NB-506, an indolocarbazole topoisomerase I inhibitor, binds preferentially to triplex DNA

A novel competition dialysis method was used to study the structural selectivity of the nucleic acid binding of NB-506, a promising indolocarbazole anticancer agent. A pronounced preference for NB-506 binding to the DNA triplex poly [dA]:(poly[dT])(2) was observed among potential binding to 12 different nucleic acid structures and sequences. Structures included in the assay ranged from single-stranded DNA, through a variety of right-handed DNA duplexes, to multistranded triplex and tetraplex forms. RNA and left-handed Z DNA were also included in the assay. The preferential binding to triplex was confirmed by UV melting experiments. The novel and unexpected structural selectivity shown by NB-506 may arise from a complementary shape between its extended aromatic ring system and the planar triplex stack.     

A high-throughput strategy to screen 2D crystallization trials of membrane proteins

Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for detergent removal and reconstitution of the protein into a lipid bilayer, and devices for testing numerous conditions in parallel are not readily available. Furthermore, the small size of resulting 2D crystals requires electron microscopy to evaluate the results and automation of the necessary steps is essential to achieve a reasonable throughput. We have designed a crystallization block, using standard microplate dimensions, by which 96 unique samples can be dialyzed simultaneously against 96 different buffers and have demonstrated that the rate of detergent dialysis is comparable to those obtained with conventional dialysis devices. A liquid-handling robot was employed to set up 2D crystallization trials with the membrane proteins CopA from Archaeoglobus fulgidus and light-harvesting complex II (LH2) from Rhodobacter sphaeroides. For CopA, 1 week of dialysis yielded tubular crystals and, for LH2, large and well-ordered vesicular 2D crystals were obtained after 24 h, illustrating the feasibility of this approach. Combined with a high-throughput procedure for preparation of EM-grids and automation of the subsequent negative staining step, the crystallization block offers a novel pipeline that promises to speed up large-scale screening of 2D crystallization and to increase the likelihood of producing well-ordered crystals for analysis by electron crystallography.     

葡萄酒发酵过程中酵母菌之间相互抑制作用的研究

目的确定葡萄酒发酵过程中不同酵母间相互抑制作用产生的原因。方法采用透析袋发酵法通过单因素实验、上清液抑菌实验和双向电泳等方法,确立溶氧、酒精度、pH、氮源、生存空间以及分泌蛋白等因素对非酿酒酵母衰亡的诱导作用。结果溶氧、酒精、pH、氮源及生存空间的竞争并不是非酿酒酵母提前衰亡的主要原因,酿酒酵母菌产生的代谢产物对非酿酒酵母的提前衰亡具有很强的诱导作用。结论酿酒酵母分泌的分子量小于10kDa的代谢产物和一些分子量超过10 kDa的蛋白类物质均对克鲁维酵母等非酿酒酵母的衰亡具有诱导作用。     

Alternative rib bone biopsy measurements to estimate changes in skeletal mineral reserves in cattle

Rib bone biopsy samples are often used to estimate changes in skeletal mineral reserves in cattle but differences in sampling procedures and the bone measurements reported often make interpretation and comparisons among experiments difficult. ‘Full-core’ rib bone biopsy samples, which included the external cortical bone, internal cortical bone and trabecular bone (CB_ext, CB_int and Trab, respectively), were obtained from cattle known to be in phosphorus (P) adequate (P_adeq) or severely P-deficient (P_defic) status. Experiments 1 and 2 examined growing steers and Experiment 3 mature breeder cows. The thickness of cortical bone, specific gravity (SG), and the amount and concentration of ash and P per unit fresh bone volume, differed among CB_ext, CB_int and Trab bone. P concentration (mg/cc) was closely correlated with both SG and ash concentrations (pooled data, r=0.99). Thickness of external cortical bone (CBT_ext) was correlated with full-core P concentration (FC-P_conc) (pooled data, r=0.87). However, an index, the amount of P in CB_ext per unit surface area of CB_ext (PSACB; mg P/mm²), was more closely correlated with the FC-P_conc (pooled data, FC-P_conc=37.0+146×PSACB; n=42, r=0.94, RSD=7.7). Results for measured or estimated FC-P_conc in 10 published studies with cattle in various physiological states and expected to be P_adeq or in various degrees of P_defic status were collated and the ranges of FC-P_conc indicative of P adequacy and P deficiency for various classes of cattle were evaluated. FC-P_conc was generally in the range 130 to 170 and 100 to 120 mg/cc fresh bone in P_adeq mature cows and young growing cattle, respectively. In conclusion, the FC-P_conc could be estimated accurately from biopsy samples of CB_ext. This allows comparisons between studies where full-core or only CB_ext biopsy samples of rib bone have been obtained to estimate changes in the skeletal P status of cattle and facilitates evaluation of the P status of cattle.     

