Chromosomal sex determination and heterochromatin structure in date palm

In the date palm, a dioecious mode (separate male and female individuals) and the late initial reproductive age (5–10 years) are major practical constraints for genetic improvement. Early selection on young seedlings could enhance breeding programmes and generate experimental male and female genetic stocks, but no cytogenetic protocol exists for sex determination in an immature date palm. Here we describe a cytological method based on chromomycin staining which demonstrates the occurrence of sexual chromosomes carrying distinctive nucleolar heterochromatin and thus offers, for the first time, the possibility of identifying male and female individuals by simple analysis of root meristems. This observation has been extended by in situ rDNA hybridization, confocal microscopy and dual-label flow cytometry of nuclei.     

Phylogenetic screening of the human genome: identification of differentially hybridizing repetitive sequence families

The phi-screen, a method of phylogenetic screening, can be employed to detect repetitive sequence families that differentially hybridize between closely related species. Such differences may involve sequence divergence or variations in copy number, including total presence versus absence of a family of repeated DNA. We present the results of a phi-screen comparing the human genome to that of the prosimian, Galago crassicaudatus. Three human repetitive families that are divergent or not present in galago have been detected. One of these families is described in detail; it is similar among the anthropoids but is present in a lower copy number and/or divergent form in prosimians. The family is clearly related to the transposon-like human element (THE) described by Paulson et al. (1985). THEs have long terminal repeats reminiscent of retroviruses but are unique in that they have no sequence similarity to known mammalian retroviruses. The sequence of a solo long terminal repeat, found unassociated with THE internal sequence, is presented. This family member, THE p2, is bordered by a 5-bp target-site repeat and is interrupted by the insertion of an Alu element. A solo THE element sequenced by Wiginton et al. (1986) contains an insertion of Alu at precisely the same position as does THE p2.      

Overexpression of Flavodoxin in Bacteroids Induces Changes in Antioxidant Metabolism Leading to Delayed Senescence and Starch Accumulation in Alfalfa Root Nodules

Sinorhizobium meliloti cells were engineered to overexpress Anabaena variabilis flavodoxin, a protein that is involved in the response to oxidative stress. Nodule natural senescence was characterized in alfalfa (Medicago sativa) plants nodulated by the flavodoxin-overexpressing rhizobia or the corresponding control bacteria. The decline of nitrogenase activity and the nodule structural and ultrastructural alterations that are associated with nodule senescence were significantly delayed in flavodoxin-expressing nodules. Substantial changes in nodule antioxidant metabolism, involving antioxidant enzymes and ascorbate-glutathione cycle enzymes and metabolites, were detected in flavodoxin-containing nodules. Lipid peroxidation was also significantly lower in flavodoxin-expressing nodules than in control nodules. The observed amelioration of the oxidative balance suggests that the delay in nodule senescence was most likely due to a role of the protein in reactive oxygen species detoxification. Flavodoxin overexpression also led to high starch accumulation in nodules, without reduction of the nitrogen-fixing activity.Symbiotic nodules have a limited functional life that varies among different legume species. Nodule senescence is the sequence of structural, molecular, biochemical, and physiological events taking place in the process that a mature and functional nodule undergoes leading to the loss of the nitrogen-fixing activity and culminating in cell death of symbiotic tissue (Swaraj and Bishnoi, 1996; Puppo et al., 2005; Van de Velde et al., 2006).Various models have been proposed to explain the mechanisms that trigger the process of natural or stress-induced nodule senescence. However, it is generally accepted that a senescence-inducing signal from the plant causes a decrease in antioxidant levels and thus an increase in reactive oxygen species (ROS) up to a point of no return. Numerous studies have shown that ROS and antioxidant systems are involved in natural (Lucas et al., 1998; Evans et al., 1999; Hernández-Jiménez et al., 2002; Puppo et al., 2005) as well as induced (Dalton et al., 1993; Becana et al., 2000; Hernández-Jiménez et al., 2002; Matamoros et al., 2003) nodule senescence. Nitrogen fixation is very sensitive to ROS, and nitrogenase activity drastically decreases during nodule senescence (Dalton et al., 1986).Antioxidant systems that protect cells from oxidative damage have been described in symbiotic nodules (Dalton et al., 1986, 1993; Evans et al., 1999; Becana et al., 2000; Matamoros et al., 2003; Puppo et al., 2005). These include the enzymes superoxide dismutase (SOD), catalase, and peroxidase. Another enzymatic system associated with ROS detoxification is the ascorbate-glutathione pathway, which includes ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR; Dalton et al., 1986, 1992; Noctor and Foyer 1998; Becana et al., 2000). Ascorbate and reduced glutathione (GSH) in this pathway can also scavenge superoxide and hydrogen peroxide.During nodule senescence, several ultrastructural alterations in the nodule tissues and cells have been observed (Lucas et al., 1998; Hernández-Jiménez et al., 2002; Puppo et al., 2005, and refs. therein; Van de Velde et al., 2006). Cytosol becomes electron dense, altered vesicles proliferate, and eventually the cytosol undergoes lysis. The number of peroxisomes increases, mitochondria form complex elongated structures, and symbiosomes change in size and shape and fuse during natural and induced senescence of nodules (Hernández-Jiménez et al., 2002). Damage of the symbiosome membrane is also detected (Puppo et al., 2005; Van de Velde et al., 2006).A strategy of delayed nodule senescence could lead to increased nitrogen fixation and legume productivity. Delayed nodule senescence together with enhanced sustainability under field conditions are among the key aims of legume improvement programs (Puppo et al., 2005). An interesting approach proposed to achieve delayed senescence is to induce nodulation in legumes using rhizobial strains with modified redox capacity (Zahran, 2001).The protein flavodoxin contains a FMN group acting as a redox center transferring electrons at low potentials (Pueyo et al., 1991; Pueyo and Gómez-Moreno, 1991). The FMN cofactor of flavodoxin can exist in three different redox states: oxidized, one-electron-reduced semiquinone, and two-electron-reduced hydroquinone. This property confers high versatility to flavodoxins in electron transport systems (Simondsen and Tollin, 1980; McIver et al., 1998). To date, flavodoxin has not been described in plants, as flavodoxin-encoding genes were lost during the transition of algae to plants (Zurbriggen et al., 2007) and, consequently, no homologs have been identified in the sequenced genome of Arabidopsis (Arabidopsis thaliana; Arabidopsis Genome Initiative, 2000). Flavodoxin is present as a constitutive or inducible protein in different microorganisms (Klugkist et al., 1986). In the nitrogen-fixing cyanobacterium Anabaena variabilis PCC 7119, flavodoxin is expressed under conditions of limited iron availability, replacing ferredoxin in the photosynthetic electron transport from PSI to NADP⁺ and in nitrogenase reduction (Sandmann et al., 1990). Reversible electron transfer from flavodoxin to NADP⁺ is catalyzed by ferredoxin NADP⁺ reductase in different pathways of oxidative metabolism (Arakaki et al., 1997). In its reduced state, flavodoxin might be able to react with ROS and revert to its original redox state in the presence of an appropriate electron source. This could probably occur without the associated molecular damage that metallic complexes in catalases or SODs suffer (Keyer et al., 1995). The presence of flavodoxin has not been documented to date in the symbiotic bacterium Sinorhizobium meliloti. In Escherichia coli, however, flavodoxin induction is linked to the oxidative stress-responsive regulon soxRS (Zheng et al., 1999). It has been suggested that flavodoxin and ferredoxin (flavodoxin) NADP⁺ reductase might be induced and have a role in reestablishing the cell redox balance under oxidative stress conditions (Liochev et al., 1994). The properties of flavodoxin suggest that its presence in the cell may have a facilitating effect on ROS detoxification. In fact, an increase in the amount of flavodoxin has been observed in some bacterial species subjected to oxidative stress (Zheng et al., 1999; Yousef et al., 2003; Singh et al., 2004), and transgenic tobacco (Nicotiana tabacum) plants expressing flavodoxin in chloroplasts show enhanced tolerance to a broad range of stresses related to oxidative damage (Tognetti et al., 2006, 2007a, 2007b).In this work, Sinorhizobium meliloti was transformed with the A. variabilis flavodoxin gene and used to nodulate alfalfa (Medicago sativa) plants. The effects of flavodoxin expression on nodulation dynamics, on nodule development and senescence processes, and on nitrogen-fixing activity were analyzed. Mechanistic insights suggesting putative roles for flavodoxin in protection from ROS and the induced delay of nodule senescence are likewise discussed.     

Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

Background

Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages.

Results

We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages.

Conclusion

Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.     

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

大鼠心肌缺血再灌注损伤模型的实验研究

目的 制备一种新型的心肌急性缺血再灌注损伤模型,以探讨一种更符合临床实际需求的实验方法.方法 将20只雌性SD(Sprague-Dawley)大鼠随机分成2组(对照组、实验组),采用结扎主动脉根部引起心肌缺血5min再灌注30 min建立心肌急性缺血再灌注模型;通过应用透射电镜观察心肌细胞超微结构的改变,同时检测心肌组织匀浆丙二醛(Maleic Dialdehyde,MDA)含量、超氧化物歧化酶(Superoxide Dismutase,SOD)活力.结果 透射电镜下超微结构显示实验组较对照组明显加重了心肌组织结构和线粒体的损害;实验组心肌组织MDA明显高于对照组(P<0.01),而SOD明显低于对照组(P<0.01).结论 本实验成功建立了方法简便、易于操作、取材范围广泛的心肌缺血再灌注损伤模型,为心肌缺血再灌注损伤研究提供了一种更为可行的模型.     

Genetic polymorphims of estrogen receptor alpha −397 PvuII (T>C) and −351 XbaI (A>G) in a portuguese population: prevalence and relation with breast cancer susceptibility

Estrogen receptor alpha (ERα), that mediates the biologic effects of estrogen in estrogen-sensitive tissues like breast, is genetically polymorphic. To evaluate the association between ?397 PvuII (T>C) and ?351 XbaI (A>G) restriction fragment length polymorphisms (RFLPs) in intron 1 of ERα gene and susceptibility of breast cancer, we undertook a case–control study in BRCA1 185delAG and 5382insC/BRCA2 6174delT negative Portuguese women. The study population consisted of 107 patients with histological diagnosis of breast cancer and 121 women with no history of breast cancer. Genomic DNA was extracted from blood samples and genotyping analyses were performed by PCR–RFLP. XbaI polymorphism was associated with a significant reduced risk of breast cancer for carriers of the x allele in homozygozity (OR 0.178; 95 % CI 0.070–0.456; P < 0.001) or heterozigozity (OR 0.223; 95 % CI 0.089–0.561; P = 0.001). The PvuII polymorphism was associated with a non-significantly reduced risk. The combined analysis of PvuII and XbaI polymorphisms revealed none synergistic effect of the two genotypes, except for simultaneous carriers of pp and xx genotypes, that have a reduced risk of breast cancer (OR 0.226; 95 % CI 0.049–1.035; P = 0.044). The combination of PvuII and XbaI genotypes into haplotypes showed that carriers of two copies of the px (ppxx) haplotype had a reduced risk of breast cancer (OR 0.405; 95 % CI 0.194–0.843; P = 0.014), compared with PX (PPXX + PPXx + PpXX + PpXx) haplotypes. PvuII and XbaI polymorphisms were in linkage disequilibrium both in cases (D = 0.044, r² = 0.049, X² = 5.216, P = 0.022) and controls (D = 0.090, r² = 0.139, X² = 16.819, P < 0.001), but not in the entire sample population analyzed as a whole (D = 0.087, r² = 0.0076, X² = 1.733, P = 0.188). In conclusion, in this case–control study we found that ERα gene XbaI polymorphism may modify individual susceptibility for breast cancer in this population.     

Germ band retraction as a landmark in glucose metabolism during <Emphasis Type="Italic">Aedes aegypti</Emphasis> embryogenesis

Background  

The mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown.