The Expression Patterns of Minor Fibrillar Collagens During Development in Zebrafish

Minor fibrillar collagens are recognized as the organizers and nucleators during collagen fibrillogenesis but likely serve additional functions. The minor fibrillar collagens include collagens type V and type XI. Mutations of collagen type V and XI can cause Ehlers Danlos, Stickler\u27s, and Marshall\u27s syndromes in human. We have characterized the spatiotemporal expression patterns of Col11a1, Col11a2, Col5a1 as well as Col5a3 in zebrafish embryos by in situ hybridization. Col5a1 is expressed in developing somites, neural crest, the head mesenchyme, developing cranial cartilage, pharyngeal arches and vertebrae. Col5a3 is detected in the notochord, mesenchyme cells in the eyes and lens. Both Col11a1 and Col11a2 have similar expression patterns, including notochord, otic vesicle, and developing cranial cartilages. Zebrafish may therefore serve as a valuable vertebrate model system for the study of diseases associated with collagens type V and XI mutations

A Validated Software Application to Measure Fiber Organization in Soft Tissue

The mechanical behavior of soft connective tissue is governed by a dense network of fibrillar proteins in the extracellular matrix. Characterization of this fibrous network requires the accurate extraction of descriptive structural parameters from imaging data, including fiber dispersion and mean fiber orientation. Common methods to quantify fiber parameters include fast Fourier transforms (FFT) and structure tensors, however, information is limited on the accuracy of these methods. In this study, we compared these two methods using test images of fiber networks with varying topology. The FFT method with a band-pass filter was the most accurate, with an error of 0.71 ± 0.43 degrees in measuring mean fiber orientation and an error of 7.4 ± 3.0% in measuring fiber dispersion in the test images. The accuracy of the structure tensor method was approximately 4 times worse than the FFT bandpass method when measuring fiber dispersion. A free software application, FiberFit, was then developed that utilizes an FFT band-pass filter to fit fiber orientations to a semicircular von Mises distribution. FiberFit was used to measure collagen fibril organization in confocal images of bovine ligament at magnifications of 63x and 20x. Grayscale conversion prior to FFT analysis gave the most accurate results, with errors of 3.3 ± 3.1 degrees for mean fiber orientation and 13.3 ± 8.2% for fiber dispersion when measuring confocal images at 63x. By developing and validating a software application that facilitates the automated analysis of fiber organization, this study can help advance a mechanistic understanding of collagen networks and help clarify the mechanobiology of soft tissue remodeling and repair

Gonadal Development and Sexual Dimorphism of Gobiomorus dormitor from the Estuarine System of Tecolutla, Veracruz, Mexico

The bigmouth sleeper, Gobiomorus dormitor, is a benthic, euryhaline species, and is very abundant in river mouths, coastal lagoons, and sites away from marine influence from south Florida to Dutch Guyana. There are few studies of its life history, ecology, and abundance, particularly within Mexican waters. Nine trips to Tecolutla estuary, Veracruz, Mexico, were taken between October 1995 and May 1998 to estimate the gonadal development and sexual dimorphism of G. dormitor. A total of 94 individuals ranging from 15–260 mm SL and 0.05–181 g were captured. Seventy-two specimens were adults (60 females, 12 males) and 22 were juveniles that did not show external sexual dimorphism. Both juvenile and adult stages of G. dormitor were captured year-round in seagrass beds and adjacent shallow, muddy or sandy areas. This study has shown that G. dormitor are resident and undergo sexual maturation in the Tecolutla estuary. Histological evidence suggests both males and females undergo gonadal recrudescence in the estuary and have an extended reproductive season from May through November. However, it is unclear if the species actually spawns in the estuary, since females in the final stages of oocyte maturation were not captured. Additional research on the reproductive biology and ecology of this under-studied species is necessary to determine its role in tropical estuaries in the southern Gulf of Mexico. Information learned from areas in the center of its distribution may aid in conserving the species at the periphery of its range in Florida, where it is considered threatened

Depletion of Beclin-1 Due to Proteolytic Cleavage by Caspases in the Alzheimer\u27s Disease Brain

The Beclin-1 protein is essential for the initiation of autophagy and recent studies suggest this function may be compromised in Alzheimer’s disease (AD). In addition, in vitro studies have supported a loss of function of Beclin-1 due to proteolytic modification by caspases. In the present study we examined whether caspase-cleavage of Beclin-1 occurs in the AD brain by designing a site-directed caspase-cleavage antibody based upon a known cleavage site within the protein at position D149. We confirmed that Beclin-1 is an excellent substrate for caspase-3 and demonstrate cleavage led to the formation of a 35 kDa C-terminal fragment labeled by our novel antibody following Western blot analysis. Application of this antibody termed Beclin-1 caspase-cleavage product antibody or BeclinCCP in frontal cortex tissue sections revealed strong immunolabeling within astrocytes that localized with plaque-regions and along blood vessels in all AD cases examined. In addition, weaker, more variable BeclinCCP labeling was also observed within neurofibrillary tangles that co-localized with the early tau conformational marker, MC-1 as well as the late tangle marker, PHF-1. Collectively, these data support a depletion of Beclin-1 in AD following caspase-cleavage

Immunolocalization of an Amino-Terminal Fragment of Apolipoprotein E in the Pick\u27s Disease Brain

Although the risk factor for apolipoprotein E (apoE) polymorphism in Alzheimer\u27s disease (AD) has been well described, the role that apoE plays in other neurodegenerative diseases, including Pick\u27s disease, is not well established. To examine a possible role of apoE in Pick\u27s disease, an immunohistochemical analysis was performed utilizing a novel site-directed antibody that is specific for an amino-terminal fragment of apoE. Application of this antibody, termed the amino-terminal apoE cleavage fragment (nApoECF) antibody, consistently labeled Pick bodies within area CA1 of the hippocampus in 4 of the 5 cases examined. Co-localization of the nApoECF antibody with PHF-1, a general marker for Pick bodies, as well as with an antibody to caspase-cleaved tau (TauC3) was evident within the hippocampus. While staining of the nApoECF antibody was robust in area CA1, little co-localization with PHF-1 in Pick bodies within the dentate gyrus was observed. A quantitative analysis indicated that approximately 86% of the Pick bodies identified in area CA1 labeled with the nApoECF antibody. The presence of truncated apoE within Pick bodies suggests a broader role of apoE beyond AD and raises the question as to whether this protein contributes to pathogenesis associated with Pick\u27s disease

Aggregatibacter Actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association

Background: A repeats-in-toxin (RTX) leukotoxin and its integrin receptor aggregate in cholesterol-rich lipid rafts. Results: The affinity of the toxin to cholesterol is driven by a cholesterol recognition/amino acid consensus (CRAC) motif. Conclusion: Leukotoxin cytotoxicity is regulated by the CRAC motif. Significance: Other RTX toxins contain this CRAC motif, suggesting a role for cholesterol recognition in RTX cytolysis. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc

Propagator of a Charged Particle with a Spin in Uniform Magnetic and Perpendicular Electric Fields

We construct an explicit solution of the Cauchy initial value problem for the time-dependent Schroedinger equation for a charged particle with a spin moving in a uniform magnetic field and a perpendicular electric field varying with time. The corresponding Green function (propagator) is given in terms of elementary functions and certain integrals of the fields with a characteristic function, which should be found as an analytic or numerical solution of the equation of motion for the classical oscillator with a time-dependent frequency. We discuss a particular solution of a related nonlinear Schroedinger equation and some special and limiting cases are outlined.Comment: 17 pages, no figure

In silico APC/C substrate discovery reveals cell cycle-dependent degradation of UHRF1 and other chromatin regulators

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease. Copyright