Sek4 and Nuk receptors cooperate in guidance of commissural axons and in palate formation.

Sek4 and Nuk are members of the Eph-related family of receptor protein-tyrosine kinases. These receptors interact with a set of cell surface ligands that have recently been implicated in axon guidance and fasciculation. We now demonstrate that the formation of the corpus callosum and anterior commissure, two major commissural axon tracts that connect the two cerebral hemispheres, is critically dependent on Sek4 and Nuk. While mice deficient in Nuk exhibit defects in pathfinding of anterior commissure axons, sek4 mutants have defects in corpus callosum formation. The phenotype in both axon tracts is markedly more severe in sek4/nuk1 double mutants, indicating that the two receptors act in a partially redundant fashion. sek4/nuk1 double mutants also exhibit specific guidance and fasciculation defects of diencephalic axon tracts. Moreover, while mice singly deficient in either Sek4 or Nuk are viable, most sek4/nuk1 double mutants die immediately after birth primarily due to a cleft palate. These results demonstrate essential and cooperative functions for Sek4 and Nuk in establishing axon pathways in the developing brain, and during the development of facial structures.     

Comparison of Dixon Sequences for Estimation of Percent Breast Fibroglandular Tissue

Objectives

To evaluate sources of error in the Magnetic Resonance Imaging (MRI) measurement of percent fibroglandular tissue (%FGT) using two-point Dixon sequences for fat-water separation.

Methods

Ten female volunteers (median age: 31 yrs, range: 23–50 yrs) gave informed consent following Research Ethics Committee approval. Each volunteer was scanned twice following repositioning to enable an estimation of measurement repeatability from high-resolution gradient-echo (GRE) proton-density (PD)-weighted Dixon sequences. Differences in measures of %FGT attributable to resolution, T₁ weighting and sequence type were assessed by comparison of this Dixon sequence with low-resolution GRE PD-weighted Dixon data, and against gradient-echo (GRE) or spin-echo (SE) based T₁-weighted Dixon datasets, respectively.

Results

%FGT measurement from high-resolution PD-weighted Dixon sequences had a coefficient of repeatability of ±4.3%. There was no significant difference in %FGT between high-resolution and low-resolution PD-weighted data. Values of %FGT from GRE and SE T₁-weighted data were strongly correlated with that derived from PD-weighted data (r = 0.995 and 0.96, respectively). However, both sequences exhibited higher mean %FGT by 2.9% (p < 0.0001) and 12.6% (p < 0.0001), respectively, in comparison with PD-weighted data; the increase in %FGT from the SE T₁-weighted sequence was significantly larger at lower breast densities.

Conclusion

Although measurement of %FGT at low resolution is feasible, T₁ weighting and sequence type impact on the accuracy of Dixon-based %FGT measurements; Dixon MRI protocols for %FGT measurement should be carefully considered, particularly for longitudinal or multi-centre studies.     

Axl and Tyro3 modulate female reproduction by influencing gonadotropin-releasing hormone neuron survival and migration

GnRH neurons must undergo a complex and precise pattern of neuronal migration to appropriately target their projections to the median eminence to trigger gonadotropin secretion and thereby control reproduction. Using NLT GnRH cells as a model of early GnRH neuronal development, we identified the potential importance of Axl and Tyro3, members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases in GnRH neuronal cell survival and migration. Silencing studies evaluated the role of Tyro3 and Axl in NLT GnRH neuronal cells and suggest that both play a role in Gas6 stimulation of GnRH neuronal survival and migration. Analysis of mice null for both Axl and Tyro3 showed normal onset of vaginal opening but delayed first estrus and persistently abnormal estrous cyclicity compared with wild-type controls. Analysis of GnRH neuronal numbers and positioning in the adult revealed a total loss of 24% of the neuronal network that was more striking (34%) when considered within specific anatomical compartments, with the largest deficit surrounding the organum vasculosum of the lamina terminalis. Analysis of GnRH neurons during embryogenesis identified a striking loss of immunoreactive cells within the context of the ventral forebrain compartment (36%) and not more rostrally. Studies using caspase 3 cleavage as a marker of apoptosis showed that Axl(-/-), Tyro3(-/-) double-knockout mice had increased cell death in the nose and dorsal forebrain, supporting the underlying mechanism of cell loss. Together these data suggest that Axl and Tyro3 mediate the survival and appropriate targeting of GnRH neurons to the ventral forebrain, thereby contributing to normal reproductive function and cyclicity in the female.     

Exercise-mediated changes in conduit artery wall thickness in humans: role of shear stress

Episodic increases in shear stress have been proposed as a mechanism that induces training-induced adaptation in arterial wall remodeling in humans. To address this hypothesis in humans, we examined bilateral brachial artery wall thickness using high-resolution ultrasound in healthy men across an 8-wk period of bilateral handgrip training. Unilaterally, shear rate was attenuated by cuff inflation around the forearm to 60 mmHg. Grip strength, forearm volume, and girth improved similarly between the limbs. Acute bouts of handgrip exercise increased shear rate (P < 0.005) in the noncuffed limb, whereas cuff inflation successfully decreased exercise-induced increases in shear. Brachial blood pressure responses similarly increased during exercise in both the cuffed and noncuffed limbs. Handgrip training had no effect on baseline brachial artery diameter, blood flow, or shear rate but significantly decreased brachial artery wall thickness after 6 and 8 wk (ANOVA, P < 0.001) and wall-to-lumen ratio after week 8 (ANOVA, P = 0.005). The magnitude of decrease in brachial artery wall thickness and wall-to-lumen ratio after exercise training was similar in the noncuffed and cuffed arms. These results suggest that exercise-induced changes in shear rate are not obligatory for arterial wall remodeling during a period of 8 wk of exercise training in healthy humans.     

