Free-radical crosslinking of specific proteins alters the function of the egg extracellular matrix at fertilization

All animal embryos begin development by modifying the egg extracellular matrix. This protein-rich matrix protects against polyspermy, microbes and mechanical stress via enzyme-dependent transformations that alter the organization of its constituents. Using the sea urchin fertilization envelope, a well-defined extracellular structure formed within minutes of fertilization, we examine the mechanisms whereby limited permeability is established within this matrix. We find that the fertilization envelope acquires a barrier filtration of 40,000 daltons within minutes of insemination via a peroxidase-dependent mechanism, with dynamics that parallel requisite production of hydrogen peroxide by the zygote. To identify the molecular targets of this free-radical modification, we developed an in vivo technique to label and isolate the modified matrix components for mass spectrometry. This method revealed that four of the six major extracellular matrix components are selectively crosslinked, discriminating even sibling proteins from the same gene. Thus, specific free-radical chemistry is essential for establishing the embryonic microenvironment of early development.     

Distributed delays stabilize neural feedback systems

We consider the effect of distributed delays in neural feedback systems. The avian optic tectum is reciprocally connected with the isthmic nuclei. Extracellular stimulation combined with intracellular recordings reveal a range of signal delays from 3 to 9 ms between isthmotectal elements. This observation together with prior mathematical analysis concerning the influence of a delay distribution on system dynamics raises the question whether a broad delay distribution can impact the dynamics of neural feedback loops. For a system of reciprocally connected model neurons, we found that distributed delays enhance system stability in the following sense. With increased distribution of delays, the system converges faster to a fixed point and converges slower toward a limit cycle. Further, the introduction of distributed delays leads to an increased range of the average delay value for which the system's equilibrium point is stable. The system dynamics are determined almost exclusively by the mean and the variance of the delay distribution and show only little dependence on the particular shape of the distribution.     

Copepods act as a switch between alternative trophic cascades in marine pelagic food webs

A recent meta‐analysis indicates that trophic cascades (indirect effects of predators on plants via herbivores) are weak in marine plankton in striking contrast to freshwater plankton ( Shurin et al. 2002 , Ecol. Lett., 5, 785–791). Here we show that in a marine plankton community consisting of jellyfish, calanoid copepods and algae, jellyfish predation consistently reduced copepods but produced two distinct, opposite responses of algal biomass. Calanoid copepods act as a switch between alternative trophic cascades along food chains of different length and with counteracting effects on algal biomass. Copepods reduced large algae but simultaneously promoted small algae by feeding on ciliates. The net effect of jellyfish on total algal biomass was positive when large algae were initially abundant in the phytoplankton, negative when small algae were dominant, but zero when experiments were analysed in combination. In contrast to marine systems, major pathways of energy flow in Daphnia‐dominated freshwater systems are of similar chain length. Thus, differences in the length of alternative, parallel food chains may explain the apparent discrepancy in trophic cascade strength between freshwater and marine planktonic systems.     

Ontogeny of the basal lamina in the sea urchin embryo

The patterns of expression for several extracellular matrix components during development of the sea urchin embryo are described. An immunofluorescence assay was employed on paraffin-sectioned material using (i) polyclonal antibodies against known vertebrate extracellular matrix components: laminin, fibronectin, heparan sulfate proteoglycan, collagen types I, III, and IV; and (ii) monoclonal antibodies generated against sea urchin embryonic components. Most extracellular matrix components studied were found localized within the unfertilized egg in granules (0.5-2.0 micron) distinct from the cortical granules. Fertilization initiated trafficking of the extracellular matrix (ECM) components from within the egg granules to the basal lamina of the developing embryo. The various ECM components arrived within the developing basal lamina at different times, and not all components were unique to the basal lamina. Two ECM components were not found within the egg. These molecules appeared de novo at the mesenchyme blastula stage, and remained specific to the mesoderm through development. The reactivity of antibodies to vertebrate ECM antigens with components of the sea urchin embryo suggests the presence of immunologically similar ECM molecules between the phyla.     

Physiological and autonomic stress responses after prolonged sleep restriction and subsequent recovery sleep in healthy young men

Sleep and Biological Rhythms - Sleep restriction is increasingly common and associated with the development of health problems. We investigated how the neuroendocrine stress systems respond to...     

Comparison of three methods for beat-to-beat-interval extraction from continuous blood pressure and electrocardiogram with respect to heart rate variability analysis.

In recent years the analysis of heart rate variability (HRV) has become a suitable method for characterizing autonomous cardiovascular regulation. The aim of this study was to investigate the differences in HRV estimated from continuous blood pressure (BP) measurement by different methods in comparison to electrocardiogram (ECG) signals. The beat-to-beat intervals (BBI) were simultaneously extracted from the ECG and blood pressure of 9 cardiac patients (10 min, Colin system, 1000-Hz sampling frequency). For both data types, slope, peak, and correlation detection algorithms were applied. The short-term variability was calculated using concurrent 10-min BP and ECG segments. The root mean square errors in comparison to ECG slope detection were: 1.74 ms for ECG correlation detection; 5.42 ms for ECG peak detection; 5.45 ms for BP slope detection; 5.75 ms for BP correlation detection; and 11.96 ms for BP peak detection. Our results show that the variability obtained with ECG is the most reliable. Moreover, slope detection is superior to peak detection and slightly superior to correlation detection. In particular, for ECG signals with higher frequency characteristics, peak detection often exhibits more artificial variability. Besides measurement noise, respiratory modulation and pulse transit time play an important role in determining BBI. The slope detection method applied to ECG should be preferred, because it is more robust as regards morphological changes in the signals, as well as physiological properties. As the ECG is not recorded in most animal studies, distal pulse wave measurement in combination with correlation or slope detection may be considered an acceptable alternative.     

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman

The sodium (Na⁺)-calcium (Ca²⁺) exchanger 1 (NCX1) is an important regulator of intracellular Ca²⁺ homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na⁺/K⁺-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca²⁺ binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X_5–8Φ₁Φ₂-X_8–9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.     

Stage Specific Glycosylation Pattern for Lactoseries Carbohydrates in the Developing Chick Retina

Based on the idea of differentiation-related changes in the glycosylation pattern of neurons, the expression of two cell surface oligosaccharide epitopes, N-acetyl-lactosamine (NALA), and its sulpho-glucuronyl derivative (HNK-1), was studied, by immunohistochemistry and Western blot experiments, in the developing chick retina beginning on day 2 of incubation (E2) until day 18 post-hatching. NALA was detectable on neuroepithelial cells as soon as the primary optic vesicles formed, and this pattern continued until E3. During subsequent retinal development NALA expression became progressively restricted in concert with the appearance of postmitotic neurons as revealed by neurite outgrowth, and with the formation of synaptic contacts until it disappeared at the end of the incubation period. The pattern of NALA expression was the inverse of HNK-1 which was detected for the first time at E3 on postmitotic ganglion cells accumulating at the vitreal surface. The number of HNK-1+ cells steadily increased until around E10, when the entire neural epithelium was labelled. Synchronously to synaptogenesis, most neurons lost their HNK-1 immunoreactivity. At the time of hatching the adult-like pattern was found, characterised by subpopulations of labelled horizontal, bipolar, amacrine, and ganglion cells. Immunoblot experiments demonstrated transient NALA glycosylation of protein bands, partially identical in their apparent molecular weight to those proteins with HNK-1 glycosylation. The observed temporospatial changes in the glycosylation patterns of distinct proteins during retinal development suggest NALA as a suitable marker for neuronal proliferation, and HNK-1 for differentiation and establishment of final synaptic configuration.