Homologous recombination: a basis for targeted genome optimization in crop species such as maize

In an attempt to understand the feasibility of future targeted genome optimization in agronomic crops, we tested the efficiency of homologous recombination-mediated sequence insertion upon induction of a targeted DNA double-strand break at the desired integration site in maize. By the development of an efficient tissue culture protocol, and with the use of an I- Sce I gene optimized for expression in maize, large numbers of precisely engineered maize events were produced in which DNA integration occurred very accurately. In a subset of events examined in detail, no additional deletions and/or insertions of short filler DNA at the integration site were observed. In 30%–40% of the recovered events, no traces of random insertions were observed. This was true for DNA delivery by both Agrobacterium and particle bombardment. These data suggest that targeted double-strand break-induced homologous recombination is a superior method to generate specific desired changes in the maize genome, and suggest targeted genome optimization of agronomic crops to be feasible.     

Permanent cardiac pacing for neurocardiogenic syncope

Vasovagal syncope is not a benign condition in the elderly population. In patients not responsive to conservative therapy and whose abrupt faints are associated with serious injuries and seriously affected quality of life, pacemaker therapy was suggested. However, the usefulness of cardiac pacing for the prevention of recurrences of vasovagal syncope remains controversial because of the dominant role of the vasodepressor component during the episode. In the Medical Center Alkmaar, the Head-Up Tilt Test (HUTT) has been used since 1996 during the work-up of patients who present with vasovagal syncope. The HUTT showed a dominant cardioinhibitory response in 4.5% of our patients; in elderly patients with vasovagal syncope without prodromal symptoms and refractory on conservative therapy, pacemaker therapy was very effective in preventing syncope during long-term follow-up. (Neth Heart J 2008;16(suppl 1):S15-S19.)     

Glossopharyngeal neuralgia with syncope: a case report

We present the case of a 60-year-old woman with known glossopharyngeal neuralgia who was admitted to hospital because of recurrent syncopes associated with episodes of painful sensations in the caudal region of her tongue. Rhythm observation showed prolonged asystole, which was accompanied by a loss of consciousness. The asystole was preceded by an episode of pain. We concluded that the bradyarrhythmia and syncopes where associated with the glossopharyngeal neuralgia. Because of the life-threatening condition, we inserted a permanent dual-chamber pacing device. After implantation of the pacemaker, the patient had no further syncopes, although she still complained of episodic tongue pain.     

Detritus, trophic dynamics and biodiversity

Traditional approaches to the study of food webs emphasize the transfer of local primary productivity in the form of living plant organic matter across trophic levels. However, dead organic matter, or detritus, a common feature of most ecosystems plays a frequently overlooked role as a dynamic heterogeneous resource and habitat for many species. We develop an integrative framework for understanding the impact of detritus that emphasizes the ontogeny and heterogeneity of detritus and the various ways that explicit inclusion of detrital dynamics alters generalizations about the structure and functioning of food webs. Through its influences on food web composition and dynamics, detritus often increases system stability and persistence, having substantial effects on trophic structure and biodiversity. Inclusion of detrital heterogeneity in models of food web dynamics is an essential new direction for ecological research.     

Inhibition of a dual chamber pacemaker by oversensing of far-field atrial depolarisation in a patient after His-bundle ablation

After His-bundle ablation, a 54-year-old pacemaker-dependent patient suffered from severe presyncopal attacks one year after pacemaker replacement. The attacks were caused by ventricular inhibition due to atrial far-field sensing during paroxysmal atrial flutter.     

Initial phase of maximal voluntary and electrically stimulated knee extension torque development at different knee angles.

We investigated the capacity for torque development and muscle activation at the onset of fast voluntary isometric knee extensions at 30, 60, and 90 degrees knee angle. Experiments were performed in subjects (n = 7) who had high levels (>90%) of activation at the plateau of maximal voluntary contractions. During maximal electrical nerve stimulation (8 pulses at 300 Hz), the maximal rate of torque development (MRTD) and torque time integral over the first 40 ms (TTI40) changed in proportion with torque at the different knee angles (highest values at 60 degrees ). At each knee angle, voluntary MRTD and stimulated MRTD were similar (P < 0.05), but time to voluntary MRTD was significantly longer. Voluntary TTI40 was independent (P > 0.05) of knee angle and on average (all subjects and angles) only 40% of stimulated TTI40. However, among subjects, the averaged (across knee angles) values ranged from 10.3 +/- 3.1 to 83.3 +/- 3.2% and were positively related (r2 = 0.75, P < 0.05) to the knee-extensor surface EMG at the start of torque development. It was concluded that, although all subjects had high levels of voluntary activation at the plateau of maximal voluntary contraction, among subjects and independent of knee angle, the capacity for fast muscle activation varied substantially. Moreover, in all subjects, torque developed considerably faster during maximal electrical stimulation than during maximal voluntary effort. At different knee angles, stimulated MRTD and TTI40 changed in proportion with stimulated torque, but voluntary MRTD and TTI40 changed less than maximal voluntary torque.     

