Determination of the thermogenesis curves and studies of the thermodynamics and thermokinetics of seed germination

The thermogenesis curves of the germination of different rice and tree seeds were determined and studied by using a newly constructed microcalorimeter. The thermogenesis curves of the germination of the seeds demonstrate the existence of physiological triphasic patterns, which include imbibition, activation and growth stages in the germination process. The thermodynamics and thermokinetics of the main growth phase of the growth stage in the germination process have been studied. The growth heat effect (deltaH), the growth rate constant (k), the growth inhibitory factor (s) and deceleration rate constant (beta) have been determined and calculated, In addition, the experimental thermokinetic equations of the growth stage in the seed germination process have been established.     

Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex

Bacteriophage T4 deoxycytidylate hydroxymethylase (EC 2.1.2.8), a homodimer of 246-residue subunits, catalyzes hydroxymethylation of the cytosine base in deoxycytidylate (dCMP) to produce 5-hydroxymethyl-dCMP. It forms part of a phage DNA protection system and appears to function in vivo as a component of a multienzyme complex called deoxyribonucleoside triphosphate (dNTP) synthetase. We have determined its crystal structure in the presence of the substrate dCMP at 1.6 A resolution. The structure reveals a subunit fold and a dimerization pattern in common with thymidylate synthases, despite low (approximately 20%) sequence identity. Among the residues that form the dCMP binding site, those interacting with the sugar and phosphate are arranged in a configuration similar to the deoxyuridylate binding site of thymidylate synthases. However, the residues interacting directly or indirectly with the cytosine base show a more divergent structure and the presumed folate cofactor binding site is more open. Our structure reveals a water molecule properly positioned near C-6 of cytosine to add to the C-7 methylene intermediate during the last step of hydroxymethylation. On the basis of sequence comparison and crystal packing analysis, a hypothetical model for the interaction between T4 deoxycytidylate hydroxymethylase and T4 thymidylate synthase in the dNTP-synthesizing complex has been built.     

Activating mutation of GS alpha in McCune-Albright syndrome causes skin pigmentation by tyrosinase gene activation on affected melanocytes

McCune-Albright syndrome (MAS) is a sporadic disease characterized by café-au-lait spots, polyostotic fibrous dysplasia and hyperfunctional endocrinopathies. To elucidate the mechanism of skin pigmentation, melanocytes, keratinocytes and fibroblasts were primary cultured from the café-au-lait spot of a MAS patient. Then, mutational analysis and morphologic evaluation were performed. Also, cAMP level and tyrosinase gene expression in cultured cells were determined. Only Gsalpha mutation was found in affected melanocytes and the cAMP level in affected melanocytes was higher than that of normal melanocytes. The mRNA expression of tyrosinase gene was increased in the affected melanocytes. This study suggests that skin pigmentation of MAS results from activating mutation of Gsalpha in melanocytes and the mechanism involves the c-AMP-mediated tyrosinase gene activation.     

微透析测试刺激皮肤和肌肉神经引起的天门冬氨酸和谷氨酸在脊髓的 …

To investigate the possible mechanisms underlying the difference of NMDA and non-NMDA receptors in spinal nociception originating in skin and muscle, release of aspartate (Asp) and glutamate (Glu) in the spinal dorsal horn was detected by stimulation of cutaneous and muscular nerves in cats using microdialysis technique. Asp and Glu were increased respectively by (323 +/- 55)% and (169 +/- 16)% following stimulation of cutaneous nerve, but by (150 +/- 16)% and (218 +/- 42)% respectively following stimulation of muscular nerve. Asp increase was approximately three times higher than that of Glu following cutaneous nerve-stimulation (P < 0.01), while Glu increase was approximately twice as high as that of Asp following muscular nerve-stimulation (P < 0.05). It is likely that nociceptive cutaneous and muscular inputs preferentially elicite release of Asp and Glu respectively, resulting in a functional differentiation of NMDA and non-NMDA receptor in the mediation of different nociceptive information.     

