The electrocardiogram of the Beagle dog: reference values and effect of sex, genetic strain, body position and heart rate

The aim of the study was to establish a database for electrocardiographic parameters of Beagle dogs used for toxicological studies and to evaluate the influence of supplier, sex, heart rate (HR) and body position for electrocardiogram (ECG) recording on ECG parameters. Peripheral ECG leads were recorded from 934 female and 946 male dogs from Marshall Farms and 27 females and 30 males from Harlan, either standing on a table or restrained in a hammock. HR, RR, PQ and QT intervals, P and QRS duration and P-wave amplitude were measured. There were no major differences between sexes for ECG parameters. The axis of the heart was shifted to the left when the animals were restrained in a hammock compared to when they were standing on a table. The PQ interval was higher (about 9%) in Harlan than in Marshall dogs. HR was negatively correlated with QT (coefficient of linear correlation: r=-0.61 to -0.74), which emphasizes the need for a formula correcting QT interval for HR when interpreting changes in QT interval. HR was also negatively correlated with PQ intervals (r=-0.26 to -0.11), whereas a positive correlation was found between HR and the amplitude of the P wave (r=0.21-0.34). The level of the respiratory sinus arrhythmia (SA) was quantified by calculating the ratio of maximum to minimum RR interval measured over a 10 s period. This ratio was negatively correlated with HR (r =-0.49 to -0.33). Therefore, at high HRs, SA was less marked than at low HRs, but it did not completely disappear. Analysis of beat-to-beat variation indicated that QT and PQ intervals and the amplitude of P wave fluctuated over time and the degree of this variability was positively correlated with the level of SA. In conclusion, we have established reference values for the duration and/or amplitude of some ECG parameters both in terms of means and variability over the recording period, and we have evaluated the influence of body position, genetic strain and HR on the ECG parameters. These data can be used as baseline for the interpretation of the ECG of Beagle dogs.     

The plant ER-Golgi interface: a highly structured and dynamic membrane complex

As compared with other eukaryotic cells, plants have developed an endoplasmic reticulum (ER)-Golgi interface with very specific structural characteristics. ER to Golgi and Golgi to ER transport appear not to be dependent on the cytoskeleton, and ER export sites have been found closely associated with Golgi bodies to constitute entire mobile units. However, the molecular machinery involved in membrane trafficking seems to be relatively conserved among eukaryotes. Therefore, a challenge for plant scientists is to determine how these molecular machineries work in a different structural and dynamic organization. This review will focus on some aspects of membrane dynamics that involve coat proteins, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment receptor proteins), lipids, and lipid-interacting proteins.     

Vacuolar transport in tobacco leaf epidermis cells involves a single route for soluble cargo and multiple routes for membrane cargo

We tested if different classes of vacuolar cargo reach the vacuole via distinct mechanisms by interference at multiple steps along the transport route. We show that nucleotide-free mutants of low molecular weight GTPases, including Rab11, the Rab5 members Rha1 and Ara6, and the tonoplast-resident Rab7, caused induced secretion of both lytic and storage vacuolar cargo. In situ analysis in leaf epidermis cells indicates a sequential action of Rab11, Rab5, and Rab7 GTPases. Compared with Rab5 members, mutant Rab11 mediates an early transport defect interfering with the arrival of cargo at prevacuoles, while mutant Rab7 inhibits the final delivery to the vacuole and increases cargo levels in prevacuoles. In contrast with soluble cargo, membrane cargo may follow different routes. Tonoplast targeting of an α-TIP chimera was impaired by nucleotide-free Rha1, Ara6, and Rab7 similar to soluble cargo. By contrast, the tail-anchored tonoplast SNARE Vam3 shares only the Rab7-mediated vacuolar deposition step. The most marked difference was observed for the calcineurin binding protein CBL6, which was insensitive to all Rab mutants tested. Unlike soluble cargo, α-TIP and Vam3, CBL6 transport to the vacuole was COPII independent. The results indicate that soluble vacuolar proteins follow a single route to vacuoles, while membrane spanning proteins may use at least three different transport mechanisms.     

Diapause-dependent changes in prothoracicotropic hormone-producing neurons of the tobacco hornworm,Manduca sexta

The prothoracicotropic hormone (PTTH), which stimulates ecdysteroid synthesis in the prothoracic glands, is produced, in the dorso-lateral protocerebrum of Manduca sexta, by paired peptidergic neurons, the lateral neurosecretory cell group III (L-NSC III). Our study revealed ultrastructural features of L-NSC III, identified by immunogold labeling, and compared developing and diapause states. In developing and early-diapause pupae, L-NSC III soma ultrastructure is similar and is characterized by numerous clusters of neurosecretory granules (NSG) and an extensive trophospongium formed by satellite-glial cells. However, as diapause progresses, the ultrastructure changes, with the NSG becoming concentrated into large clusters separated by highly organized rough endoplasmic reticulum. Most conspicuous is a substantial reduction in the number of Golgi complexes and the glial trophospongium, and the presence of stacked plasma membrane separating the glia and neuron somata. The deep-diapause soma also has abundant glycogen deposits and autophagic vacuoles. With diapause termination, this morphology reverts to the nondiapause ultrastructure within three days, i.e. just before PTTH release that evokes development to the adult. During PTTH release the abundance of NSG in the soma does not change, suggesting that NSG depletion in the perikarya is not a marker for neurosecretion by the L-NSC III.     

Continuous inhalation of carbon monoxide induces right ventricle ischemia and dysfunction in rats with hypoxic pulmonary hypertension

We aimed to investigate the toxicity of carbon monoxide (CO) in rats with right ventricle (RV) remodeling induced by hypoxic pulmonary hypertension (PHT). A group of Wistar rats was exposed to 3-wk hypobaric hypoxia (H). A second group was exposed to 50 ppm CO for 1 wk (CO). A third group was exposed to chronic hypoxia including 50 ppm CO during the third week (H+CO). These groups were compared with controls. RV and left ventricle (LV) functions were assessed by echocardiography and transparietal catheterization. Ventricular perfusion was estimated with the fluorescent microsphere method. Results were confirmed by histology. PHT induced RV hypertrophy and function enhancement. In the H group, RV shortening fraction (RVSF; 71 +/- 12% vs. 41 +/- 2%) and RV end-systolic pressure (RVESP; 54 +/- 6 vs. 19 +/- 2 mmHg) were increased compared with controls. Moreover, myocardial perfusion was increased in the RV (36 +/- 2% vs. 22 +/- 2%) and decreased in the LV (64 +/- 3% vs. 78 +/- 2%). In the H+CO group, RVSF (45 +/- 3% vs. 71 +/- 12%) and RVESP (38 +/- 3 vs. 54 +/- 6 mmHg) were decreased compared with the H group. RV perfusion was decreased in the H+CO group compared with the H group (21 +/- 5% vs. 36 +/- 2%), and LV perfusion was increased (79 +/- 5% vs. 64 +/- 3%). PHT and RV hypertrophy were still present in the H+CO group, and fibroses localized in the RV were detected. Similar lesions were observed in an additional group exposed simultaneously to hypoxia and 50 ppm CO over 3 wk. We demonstrated that rats with established PHT were more sensitive to CO, which dramatically alters the RV adaptive response to PHT, leading to ischemic lesions.     

Common requirements for melanocortin-4 receptor selectivity of structurally unrelated melanocortin agonist and endogenous antagonist, Agouti protein

The activity of melanocortin receptors (MCR) is regulated by melanocortin peptide agonists and by the endogenous antagonists, Agouti protein and AgRP (Agouti-related protein). To understand how the selectivity for these structurally unrelated agonists and antagonist is achieved, chimeric and mutants MC3R and MC4R were expressed in cell lines and pharmacologically analyzed. A region containing the third extracellular loop, EC3, of MC4R was essential for selective Agouti protein antagonism. In addition, this part of MC4R, when introduced in MC3R, conferred Agouti protein antagonism. Further mutational analysis of this region of MC4R demonstrated that Tyr(268) was required for the selective interaction with Agouti protein, because a profound loss of the ability of Agouti protein to inhibit (125)I-labeled [Nle(4),d-Phe(7)]alpha-melanocyte-stimulating hormone (MSH) binding was observed by the single mutation of Tyr(268) to Ile. This same residue conferred selectivity for the MC4R selective agonist, [d-Tyr(4)]MT-II, whereas it inhibited interaction with the MC3R-selective agonist, [Nle(4)]Lys-gamma(2)-MSH. Conversely, mutation of Ile(265) in MC3 (the corresponding residue of Tyr(268)) to Tyr displayed a gain of affinity for [d-Tyr(4)]MT-II, but not for Agouti protein, and a loss of affinity for [Nle(4)]Lys-gamma(2)-MSH as compared with wild-type MC3R. This single amino acid mutation thus confers the selectivity of MC3R toward a pharmacological profile like that observed for MC4R agonists but not for the antagonist, Agouti protein. Thus, selectivity for structurally unrelated ligands with opposite activities is achieved in a similar manner for MC4R but not for MC3R.     

Synthesis and characterisation of chitosan-graft-poly(OEGMA) copolymers prepared by ATRP

Two synthetic routes, “grafting-from” and “grafting-to” the chitosan backbone, were investigated to prepare chitosan-graft-poly(OEGMA) copolymers by ATRP. The copolymers were characterised by ¹H NMR and FT-IR spectroscopy, elemental analyses and GPC. Estimates of the degree of grafting were quantitatively found from integration of the ¹H NMR spectra. The GPC data indicated that the hydrodynamic volumes of the polymers were significantly altered by the incorporation of poly(OEGMA). A comparison of the two synthetic methods showed differences in the ratio of synthetic and natural polymers present in the resulting combs. The “grafting-to” synthetic route was preferable as the polymers were of higher purity and more defined.     

Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting

We have characterized the requirements to inhibit the function of the plant vacuolar sorting receptor BP80 in vivo and gained insight into the crucial role of receptor recycling between the prevacuolar compartment and the Golgi apparatus. The drug wortmannin interferes with the BP80-mediated route to the vacuole and induces hypersecretion of a soluble BP80-ligand. Wortmannin does not prevent receptor-ligand binding itself but causes BP80 levels to be limiting. Consequently, overexpression of BP80 partially restores vacuolar cargo transport. To simulate receptor traffic, we tested a truncated BP80 derivative in which the entire lumenal domain of BP80 has been replaced by the green fluorescent protein (GFP). The resulting chimeric protein (GFP-BP80) accumulates in the prevacuolar compartment as expected, but a soluble GFP fragment can also be detected in purified vacuoles. Interestingly, GFP-BP80 coexpression interferes with the correct sorting of a BP80-ligand and causes hypersecretion that is reversible by expressing a 10-fold excess of full-length BP80. This suggests that GFP-BP80 competes with endogenous BP80 mainly at the retrograde transport route that rescues receptors from the prevacuolar compartment. Treatment with wortmannin causes further leakage of GFP-BP80 from the prevacuolar compartment to the vacuoles, whereas BP80-ligands are secreted. We propose that recycling of the vacuolar sorting receptor from the prevacuolar compartment to the Golgi apparatus is an essential process that is saturable and wortmannin sensitive.     

Crossing the divide--transport between the endoplasmic reticulum and Golgi apparatus in plants

The transport of proteins between the endoplasmic reticulum (ER) and the Golgi apparatus in plants is an exciting and constantly expanding topic, which has attracted much attention in recent years. The study of protein transport within the secretory pathway is a relatively new field, dating back to the 1970s for mammalian cells and considerably later for plants. This may explain why COPI- and COPII-mediated transport between the ER and the Golgi in plants is only now becoming clear, while the existence of these pathways in other organisms is relatively well documented. We summarize current knowledge of these protein transport routes, as well as highlighting key differences between those of plant systems and those of mammals and yeast. These differences have necessitated the study of plant-specific aspects of protein transport in the early secretory pathway, and this review discusses recent developments in this area. Advances in live-cell-imaging technology have allowed the observation of protein movement in vivo, giving a new insight into many of the processes involved in vesicle formation and protein trafficking. The use of these new technologies has been combined with more traditional methods, such as protein biochemistry and electron microscopy, to increase our understanding of the transport routes in the cell.