Identifying patterns of DNA for tumor diagnosis using capillary electrophoresis-amplified fragment length polymorphism (CE-AFLP) screening

Amplified Fragment Length Polymorphism (AFLP) screening is a genome-wide genotyping strategy that has been widely used in plants and bacteria, but little has been reported concerning its use in humans. We investigated if the AFLP procedure could be coupled with high-throughput capillary electrophoresis (CE) for use in tumor diagnosis and classification. Using CE-AFLP, a series of molecular 'fingerprints' were generated for a set of gastric tumor and normal genomic DNA samples. The CE-AFLP procedure was qualitatively and quantitatively robust, and a variety of clustering tools were used to identify a specific DNA marker 'pattern' of 20 features that classified the tumor and normal samples to reasonable degrees of accuracy (Sensitivity 95%, Specificity 80%). The CE-AFLP-based approach also correctly classified 16 tumor samples, which in a previous study had exhibited no detectable genomic aberrations by comparative genome hybridization (CGH). This is the first reported application of CE-AFLP screening in tumor diagnosis. As the procedure is relatively inexpensive and requires minimal prior sequence knowledge and biological material, we suggest that CE-AFLP-based protocols may represent a promising new approach for DNA-based cancer screening and diagnosis.     

Limitations to net photosynthesis as affected by nitrogen status in jack pine (Pinus banksiana Lamb.) seedlings

Relative limitations of nitrogen (N) status on the processescontributing to photosynthetic rate (A) were investigated. Jackpine {Pinus banksiana Lamb.) seedlings from seeds grown in sandculture were supplied with four different N treatments for 6weeks, which resulted in a needle N content ranging from 50–85mmol m^–2 (14–32 mg g^–1 dry weight). Leaf gasexchange at varying CO₂ levels was measured and limitationson A₃₅₀ (A at ambient CO₂ level) caused by finite, limitingcarboxylation efficiency (c.e.), maximum A (A_max)and stomatalconductance were estimated from an analysis of the responseof A to internal CO₂ concentration. Although c.e. and A_max decreasedlinearly with the decline in needle N, the magnitudes of theirchanges relative to A₃₅₀ differed. A_max varied with A₃₅₀ andalways exceeded A₃₅₀ by 37–38% c.e., however, declinedfaster than A₃₅₀, as needle N level decreased. Consequently,relative limitation on A₃₅₀ caused by inefficient A_max remainedconstant, but limitations caused by c.e. increased by 10–15%at low N levels. In contrast, the limitation by stomatal conductancedeclined initially, but remained stable when N content droppedbelow 75 mmol m^–2. The results suggest: (1) a decreasein biochemical capacity, but not stomatal conductance, contributedto the reduction of A₃₅₀ induced by N-deficiency in jack pineseedlings; and (2) the capacity of carboxylation appeared tobe impaired more than that of electron transport and/or photophosphorylationand its reduction may be the major reason for the reductionin A₃₅₀. Key words: A–C_i analysis, carboxylation efficiency, electron transport, nitrogen deficiency, stomatal conductance     

The effects of delayed activation and MG132 treatment on nuclear remodeling and preimplantation development of embryos cloned by electrofusion are correlated with the age of recipient cytoplasts

The electrofusion method, used extensively in livestock cloning, cannot be used in mice, because it is believed that the mouse oocytes are more susceptible to detrimental effects of electrical stimulus than oocytes from other species. Reports on whether a delayed activation after electrofusion and a premature chromosome condensation (PCC) is essential for efficient cloning are inconclusive. To address these issues, effects of pulsing on activation and MPF activity of nonenucleated oocytes and effects of delayed activation and MG132 treatment on donor nuclear PCC and preimplantation development of embryos cloned by electrofusion or nuclear injection were compared between different cytoplast ages in mice and goats. The results indicated that the use of oocytes collected early after donor stimulation would make it possible to conduct somatic cell nuclear transfer in mice by electrofusion. Whether a delayed activation is essential was dependent upon the age, or rather, the level, of MPF activity of the cytoplasts at the time of electrofusion, as was the requirement for MG132 treatment. The competence for blastocyst formation of cloned embryos was highly correlated with the level of donor nuclear PCC in recipient cytoplasts. The nuclear injection technique was more adaptable to older cytoplast ages, and hence less dependent on drugs for inhibition of MPF inactivation, compared to electrofusion.     

The stability of impulsive stochastic Cohen–Grossberg neural networks with mixed delays and reaction–diffusion terms

The global asymptotic stability of impulsive stochastic Cohen–Grossberg neural networks with mixed delays and reaction–diffusion terms is investigated. Under some suitable assumptions and using Lyapunov–Krasovskii functional method, we apply the linear matrix inequality technique to propose some new sufficient conditions for the global asymptotic stability of the addressed model in the stochastic sense. The mixed time delays comprise both the time-varying and continuously distributed delays. The effectiveness of the theoretical result is illustrated by a numerical example.     

Identification and characterization of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase

UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo.     

发酵法生产S-腺苷蛋氨酸前体蛋氨酸补加策略

利用酿酒酵母菌株高密度发酵法生产S-腺苷蛋氨酸关键的影响因素之一是前体L-蛋氨酸的补加策略.本研究采用一支经过常规诱变处理的S-腺苷蛋氨酸优势积累菌株酿酒酵母SAM0801,通过5 L发酵罐高密度发酵实验研究,考察了6种补加策略,最终确定了L-蛋氨酸的加入时机为30h左右,当茵体干重达到100g/L时,补加量为每罐40gL-蛋氨酸,发酵58 h左右达到最高生物量干重168 g/L,产量14.48 g/L.