell lysate with H₆TLHS1 were prevented from thermally induced aggregation at up to 90°C. We compared the viability of E. coli cells expressing H₆TLHS1 to the E. coli cells without H₆TLHS1 at a temperature of 50°C. After 8 h of high temperature treatment, E. coli cells with H₆TLHS1 survived about three thousand times more than the bacterial cells without H₆TLHS1. These results showed that a plant class I LMW HSP, TLHS1, can protect proteins of E. coli from heat denaturation, which could lead to a higher survival rate of the bacterial cells at high temperature.     

Tumor Suppressor WWOX Contributes to the Elimination of Tumorigenic Cells in Drosophila melanogaster

WWOX is a >1Mb gene spanning FRA16D Common Chromosomal Fragile Site, a region of DNA instability in cancer. Consequently, altered WWOX levels have been observed in a wide variety of cancers. In vitro studies have identified a large number and variety of potential roles for WWOX. Although its normal role in vivo and functional contribution to cancer have not been fully defined, WWOX does have an integral role in metabolism and can suppress tumor growth. Using Drosophila melanogaster as an in vivo model system, we find that WWOX is a modulator of TNFα/Egr-mediated cell death. We found that altered levels of WWOX can modify phenotypes generated by low level ectopic expression of TNFα/Egr and this corresponds to altered levels of Caspase 3 activity. These results demonstrate an in vivo role for WWOX in promoting cell death. This form of cell death is accompanied by an increase in levels of reactive oxygen species, the regulation of which we have previously shown can also be modified by altered WWOX activity. We now hypothesise that, through regulation of reactive oxygen species, WWOX constitutes a link between alterations in cellular metabolism observed in cancer cells and their ability to evade normal cell death pathways. We have further shown that WWOX activity is required for the efficient removal of tumorigenic cells from a developing epithelial tissue. Together these results provide a molecular basis for the tumor suppressor functions of WWOX and the better prognosis observed in cancer patients with higher levels of WWOX activity. Understanding the conserved cellular pathways to which WWOX contributes provides novel possibilities for the development of therapeutic approaches to restore WWOX function in cancer.     

Radioprotective Effect of Aminothiol PrC-210 on Irradiated Inner Ear of Guinea Pig

Radiotherapy of individuals suffering with head & neck or brain tumors subserve the risk of sensorineural hearing loss. Here, we evaluated the protective effect of Aminothiol PrC-210 (3-(methyl-amino)-2-((methylamino)methyl)propane-1-thiol) on the irradiated inner ear of guinea pigs. An intra-peritoneal or intra-tympanic dose of PrC-210 was administered prior to receiving a dose of gamma radiation (3000 cGy) to each ear. Auditory Brainstem Responses (ABRs) were recorded one week and two weeks after the radiation and compared with the sham animal group. ABR thresholds of guinea pigs that received an intra-peritoneal dose of PrC-210 were significantly better compared to the non-treated, control animals at one week post-radiation. Morphologic analysis of the inner ear revealed significant inflammation and degeneration of the spiral ganglion in the irradiated animals not treated with PrC-210. In contrast, when treated with PrC-210 the radiation effect and injury to the spiral ganglion was significantly alleviated. PrC-210 had no apparent cytotoxic effect in vivo and did not affect the morphology or count of cochlear hair cells. These findings suggest that aminothiol PrC-210 attenuated radiation-induced cochlea damage for at least one week and protected hearing.     

Temperature and dose effects on the pathogenicity and reproduction of two Korean isolates of Heterorhabditis bacteriophora (Nematoda: Heterorhabditidae)

Out of some isolated Heterorhabditis bacteriophora from Korea, ecological study on two isolates which had different geographical features was investigated. That is, effects of temperature and dose on the pathogenicity and reproduction of two Korean isolates of H. bacteriophora were investigated using Galleria mellonella larvae in the laboratory. The median lethal dose (LD₅₀) decreased with increasing temperature, but increased at 35 °C. The optimal temperatures for infection were 30 °C for H. bacteriophora Jeju strain and 24 °C for H. bacteriophora Hamyang strain. The median lethal time, LT₅₀ of H. bacteriophora Hamyang strain was recorded at 13 °C to 35 °C and that of H. bacteriophora Jeju strain was recorded at 18 °C to 30 °C. The number of established nematodes in G. mellonella larvae was significantly different depending on temperature and dose. When G. mellonella larvae were exposed to 300 infective juveniles (IJs), mortality of G. mellonella gradually increased with exposure time with H. bacteriophora Jeju strain but not with H. bacteriophora Hamyang strain. 87.5% mortality of G. mellonella was recorded by H. bacteriophora Hamyang strain after 1440 min whereas 100% mortality was recorded by H. bacteriophora Jeju strain after 4320 min. The time from infection to the first emergence of nematodes decreased with increasing temperature. Duration of emergence of the two strains in the White traps also decreased with increasing temperature. The highest progeny numbers of H. bacteriophora Jeju strain were 264,602 while those of H. bacteriophora Hamyang strain were 275,744 at the rate of 160 IJs at 24 °C.     

Rapid molecular authentication of three medicinal plant species, Cynanchum wilfordii, Cynanchum auriculatum, and Polygonum multiflorum (Fallopia multiflorum), by the development of RAPD-derived SCAR markers and multiplex-PCR

Definitive identification of original plant species is important for standardizing herbal medicine. The herbal medicines Cynanchi Wilfordii Radix (Baekshuoh in Korean and Beishuwu in Chinese) and Polygoni Multiflori Radix (Hashuoh in Korean and Heshuwu in Chinese) are often misidentified in the Korean herbal market due to morphological similarities and similar names. Therefore, we developed a reliable molecular marker for the identification of Cynanchi Wilfordii Radix and Polygoni Multiflori Radix. We used random amplified polymorphic DNA (RAPD) analysis of three plant species, Polygoni multiflorum, Cynanchum wilfordii, and Cynanchum auriculatum, to obtain several species-specific RAPD amplicons. From nucleotide sequences of these RAPD amplicons, we developed six sequence characterized amplification region (SCAR) markers for distinguishing Polygoni Multiflori Radix and Cynanchi Wilfordii Radix. Furthermore, we established SCAR markers for the simultaneous discrimination of the three species within a single reaction by using multiplex-PCR. These SCAR markers can be used for efficient and rapid authentication of these closely related species, and will be useful for preventing the distribution of adulterants.     

Hydrogen metabolism in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 hydrogenases, hydA, encoding a periplasmic [Fe-Fe] hydrogenase, and hyaB, encoding a periplasmic [Ni-Fe] hydrogenase, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of DeltahydA, DeltahyaB, and DeltahydA DeltahyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming hydrogenase while HyaB has a bifunctional role and represents the dominant hydrogenase activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA hydrogenase and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions.