ENOD12, an early nodulin gene, is not required for nodule formation and efficient nitrogen fixation in alfalfa.

To demonstrate the importance of an extensively studied early nodulin gene ENOD12 in symbiotic nodule development, plants of different Medicago sativa subspecies were tested for the presence or absence of ENOD12 alleles. In M. s. ssp coerulea w2 (Mcw2), two ENOD12 genes were detected, whereas in M. s. ssp quasifalcata k93 (Mqk93) only one gene was present. In both plants, the ENOD12 genes were expressed in nodules induced by Rhizobium meliloti. The nucleotide sequence of the ENOD12 genes showed that the two Mcw2-specific genes were similar to the ENOD12A and ENOD12B genes of the tetraploid M. s. ssp sativa. ENOD12 from Mqk93 was similar to the corresponding gene found in M. truncatula. From the aligned ENOD12 sequences, an evolutionary tree was constructed. Genetic analysis of the progenies of a cross between Mqk93 and Mcw2 showed that several offspring in F1 carried a null allele originating from Mcw2, and among the F2 progenies, plants with the null allele only lacking the ENOD12 gene appeared. Surprisingly, the ENOD12-deficient plants were similar to their wild-type parents in viability, nodule development, nodule structure, and nitrogen fixation efficiency. Therefore, we concluded that in Medicago the ENOD12 gene is not required for symbiotic nitrogen fixation. Furthermore, we proposed that the heterozygous nature of these legumes can be exploited for the identification of mutated alleles of other known nodulin genes; this will permit the construction of plant mutants deficient in these genes.     

Comparison of a New P2Y12 Receptor Specific Platelet Aggregation Test with Other Laboratory Methods in Stroke Patients on Clopidogrel Monotherapy

Background

Clinical studies suggest that 10-50% of patients are resistant to clopidogrel therapy. ADP induced platelet aggregation, a widely used test to monitor clopidogrel therapy, is affected by aspirin and is not specific for the P2Y12 receptor inhibited by clopidogrel.

Objectives

To develop a P2Y12-specific platelet aggregation test and to compare it with other methods used for monitoring clopidogrel therapy.

Patients/Methods

Study population included 111 patients with the history of ischemic stroke being on clopidogrel monotherapy and 140 controls. The effect of clopidogrel was tested by a newly developed ADP(PGE1) aggregation test in which prostaglandin E1 treated platelets are used. Results of conventional ADP induced platelet aggregation, VerifyNow P2Y12 assay and ADP(PGE1) aggregation were compared to those obtained by flow cytometric analysis of vasodilator stimulated phosphoprotein (VASP) phosphorylation. Reference intervals for all assays were determined according to the guidelines of Clinical Laboratory Standards Institute.

Results

The P2Y12-specificity of ADP(PGE1) test was proven by comparing it with ADP aggregation in the presence of P2Y1 antagonist, adenosine 3’, 5’-diphosphate. The method was not influenced by aspirin treatment. Approximately 50% of patients were clopidogrel resistant by conventional ADP aggregation and VerifyNow tests. The ADP(PGE1) method and the VASP phosphorylation assay identified 25.9% and 11.7% of patients as non-responders, respectively. ADP(PGE1) aggregation showed good correlation with VASP phosphorylation and had high diagnostic efficiency.

Conclusion

The new ADP(PGE1) method is a reliable test for monitoring P2Y12 receptor inhibition by platelet aggregation. As a subset of patients are non-responders, monitoring clopidogrel therapy by adequate methods is essential.     

The pseudokinase tribbles homologue-3 plays a crucial role in cannabinoid anticancer action

Δ⁹-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer. This effect relies, at least in part, on the up-regulation of several endoplasmic reticulum stress-related proteins including the pseudokinase tribbles homologue-3 (TRIB3), which leads in turn to the inhibition of the AKT/mTORC1 axis and the subsequent stimulation of autophagy-mediated apoptosis in tumor cells. Here, we took advantage of the use of cells derived from Trib3-deficient mice to investigate the precise mechanisms by which TRIB3 regulates the anti-cancer action of THC. Our data show that RasV¹²/E1A-transformed embryonic fibroblasts derived from Trib3-deficient mice are resistant to THC-induced cell death. We also show that genetic inactivation of this protein abolishes the ability of THC to inhibit the phosphorylation of AKT and several of its downstream targets, including those involved in the regulation of the AKT/mammalian target of rapamycin complex 1 (mTORC1) axis. Our data support the idea that THC-induced TRIB3 up-regulation inhibits AKT phosphorylation by regulating the accessibility of AKT to its upstream activatory kinase (the mammalian target of rapamycin complex 2; mTORC2). Finally, we found that tumors generated by inoculation of Trib3-deficient cells in nude mice are resistant to THC anticancer action. Altogether, the observations presented here strongly support that TRIB3 plays a crucial role on THC anti-neoplastic activity. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.     

Hyaluronan conformations on surfaces: effect of surface charge and hydrophobicity

Extended, relaxed, condensed, and interacting forms of the polysaccharide hyaluronan have been observed by atomic force microscopy (AFM). The types of images obtained depend on the properties of the surfaces used. We have investigated several different surface conditions for HA imaging, including unmodified mica, mica chemically modified with two different kinds of amino-terminated silanes (3-aminopropyltriethoxysilane and N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride), and highly oriented pyrolytic graphite. We found the degree of HA molecular extension or condensation to be variable, and the number of bound chains per unit area was low, for all of the mica-based surfaces. HA was more easily imaged on graphite, a hydrophobic surface. Chains were frequently observed in high degrees of extension, maintained by favorable interaction with the surface after molecular combing. This observation suggests that the HA macromolecule interacts with graphite through hydrophobic patches along its surface. AFM studies of HA behavior on differing surfaces under well-controlled environmental conditions provides useful insight into the variety of conformations and interactions likely to be found under differing physiological conditions.     

Challenging claims in the study of migratory birds and climate change

Recent shifts in phenology in response to climate change are well established but often poorly understood. Many animals integrate climate change across a spatially and temporally dispersed annual life cycle, and effects are modulated by ecological interactions, evolutionary change and endogenous control mechanisms. Here we assess and discuss key statements emerging from the rapidly developing study of changing spring phenology in migratory birds. These well‐studied organisms have been instrumental for understanding climate‐change effects, but research is developing rapidly and there is a need to attack the big issues rather than risking affirmative science. Although we agree poorly on the support for most claims, agreement regarding the knowledge basis enables consensus regarding broad patterns and likely causes. Empirical data needed for disentangling mechanisms are still scarce, and consequences at a population level and on community composition remain unclear. With increasing knowledge, the overall support (‘consensus view’) for a claim increased and between‐researcher variability in support (‘expert opinions') decreased, indicating the importance of assessing and communicating the knowledge basis. A proper integration across biological disciplines seems essential for the field's transition from affirming patterns to understanding mechanisms and making robust predictions regarding future consequences of shifting phenologies.     

Bcl-2 genes and growth factors in the pathology of ischaemic acute renal failure.

For the past decade, an attempt has been made by many research groups to define the roles of the growing number of Bcl-2 gene family proteins in the apoptotic process. The Bcl-2 family consists of pro-apoptotic (or cell death) and anti-apoptotic (or cell survival) genes and it is the balance in expression between these gene lineages that may determine the death or survival of a cell. The majority of studies have analysed the role/s of the Bcl-2 genes in cancer development. Equally important is their role in normal tissue development, homeostasis and non-cancer disease states. Bcl-2 is crucial for normal development in the kidney, with a deficiency in Bcl-2 producing such malformation that renal failure and death result. As a corollary, its role in renal disease states in the adult has been sought. Ischaemia is one of the most common causes of both acute and chronic renal failure. The section of the kidney that is most susceptible to ischaemic damage is the outer zone of the outer medulla. Within this zone the proximal tubules are most sensitive and often die by necrosis or desquamate. In the distal nephron, apoptosis is the more common form of cell death. Recent results from our laboratory have indicated that ischaemia-induced acute renal failure is associated with up-regulation of two anti-apoptotic Bcl-2 proteins (Bcl-2 and Bcl-XL) in the damaged distal tubule and occasional up-regulation of Bax in the proximal tubule. The distal tubule is a known reservoir for several growth factors important to renal growth and repair, such as insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF). One of the likely possibilities for the anti-cell death action of the Bcl-2 genes is that the protected distal cells may be able to produce growth factors that have a further reparative or protective role via an autocrine mechanism in the distal segment and a paracrine mechanism in the proximal cells. Both EGF and IGF-1 are also up-regulated in the surviving distal tubules and are detected in the surviving proximal tubules, where these growth factors are not usually synthesized. As a result, we have been using in vitro methods to test: (i) the relative sensitivities of renal distal and proximal epithelial cell populations to injury caused by mechanisms known to act in ischaemia-reperfusion; (ii) whether a Bcl-2 anti-apoptotic mechanism acts in these cells; and (iii) whether an autocrine and/or paracrine growth factor mechanism is initiated. The following review discusses the background to these studies as well as some of our preliminary results.     

Construction of a bacterial artificial chromosome library of Medicago truncatula and identification of clones containing ethylene-response genes

 To facilitate genome analysis and map-based cloning of symbiotic genes in the model legume Medicago truncatula, a bacterial artificial chromosome (BAC) library was constructed. The library consists of 30 720 clones with an average insert size of approximately 100 kb, representing approximately five haploid-genome equivalents. The frequency of BAC clones carrying inserts of chloroplast DNA was estimated to be 1.4%. Screening of the library with single- or low-copy genes as hybridization probes resulted in the detection of 1–12 clones per gene. Hybridization of the library with repeated sequences such as rDNA genes and transposon-like elements of M. truncatula revealed the presence of 60 and 374 BAC clones containing the two sequences, respectively. The BAC library was pooled for screening by polymerase chain reaction (PCR)-amplification. To demonstrate the utility of this system, we used primers designed from a conserved region of the ein3-like loci of Arabidopsis thaliana and isolated six unique BAC clones from the library. DNA gel-blot and sequence analyses showed that these ein3-like clones could be grouped into three classes, an observation consistent with the presence of multiple ein3-like loci in M. truncatula. These results indicate that the BAC library represents a central resource for the map-based cloning and physical mapping in M. truncatula and other legumes. Received: 27 July 1998 / Accepted: 5 August 1998     

Escape from apoptosis after prolonged serum deprivation is associated with the regulation of the mitochondrial death pathway by Bcl-x(l)

Bcl-x(l) and Bax play important roles in the regulation of apoptosis. This study investigated the involvement of the mitochondrial death pathway and the role of Bcl-x(l) and Bax in the escape from apoptosis after prolonged serum deprivation in Madin-Darby canine kidney (MDCK) cells. Low level apoptosis and basal activity of the mitochondrial death pathway were detectable in normal cell growth. In serum deprivation, mitosis was partially suppressed, and the mitochondrial activity was stimulated. The level of apoptosis continuously rose over 48 h. This rise was concomitant with the increasing presence of cytochrome c in cytosol. However, both apoptosis and cytosolic cytochrome c fell dramatically at 72 h. Elevation of whole cell Bcl-x(l) and redistribution of Bcl-x(l) protein from cytosol to the membrane at 48 h and 72 h was observed. Redistribution of Bax protein from the membrane to cytosol occurred at 24 h, and remained steady to 72 h. Bax/Bcl-x(l) coimmunoprecipitation by anti-Bax antibody showed reduced Bax/Bcl-x(l) interaction at the membrane at 72 h, but not at 24 or 48 h. These results suggest that apoptosis upon serum withdrawal results from the leakage of cytochrome c to cytosol. Amelioration of the leakage of cytochrome c and apoptosis requires not only the increase of Bcl-x(l)/Bax ratio, but also the release of Bcl-x(l) from Bax at the membrane.     

CHANGES IN THE BASE COMPOSITION OF NUCLEAR RIBONUCLEIC ACID OF NEURONS DURING A SHORT PERIOD OF ENHANCED PROTEIN PRODUCTION

Nuclei from isolated nerve cells were sampled by microdissection. The content and composition of the nuclear RNA was studied and compared with that of the cytoplasmic RNA of Deiters' nerve cells of rabbits. Analyses were made of control nerve cells and of cells in which an enhanced RNA and protein production had been induced by chemical means, tricyano-amino-propene, for 60 minutes. The nuclear RNA content of the control nerve cells was 56 µµg, i.e. 3 per cent of the total RNA content of the nerve cell. The base ratios were: adenine 21.3, guanine 26.6, cytosine 30.8, uracil 21.3. Purine-pyrimidine analyses showed that the nuclear RNA differed significantly from the cytoplasmic RNA in having higher adenine and uracil values. The guanine and cytosine values were high, however, and the ratio G/C was 0.86 as compared with 1.16 for the cytoplasmic RNA. The composition of the nuclear RNA was interpreted as reflecting the extraordinarily strong development of the nucleolus in these neurons. During the 60 minutes of enhanced neuronal RNA production (+25 per cent) the guanine value increased and the uracil value decreased significantly in the nuclear RNA. In the cytoplasmic RNA the guanine value also increased although not so much as the nuclear guanine. The cytoplasmic cytosine value decreased. The result indicated that the production of the characteristic cytoplasmic RNA had been influenced by the change in the nuclear RNA