Functional specialization of presynaptic Cav2.3 Ca2+ channels

Ca2+ influx into presynaptic terminals via voltage-dependent Ca2+ channels triggers fast neurotransmitter release as well as different forms of synaptic plasticity. Using electrophysiological and genetic techniques we demonstrate that presynaptic Ca2+ entry through Cav2.3 subunits contributes to the induction of mossy fiber LTP and posttetanic potentiation by brief trains of presynaptic action potentials while they do not play a role in fast synaptic transmission, paired-pulse facilitation, or frequency facilitation. This functional specialization is most likely achieved by a localization remote from the release machinery and by a Cav2.3 channel-dependent facilitation of presynaptic Ca2+ influx. Thus, the presence of Cav2.3 channels boosts the accumulation of presynaptic Ca2+ triggering presynaptic LTP and posttetanic potentiation without affecting the low release probability that is a prerequisite for the enormous plasticity displayed by mossy fiber synapses.     

Melittin-induced leakage from phosphatidylcholine vesicles is modulated by cholesterol: a property used for membrane targeting

Melittin, an amphiphathic peptide, affects the permeability of vesicles. This can be demonstrated using the dye release technique. Calcein, a fluorescent marker, is trapped in large unilamellar 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) vesicles and melittin-induced leakage of the dye can be monitored directly by increasing fluorescence intensity. First, we characterized the effect of increasing cholesterol content in the membrane on melittin-induced leakage and our results reveal that cholesterol inhibits the lytic activity of the peptide. Using intrinsic fluorescence of the single tryptophan of melittin and ²H-NMR of headgroup deuterated phosphatidylcholine, we demonstrated that the affinity of melittin for phosphatidylcholine vesicles is reduced in the presence of cholesterol; this is associated with the tighter lipid packing of the cholesterol-containing bilayer. This reduced binding is responsible for the reduced melittin-induced leakage from cholesterol-containing membranes. The pathway of release was determined to be an all-or-none mechanism. Finally, we investigated the possibility of achieving specific membrane targeting with melittin, when vesicles of different lipid composition are simultaneously present. Melittin incubated together with vesicles made of pure POPC and POPC containing 30(mol)% cholesterol can empty nearly all the cholesterol-free vesicles while the cholesterol-containing vesicles remain almost intact. Owing to the preferential interaction of melittin with the pure POPC vesicles, we were able to achieve controlled release of encapsulated material from a specific vesicle population. Received: 8 May 1996 / Accepted: 12 September 1996     

Simultaneous RP‐HPLC‐DAD Separation,and Determination of Flavonoids and Phenolic Acids in Plantago L. Species

A rapid reversed‐phase (RP) high‐performance liquid chromatography method was developed and applied for simultaneous separation, and determination of flavonoids and phenolic acids in eight Plantago L. taxa (P. altissima L., P. argentea Chaix , P. coronopus L., P. holosteum Scop . ssp. depauperata Pilger , P. holosteum ssp. holosteum, P. holosteum ssp. scopulorum (Degen) Horvati? , P. lagopus L., and P. maritima L.) growing in Croatia. Chromatographic separation was carried out on Zorbax Eclipse XDB‐C18 using gradient elution with a H₂O (pH 2.5, adjusted with CF₃COOH) and MeCN mixture at 30°. The contents of analyzed phenolic compounds (% of the dry weight of the leaves, dw) varied among examined species: rutin (max. 0.024%, P. argentea), hyperoside (max. 0.020%, P. lagopus), quercitrin (max. 0.013%, P. holosteum ssp. holosteum), quercetin (max. 0.028%, P. holosteum ssp. scopulorum), chlorogenic acid (max. 0.115%, P. lagopus), and caffeic acid (max. 0.046%, P. coronopus). Isoquercitrin was detected only in P. argentea (0.020%), while isochlorogenic acid content was below limit of quantification in all investigated species. Multivariate analyses (UPGMA and PCA) showed significant differences in contents of investigated polyphenolic compounds between different Plantago taxa. Accordingly, investigated substances might be employed as chemotaxonomic markers in the study of the complex genus Plantago.     

ADAM17 overexpression promotes angiogenesis by increasing blood vessel sprouting and pericyte number during brain microvessel development

The angiogenic process is precisely regulated by different molecular mechanisms, with a balance between stimulatory and inhibitory factors in embryonic development. Transmembrane proteins of the ADAM (a disintegrin and metalloprotease) family play a critical role in embryogenesis and are involved in protein ectodomain shedding, as well as cell-cell and cell-matrix interactions. In the present study, we found that ADAM17 is expressed spatiotemporally in the tectal layers during chicken embryonic development. To investigate the effect of ADAM17 overexpression on angiogenesis, chicken ADAM17 plasmids were transfected into the developing tectum in vivo by electroporation. Results showed that overexpression of ADAM17 induces morphological changes of brain microvessels, such as an increase in diameter, of capillary sprouting from radial microvessels and an increase in the number of pericytes, but not of endothelial cells. Our data suggest that overexpression of ADAM17 in the developing tectum promotes angiogenesis by increasing the number of pericytes and capillary sprouting in the radial vessels.