Investigation of extended-spectrum beta-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli in Korea

AIMS: Isolates obtained from various regions in Korea in 2002 were identified and their susceptibility to extended-spectrum cephalosporins, monobactams and/or cephamycins was studied along with any production of extended-spectrum beta-lactamases (ESBLs). METHODS AND RESULTS: Bacteria identified by the conventional techniques and Vitek GNI card were Klebsiella pneumoniae and Escherichia coli. Using disk diffusion and double-disk synergy tests, we found that 39.2% of strains produced ESBLs. About 52% of isolates transferred resistance to ceftazidime by conjugation. Banding patterns of PCR amplification with the designed primers showed that 837- and 259-bp fragments specific to bla(TEM) genes were amplified in 63.3% of strains. 929- and 231-bp fragments (bla(SHV)), 847- and 520-bp fragments (bla(CMY)), 597- and 858-bp fragments (bla(CTX-M)) were amplified in 61.5, 17.3 and 7.7% of strains respectively. About 51.9% of strains contained more than two types of beta-lactamase genes. Especially, one strain contained bla(TEM), bla(CMY) and bla(CTX-M) genes. SIGNIFICANCE: Resistance mechanisms to beta-lactams, comprising mostly ESBL production, lead to the resistance against even recently developed beta-lactams in enterobacteria, which is now a serious threat to antibiotic therapy. The high prevalence of bla(CMY) genes and multidrug-resistant genes may also make therapeutic failure and lack of eradiation of these strains by extended-spectrum cephalosporins or cephamycins.     

Propagation of Saccharomyces cerevisiae [PSI+] prion is impaired by factors that regulate Hsp70 substrate binding

The Saccharomyces cerevisiae [PSI(+)] prion is believed to be a self-propagating cytoplasmic amyloid. Earlier characterization of HSP70 (SSA1) mutations suggested that [PSI(+)] propagation is impaired by alterations that enhance Ssa1p's substrate binding. This impairment is overcome by second-site mutations in Ssa1p's conserved C-terminal motif (GPTVEEVD), which mediates interactions with tetratricopeptide repeat (TPR) cochaperones. Sti1p, a TPR cochaperone homolog of mammalian Hop1 (Hsp70/90 organizing protein), activates Ssa1p ATPase, which promotes substrate binding by Ssa1p. Here we find that in SSA1-21 cells depletion of Sti1p improved [PSI(+)] propagation, while excess Sti1p weakened it. In contrast, depletion of Fes1p, a nucleotide exchange factor for Ssa1p that facilitates substrate release, weakened [PSI(+)] propagation, while overproducing Fes1p improved it. Therefore, alterations of Hsp70 cochaperones that promote or prolong Hsp70 substrate binding impair [PSI(+)] propagation. We also find that the GPTVEEVD motif is important for physical interaction with Hsp40 (Ydj1p), another Hsp70 cochaperone that promotes substrate binding but is dispensable for viability. We further find that depleting Cpr7p, an Hsp90 TPR cochaperone and CyP-40 cyclophilin homolog, improved [PSI(+)] propagation in SSA1 mutants. Although Cpr7p and Sti1p are Hsp90 cochaperones, we provide evidence that Hsp90 is not involved in [PSI(+)] propagation, suggesting that Sti1p and Cpr7p functionally interact with Hsp70 independently of Hsp90.     

Fitness estimation through performance comparison of F1 hybrids with their parental species Oryza rufipogon and O. sativa

BACKGROUND AND AIMS: Introgression of crop genes into populations of wild relatives has important implications for germplasm conservation as well as for the persistence of novel transgenes in wild populations. Studies of hybrid fitness can be used to evaluate the potential for introgression to occur following episodes of interspecific hybridization. METHODS: This study estimated relative fitness of interspecific hybrids through performance comparison of F(1) hybrids with their parental species, a cultivated rice (Oryza sativa) Minghui-63 and perennial common wild rice (O. rufipogon) under the cultivation conditions. KEY RESULTS: Compared with their parents, the hybrids had the lowest values of seedling survival ability, pollen viability and seed production; intermediate values of seed germination, spikelet production and flag leaf areas; and the highest values of plant height, number of tillers and panicles. The hybrids performed poorly at the stage of sexual reproduction, although they had a slightly higher hybrid vigour at the vegetative growth stage and better tillering ability than their wild parent. There were no significant differences in composite fitness across the whole life-history between the hybrids and their wild parental species. CONCLUSIONS: Rice genes, including transgenes, might persist in wild rice populations through vegetative and sexual reproduction. Further studies are needed to examine whether the extent of gene flow from rice is sufficiently significant to influence genetic diversity in wild populations of O. rufipogon, a species that has become endangered in some regions of south-east Asia.