Selective Release of Spermine and Spermidine from the Rat Striatum by N-Methyl-d-Aspartate Receptor Activation In Vivo

The intrastriatal infusion of N-methyl-D-aspartate (NMDA; 250-1,000 microM) via a dialysis cannula in anesthetized rats resulted in a marked and rapid increase in the concentrations of spermine and spermidine recovered in the dialysate. Extracellular concentrations of NMDA-released spermine and spermidine were calculated to be in the low micromolar range. Putrescine levels were not significantly affected by NMDA. The effects of NMDA (500 microM) were blocked by the previous systemic injection of MK-801 (3 mg/kg, i.p.) but were insensitive to the intrastriatal infusion of tetrodotoxin (1 microM). Intrastriatally infused kainate or quisqualate (1,000 microM) did not increase polyamine levels in the dialysate. Spermine and spermidine dialysate levels were also significantly increased by the infusion of high concentrations of K+ (greater than 100 mM), although the effects of K+ were considerably less marked than those of NMDA. Striatal polyamines are released into the extracellular space specifically by NMDA receptor activation. Because of their multiple effects on receptor- and voltage-operated cation channels, polyamines that are released by NMDA receptor activation may play an important role in phenomena already attributed to NMDA receptor stimulation, such as long-term potentiation, synaptic plasticity, and neurotoxicity.     

Inadequate cervical cytology—the need to audit individual smear takers'' inadequate rates

In order to assess the performance of individual smear takers, their rates of inadequate smears, due to insufficient material, were monitored between 1992 and 1996. Although the yearly average clinic inadequate rates were all at acceptable levels, there was great variation of rates between smear takers, ranging from 0% to 19.5%. Identification of individuals with unacceptably high inadequate rates allowed them to undergo further training. Continuous monitoring of these inadequate rates can be used to measure levels of performance amongst all smear takers, but is particularly useful for new smear takers, or those who have required retraining.     

The importance of species range attributes and reserve configuration for the conservation of angiosperm diversity in Western Australia

In order to better understand the relationship between reserve design and the species represented by such designs, we examined the effectiveness of the Western Australian reserve system for conserving angiosperm diversity, and examined the characteristics of those species conserved. We overlayed species distribution data for 14 plant lineages with the distribution of the reserve system (8.5% of the State’s area) and identified the species that remained unprotected. We found that, depending upon the method employed, between 174 (5.7%) and 570 (18.7%) of species were not included within the reserve system. Two main unprotected regions were identified, one of which was also a centre of high diversity. Geographical range sizes of unprotected species were six times smaller than those species that were protected, while species richness of small-ranged endemic species coincided with general patterns of species richness. At the level of Western Australia’s bioregions we found that conservation effectiveness was most dependent on characteristics of the reserve system rather than characteristics (size and positioning) of species ranges. At this scale, the most effective way to conserve more species in Western Australia would be to conserve more land, while conservation would be most successful in a uniformly dispersed reserve system. Our results highlight the fact that reserve systems may take on two design approaches based on scale––at continental scales, reserves should be clustered around the hotspots of endemic species, while within regions, an evenly distributed reserve system will most adequately sample species.