Linoleic and α-linolenic acid as precursor and inhibitor for the synthesis of long-chain polyunsaturated fatty acids in liver and brain of growing pigs

Studies suggested that in human adults, linoleic acid (LA) inhibits the biosynthesis of n-3 long-chain polyunsaturated fatty acids (LC-PUFA), but their effects in growing subjects are largely unknown. We used growing pigs as a model to investigate whether high LA intake affects the conversion of n-3 LC-PUFA by determining fatty acid composition and mRNA levels of Δ5- and Δ6 desaturase and elongase 2 and -5 in liver and brain. In a 2 × 2 factorial arrangement, 32 gilts from eight litters were assigned to one of the four dietary treatments, varying in LA and α-linolenic acid (ALA) intakes. Low ALA and LA intakes were 0.15 and 1.31, and high ALA and LA intakes were 1.48 and 2.65 g/kg BW0.75 per day, respectively. LA intake increased arachidonic acid (ARA) in liver. ALA intake increased eicosapentaenoic acid (EPA) concentrations, but decreased docosahexaenoic acid (DHA) (all P < 0.01) in liver. Competition between the n-3 and n-6 LC-PUFA biosynthetic pathways was evidenced by reductions of ARA (>40%) at high ALA intakes. Concentration of EPA (>35%) and DHA (>20%) was decreased by high LA intake (all P < 0.001). Liver mRNA levels of Δ5- and Δ6 desaturase were increased by LA, and that of elongase 2 by both ALA and LA intakes. In contrast, brain DHA was virtually unaffected by dietary LA and ALA. Generally, dietary LA inhibited the biosynthesis of n-3 LC-PUFA in liver. ALA strongly affects the conversion of both hepatic n-3 and n-6 LC-PUFA. DHA levels in brain were irresponsive to these diets. Apart from Δ6 desaturase, elongase 2 may be a rate-limiting enzyme in the formation of DHA.     

Metastatic behavior of human melanoma cell lines in nude mice correlates with urokinase-type plasminogen activator, its type-1 inhibitor, and urokinase-mediated matrix degradation

Five out of six human melanoma cell lines tested were able to degrade in vitro a smooth muscle cell extracellular matrix in a plasmin-dependent way. In three of these five cell lines, this process was mediated by tissue-type plasminogen activator (t-PA) and in the other two cell lines by urokinase-type plasminogen activator (u-PA). All melanoma cell lines produced t-PA mRNA and protein, whereas only the two cell lines showing u-PA-mediated matrix degradation produced u-PA mRNA and protein. These latter cell lines also produced plasminogen activator inhibitor type-1 (PAI-1) and type-2 (PAI-2) mRNA and protein. u-PA receptor (u-PA-R) mRNA and binding of radiolabeled u-PA was found in all melanoma cell lines. The metastatic capacity of these cell lines was studied in nude mice. All cell lines were able to develop primary tumors at the subcutaneous inoculation site. The production of plasminogen activators, their inhibitors and urokinase receptor by subcutaneous tumors corresponded with the production by the parental cell lines in vitro. The two u-PA and PAI-1 producing cell lines showed the highest frequency to form spontaneous lung metastases after subcutaneous inoculation, whereas five of the six cell lines formed lung colonies after intravenous inoculation. In conclusion, u-PA mediated matrix degradation in vitro and production of u-PA and PAI-1 by human melanoma cell lines correlated with their ability to form spontaneous lung metastasis in nude mice. No correlation was found with the ability to form lung colonies after intravenous injection. These findings suggest a role for u-PA and PAI-1 in a relatively early stage of melanoma metastasis.     

New structural features of the antigenic extracellular polysaccharides of Penicillium and Aspergillus species revealed with exo-beta-D-galactofuranosidase.

To study the structures of the epitopes of the extracellular polysaccharides from Penicillium and Aspergillus species, an exo-beta-D-galactofuranosidase was purified from a commercial crude enzyme preparation from Trichoderma harzianum. Analysis of ring size and linkage position of the galactose residues of the extracellular polysaccharide of Penicillium digitatum, before and after enzymatic treatment, was determined by the reductive-cleavage technique. In addition to terminal and beta (1-5)-linked galactofuranosides, beta (1-6)-linked and beta (1,5,6)-linked branched galactofuranose residues could be identified. After degradation with the purified exo-beta-D-galactofuranosidase, all initial linkages of the galactofuranose residues were still present, but the amount of beta (1-5)-linked galactofuranose residues had decreased considerably. Treatment of the extracellular polysaccharides of Penicillium and Aspergillus species with the purified exo-beta-D-galactofuranosidase resulted in complete disappearance of the enzyme-linked immunosorbent assay reactivity of these polysaccharides, using immunoglobulin G antibodies raised against P. digitatum. Therefore, with the use of this enzyme, it was proved that the beta (1-5)-linked galactofuranosyl residues only are responsible for the antigenicity of the extracellular polysaccharides of Penicillium and Aspergillus molds. A new structural model for the antigenic galactofuranose side chains of the galactomannan from P. digitatum is proposed.